Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2τ. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.     

Structural properties of bombesin-like peptides revealed by surface-enhanced Raman scattering on roughened silver electrodes

This work presents a Fourier-transform absorption infrared, Fourier-transform Raman, and surface-enhanced Raman scattering (SERS) study of the following peptides belonging to the bombesin-like family: phyllolitorin, [Leu(8)]phyllolitorin, NMB, NMC, and PG-L. The SERS study was undertaken to understand the adsorption mechanism of bombesin-like peptides on an electrochemically roughened silver electrode surface and to show changes in the adsorption mechanism with alterations in amino acids and small tertiary structures. The SERS spectra presented here shows bands mainly associated with the Trp(8) residue vibrations. The presence of mainly pyrrole coring vibrations for phyllolitorin and [Leu(8)]phyllolitorin and mainly benzene coring modes for NMB and NMC indicated that these groups interact with the roughened silver electrode surface. Furthermore, N(1)--C(8) and C(3)--C(9) bonds of the PG-L indole ring seemed to have nearly a vertical orientation on the electrode surface. In addition, distinct vibrations of the C--S fragment were observed in the SERS spectra of [Leu(8)]phyllolitorin and PG-L. The strong enhancement of the nu(C==O) vibration in the [Leu(8)]phyllolitorin SERS spectrum yielded evidence that the intact C==O bond(s) bind strongly to the silver electrode surface, whereas NMC, phyllolitorin, and NMB were located near the silver surface. This finding was supported by the presence of the nu(C--C(==O)) mode. The amide I band observed at 1642 and 1634 cm(-1) for NMB and NMC, respectively, and the Raman amide III band seen in the 1282-1249 cm(-1) range for all peptides except PG-L, indicate that the strongly hydrogen-bonded alpha-helical conformation and random-coil structure are favored for binding to the surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 980-992, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Temperature dependent vibrational modes of glycosidic bond in disaccharide sugars

We studied the temperature dependent vibrational modes of the glycosidic bond in trehalose, sucrose, and maltose at wavenumbers ranging from 1000 to 1200 cm(-1). We found that the slope of temperature dependent Raman shifts of the glycosidic bond in trehalose and sucrose changed at temperatures around 120 degrees C, indicating a bond length or a bond angle (dihedral and torsional angles) change. However, we did not observe any slope change in maltose because the melting temperature of maltose is very close to 120 degrees C. We also found, at temperatures below 120 degrees C, that Raman shifts of the vibrational modes of the glycosidic bond in trehalose showed the strongest temperature dependence among the three disaccharides.     

Clostridium difficile cell-surface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units

Clostridium difficile is a Gram-positive bacterium that is known to be a cause of enteric diseases in humans. It is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. Recently, large outbreaks of C. difficile-associated diarrhea have been reported internationally, and there have been reports of increases in severe disease, mortality and relapse rates. At the moment, there is no vaccine against C. difficile, and the medical prevention of C. difficile infection is mostly based on the prophylactic use of antibiotics; however, this has led to an increase in the incidence of the disease. Here, we describe the chemical structure of C. difficile cell-surface polysaccharides. The polysaccharides of three C. difficile strains were structurally analyzed; ribotype 027 (North American pulsotype 1) strain was observed to express two polysaccharides, one was composed of a branched pentaglycosyl phosphate repeating unit: [-->4)-alpha-l-Rhap-(1-->3)-beta-D-Glcp-(1-->4)-[alpha-l-Rhap-(1-->3]-alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->P] and the other was composed of a hexaglycosyl phosphate repeating unit: [-->6)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->4)-alpha-D-Glcp-(1-->4)-[beta-D-Glcp-(1-->]-beta-D-GalpNAc-(1-->3)-alpha-D-Manp-(1-->P]. The latter polysaccharide was also observed to be produced by strains MOH900 and MOH718. The results described here represent the first literature report describing the covalent chemical structures of C. difficile cell-surface polysaccharides, of which PS-II appears to be a regular C. difficile antigen. These C. difficile teichoic-acid-like polysaccharides will be tested as immunogens in vaccine preparations in a rat and horse model.     

