Interfacial structure of wild-type and mutant forms of puroindoline-b bound to DPPG monolayers

The interaction of wild-type puroindoline-b (Pin-b+) and two mutant forms having single residue substitutions (G46S or W44R) with L-alpha-dipalmitoylphosphatidyl-dl-glycerol (DPPG) as a Langmuir monolayer at the air/water interface was investigated by neutron reflectivity (NR) and Brewster angle microscopy (BAM). NR profiles were fitted using a three-layer model to enable differences in penetration of protein between the lipid headgroup and acyl regions to be determined. The data showed similar surface excesses for each of the three proteins at the interface; however, it was revealed that the depth of penetration of protein into the lipid region differed for each protein with Pin-b+ penetrating further into the acyl region of the lipid compared to the mutant forms of the protein that interacted with the headgroup region only. BAM images revealed that the domain structure of the DPPG monolayers was disrupted when Pin-b+ adsorption had reached equilibrium, suggesting protein penetration had led to compression of the lipid region. In contrast, the domain structure was unaffected by the W44R mutant, suggesting no change in compression of the lipid region and hence little or no penetration of protein into the lipid layer.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Synthesis and crystal structure of [(BU4N)-B-n][Er(pic)(4)] center dot 5.5H(2)O: a new infrared emitter

The new erbium(III) complex of picolinic acid (Hpic), ["Bu4N][Er(pic)(4)].5.5H(2)O, was synthesized and the crystal structure determined by single-crystal X-ray diffraction. The compound was further characterized using IR, Raman, H-1 NMR and elemental analysis. The picolinate ligands (pic(-)) are coordinated through N,O-chelation to the erbium cations, as shown by X-ray diffraction and spectroscopic results, leading to an eight coordinate complex. Photoluminescence measurements were performed for this compound which exhibits infrared emission. (C) 2003 Elsevier B.V. All rights reserved.

Identification of Bartonella species in rodents, shrews and cats in Denmark: detection of two B-henselae variants, one in cats and the other in the long-tailed field mouse

Small mammals and stray cats were trapped in two areas of North Zealand, Denmark, and their blood cultured for hemotrophic bacteria. Bacterial isolates were recovered in pure culture and subjected to 16S rDNA gene sequencing. Bartonella species were isolated from five mammalian species: B. grahamii from Microtus agrestis (field vole) and Apodemus flavicollis (yellow-necked field mouse); B. taylorii from M. agrestis, A. flavicollis and A. sylvaticus (long-tailed field mouse); B. tribocorum from A. flavicollis; R vinsonii subsp. vinsonii from M. agrestis and A. sylvaticus; and B. birtlesii from Sorex vulgaris (common shrew). In addition, two variant types of B. henselae were identified: variant I was recovered from three specimens of A. sylvaticus, and B. henselae variant 11 from I I cats; in each case this was the only B. henselae variant found. No Bartonella species was isolated from Clethrionomys glareolus (bank vole) or Micromys minutus (harvest mouse). These results suggest that B. henselae occurs in two animal reservoirs in this region, one of variant I in A. sylvaticus, which may be transmitted between mice by the tick Ixodes ricinus, and another of variant 11 in cats, which may be transmitted by the cat flea (Ctenocephalides felis). To our knowledge, this is the first report of the occurrence of B. henselae and B. tribocorum in Apodemus mice.

Operator Theory and Its Applications: In Memory of V. B. Lidskii (1924-2008)

This book is a collection of articles devoted to the theory of linear operators in Hilbert spaces and its applications. The subjects covered range from the abstract theory of Toeplitz operators to the analysis of very specific differential operators arising in quantum mechanics, electromagnetism, and the theory of elasticity; the stability of numerical methods is also discussed. Many of the articles deal with spectral problems for not necessarily selfadjoint operators. Some of the articles are surveys outlining the current state of the subject and presenting open problems.