A polysaccharide of Alloiococcus otitidis, a new pathogen of otitis media: chemical structure and synthesis of a neoglycoconjugate thereof

Alloiococcus otitidis is a recently discovered Gram-positive bacterium that has been linked with otitis media (middle ear infections). In this study, we describe the structure of a novel capsular polysaccharide (PS) expressed by the type-strain of A. otitidis, ATCC 51267, and the synthesis of a glycoconjugate composed of the capsule PS and bovine serum albumin (BSA). The capsule PS of A. otitidis type-strain was determined to be a repeating trisaccharide composed of 3-substituted N-acetyl-D-glucosamine (GlcpNAc), 6-substituted N-acetyl-D-galactosamine (GalpNAc), and 4-substituted D-glucuronic acid (GlcpA), of which the majority was amidically decorated with L-glutamic acid (Glu): {-->6)-beta-GalpNAc-(1-->4)-[Glup-->6]-beta-GlcpA-(1-->3)-beta-GlcpNAc-(1}n. Monomeric analysis performed on other A. otitidis strains revealed that similar components were variably expressed, but Glu appeared to be a regular constituent in all the strains examined. Due to the suitable presence of GlcpA and Glu, our approach for glycoconjugate synthesis employed a carbodiimide-based strategy with activation of available carboxyl groups by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), which afforded direct coupling between the capsule PS and BSA. Analysis by mass spectrometry indicated that this A. otitidis capsule PS-BSA conjugate was composed of BSA units that carried up to seven capsule PSs. This work represents the first report in the literature describing an A. otitidis cell-surface carbohydrate and the synthesis of a glycoconjugate preparation thereof. Presently, we are formulating plans to immunologically evaluate this A. otitidis glycoconjugate vaccine in animals.     

枯盘斑多毛孢Pf-毒素组分的研究Ⅰ．活性组分Ⅰ的结构分析

以正丁醇：水：甲醇(4：2：1)作洗脱剂,通过硅胶H60型柱反复柱层析,将3种有致病活性的物质充分纯化,在-40℃下冷冻干燥后,它们为褐色深浅不一的蓬松状物质,且极易吸潮。活性组分Ⅰ(Rf0．83)、活性组分Ⅱ(Rf0．79)和活性组分Ⅲ(Rf0．80)对马尾松切根幼苗和湿地松切根幼苗针叶都有致萎作用,通过质谱(MS)、核磁共振谱(^1HNMR、)和红外光谱(IR)等分析手段确定出所分离的活性组分Ⅰ的化学组成为C5H11O5N(M=165)。     

Association of sugar-based carboranes with cationic liposomes: an electron spin resonance and light scattering study

The possibility of cationic (di-oleoyltrimethylammonium propane, DOTAP)/(l-α-dioleoylphosphatidyl-ethanolamine, DOPE) liposomes to act as carriers of boronated compounds such as 1,2-dicarba-closo-dodecaboran(12)-1-ylmethyl](β-d-galactopyranosyl)-(1→4)-β-d-glucopyranoside and 1,2-di-(β-d-gluco-pyranosyl-ox)methyl-1,2-dicarba-closo-dodeca-borane(12) has been investigated by Electron Spin Resonance (ESR) of n-doxyl stearic acids (n-DSA) and Quasi-Elastic Light Scattering (QELS). Both these carboranes have potential use in Boron Neutron Capture Therapy (BNCT), which is a targeted therapy for the treatment of radiation resistant tumors. They were shown to give aggregation both in plain water and in saline solution. Carborane aggregates were, however, disrupted when DOTAP/DOPE liposome solutions were used as dispersing agents. The computer analysis of the ESR spectra from carborane-loaded liposomes allowed to establish an increase of the order degree in the liposome bilayer with increasing carborane concentration, together with a decreased mobility. The same discontinuities of both correlation time and order parameter with respect to temperature variations were observed in carborane-containing and carborane-free liposomes. This suggested that a homogeneous dispersion of nitroxides and carboranes occurred in the liposome bilayer. The ESR line shape analysis proved that no dramatic changes were induced in the liposome environment by carborane insertion. QELS data showed that the overall liposome structure was preserved, with a slight decrease in the mean hydrodynamic radius and increase in polydispersity caused by the guest molecules.     