Measurement of apolipoprotein B-48 in the Svedberg flotation rate (Sf) > 400, Sf 60 – 400 and Sf 20 – 60 lipoprotein fractions reveals novel findings with respect to the effects of dietary fatty acids on triacylglycerol-rich lipoproteins in postmenopausal women

The present study was designed to examine whether the type of fat ingested in an initial test meal influences the response and density distribution of dietary-derived lipoproteins in the Svedberg flotation rate (Sf)>400, Sf 60 - 400 and Sf 20 - 60 lipoprotein fractions. A single-blind randomized within-subject crossover design was used to study the effects of palm oil, safflower oil, a mixture of fish and safflower oil, and olive oil on postprandial apolipoprotein (apo) B-48, retinyl ester and triacylglycerol responses in each lipoprotein fraction following an initial test meal containing one of the oils and a second standardized test meal. For all dietary oils, late postprandial (300min) concentrations of triacylglycerol and apo B-48 were significantly higher in the Sf 60 - 400 fraction than in the Sf>400 fraction (P<0.02). Significantly greater apo B-48 incremental areas under the curve (IAUCs) were also observed in the Sf 60 - 400 fraction than in the Sf>400 fraction following palm oil, safflower oil and olive oil (P<0.04), with a similar non-significant trend for fish/safflower oil. Olive oil resulted in a significantly greater apo B-48 IAUC in the Sf>400 fraction (P<0.02) than did any of the other dietary oils, as well as a tendency for a higher IAUC in the Sf 60 - 400 fraction compared with the palm, safflower and fish/safflower oils. In conclusion, we have found that the majority of intestinally derived lipoproteins present in the circulation following meals enriched with saturated, polyunsaturated or monounsaturated fatty acids are of the density and size of small chylomicrons and chylomicron remnants. Olive oil resulted in a greater apo B-48 response compared with the other dietary oils following sequential test meals, suggesting the formation of a greater number of small (Sf 60 - 400) and large (Sf>400) apo B-48-containing lipoproteins in response to this dietary oil.

Quantitation of apolipoprotein B-48 in triacylglycerol-rich lipoproteins by a specific enzyme-linked immunosorbent assay

This paper describes the use of an antiserum, specific for apolipoprotein (apo) B-48, in a competitive, enzyme-linked immunosorbent assay (ELISA) for apo B-48 in triacylglycerol-rich lipoprotein (TRL) fractions prepared from fasting and post-prandial plasma samples. Previously we showed the antiserum to act as an effective immunoblotting agent following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its use in this ELISA indicates that the antiserum recognises the C-terminal region of the protein on the surface of lipoprotein particles. The ELISA had a sensitivity of less than 37 ng/ml and intra- and inter-assay coefficients of variation of 3.8% and 8.6%, respectively. There was no cross-reaction in the ELISA against serum albumin, ovalbumin, thyroglobulin, or apo B-100 (purified by immunoaffinity chromatography), and high lipid concentrations (as Intralipid) did not interfere. A low density lipoprotein fraction reacted in the ELISA but SDS-PAGE-Western blot analysis confirmed the presence, in the fraction, of a small amount of apo B-48, indicating the existence of low density dietary-derived lipoprotein particles. ELISA and SDS-PAGE-Western blot analysis were used to measure apo B-48 in 12 series of postprandial samples collected from 4 diabetic and 8 normal subjects, following test meals of varying fat content. The mean correlation between the two methods was r = 0.74. The mean fasting concentration of apo B-48 in the TRL fractions from 15 healthy men was 0.46 μg/ml of plasma.

Regulation of NF-κB activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Sustained activation of nuclear transcription factor κB (NF-κB) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-κB signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNFα, 150 ng/ml) increased NF-κB-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative IκBα construct. In addition, TNFα increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-κB activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((−)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1–1 μM) for 18 h. None of the flavonoids modulated constitutive or TNFα-induced NF-κB activity. Therefore, we conclude that NF-κB signalling in astrocytes is not a major target for flavonoids.

Towards the total synthesis of mycaperoxide B: probing biosynthetic rationale

Studies towards the biomimetic synthesis of mycaperoxide B (1) are described. We have established the synthesis of four diastereoisomers of mycaperoxide B methyl ester (1a) by employing a Michael addition across an α,β-unsaturated ester precursor 2 as the key step. This result strongly suggestsstereocontrol in the addition of the hydroperoxide functionality to the E double bond and discloses the importance of choosing the correct geometry of the α,β-unsaturated double bond when attempting to synthesise mycaperoxide B. Four diastereoisomeric tetrahydrofurans derived from an intramolecular rearrangement of the 1,2-dioxolane enolate 12 were also isolated and characterised.