神经放电加周期分岔中由随机自共振引起一类新节律

当改变实验性神经起步点细胞外[Ca^2 ]时,放电节律表现出从周期1节律转换为周期4节律的加周期分岔序列。其中,周期n节律转换为周期n 1节律的过程中(n=1,2,3)存在一种新的具有交替特征的节律,该新节律为周期n簇与周期n 1簇放电的交替,并且周期n 1簇的时间间隔序列呈现出整数倍特征。确定性神经放电理论模型(chay模型)只能模拟周期n节律直接到周期n 1节律的加周期分岔序列;而随机chay模型可以模拟实验中的加周期分岔过程和新节律。进一步,新节律被确认是经随机自共振机制产生的。这不仅解释了实验现象,也将随机自共振的产生区间从以前认识到的Hopf分岔点附近扩大到加周期分岔点附近,同时扩大了噪声在神经放电和神经编码中起重要作用的参数区间。     

The inhibitory effects of UV-B radiation (280–315 nm) on Gunnera magellanica growth correlate with increased DNA damage but not with oxidative damage to lipids

Damage to DNA and disruption of membrane integrity by lipid peroxidation processes are two of the proposed causes of UV‐B‐induced growth inhibition in plants. However, the relative significance of these different types of molecular damage has not been established in experiments carried out under realistic physiological conditions. Plants of Gunnera magellanica (a native herb from southern Patagonia) were exposed to a gradient of biologically effective UV‐B doses (from 0 to 6.5 kJ m^?2 d^?1 of UV‐B_be) in a greenhouse study. Leaf expansion was measured and sensitive techniques were used to detect damage to DNA (in the form of cyclobutane pyrimidine dimers; CPDs) and lipid peroxidation (via electronic‐paramagnetic resonance; EPR). Leaf expansion decreased and the CPD density increased with increasing UV‐B doses, but the degree of lipid peroxidation remained unaffected. The highest UV‐B dose induced a transient oxidative stress situation (as evaluated using the ratio of ascorbyl radical to ascorbate, A^·/AH^–), which was rapidly controlled by an increase in the ascorbate pool. The present results suggest that under a range of UV‐B_be doses that overlaps the range of doses that G. magellanica plants experience in their natural environment, growth inhibition is better explained by DNA damage than by increased lipid peroxidation.     

Binding characterization of the interleukin-13 signaling complex and development of a ternary time-resolved fluorescence resonance energy transfer assay

Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Rα1 and IL-4R. Genetic variants of IL-13 and of its receptor components have been linked to asthma. One in particular, IL-13R110Q, is associated with increased IgE levels and asthma. We characterized the interactions of the binary complexes composed of IL-13 or IL-13R110Q with IL-13Rα1 and the ternary complexes composed of IL-13 or IL-13R110Q and IL-13Rα1 with IL-4R using surface plasmon resonance and time-resolved fluorescence resonance energy transfer (TR-FRET). By both biophysical methods, we found no differences between IL-13 and IL-13R110Q binding in either the binary or the ternary complex. IL-4R bound to the IL-13/IL-13Rα1 complex with slow on and off rates, resulting in a relatively weak affinity of about 100 nM. We developed a TR-FRET assay targeting the interaction between the IL-4R and the binary complex. Two antibodies with known binding epitopes to IL-13 that block binding to either IL-13Rα1 or IL-4R inhibited the TR-FRET signal formed by the ternary complex. This assay will be useful to identify and characterize inhibitory molecules of IL-13 function.