Proteomic analyses of a Listeria monocytogenes mutant lacking σB identify new components of the σB regulon and highlight a role for σB in the utilization of glycerol

In Listeria monocytogenes the alternative sigma factor σB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σB dependent; 17 of these proteins were found to require the presence of σB for full expression, while 21 were expressed at a higher level in the ΔsigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σB in the general stress response and highlighted a probable role for σB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σB in glycerol metabolism. Five newly identified members of the σB regulon were shown to be under direct regulation of σB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σB was shown to play a major role in the stationary phase of growth in complex media.

Nano structures through self-assembly of protected hydrophobic amino acids: encapsulation of rhodamine B dye by proline-based nanovesicles

The development of novel molecules for the creation of nanometer structures with specific properties has been the current interest of this research. We have developed a set of molecules from hydrophobic omega- and alpha-amino acids by protecting the -NH(2) with Boc (t-butyloxycarbonyl) group and -CO(2)H with para-nitroanilide such as BocHN-Xx-CONH-(p-NO(2))center dot C(6)H(4), where Xx is gamma-aminobutyric acid (gamma-Abu), (L)-isoleucine, alpha-aminoisobutyric acid, proline, etc. These molecules generate various nanometer structures, such as nanofibrils, nanotubes and nanovesicles, in methanol/water through the self-assembly of bilayers in which the nitro benzene moieties are stacked in the middle and the Boc-protected amino acids parts are packed in the outer surface. The bilayers can be further stacked one over the other through hydrophobic interactions to form multilayer structure, which helps to generate different kinds of nanoscopic structures. The formation of the nanostructures has been facilitated through the participation of various noncovalent interactions, such as hydrophobic interactions, hydrogen bonding and aromatic p-stacking interactions. Fluorescence microscopy and UV studies reveal that the nanovesicles generated from pro-based molecule can encapsulate dye molecules which can be released by addition of acid (at pH 2). These single amino acid based molecules are both easy to synthesize and cost-effective and therefore offer novel scaffolds for the future design of nanoscale structures.

Quantification of UV-B flux through time using UV-B-absorbing compounds contained in fossil Pinus sporopollenin

• UV-B radiation currently represents c. 1.5% of incoming solar radiation. However, significant changes are known to have occurred in the amount of incoming radiation both on recent and on geological timescales. Until now it has not been possible to reconstruct a detailed measure of UV-B radiation beyond c. 150 yr ago. • Here, we studied the suitability of fossil Pinus spp. pollen to record variations in UV-B flux through time. In view of the large size of the grain and its long fossil history, we hypothesized that this grain could provide a good proxy for recording past variations in UV-B flux. • Two key objectives were addressed: to determine whether there was, similar to other studied species, a clear relationship between UV-B-absorbing compounds in the sporopollenin of extant pollen and the magnitude of UV-B radiation to which it had been exposed; and to determine whether these compounds could be extracted from a small enough sample size of fossil pollen to make reconstruction of a continuous record through time a realistic prospect. • Preliminary results indicate the excellent potential of this species for providing a quantitative record of UV-B through time. Using this technique, we present the first record of UV-B flux during the last 9500 yr from a site near Bergen, Norway.

1,8-Cineol inhibits nuclear translocation of NF-κB p65 and NF-κB-dependent transcriptional activity

Natural plant-derived products are commonly applied to treat a broad range of human diseases, including cancer as well as chronic and acute airway inflammation. In this regard, the monoterpene oxide 1,8-cineol, the active ingredient of the clinically approved drug Soledum®, is well-established for the therapy of airway diseases, such as chronic sinusitis and bronchitis, chronic obstructive pulmonary disease and bronchial asthma. Although clinical trials underline the beneficial effects of 1,8-cineol in treating inflammatory diseases, the molecular mode of action still remains unclear. Here, we demonstrate for the first time a 1,8-cineol-depending reduction of NF-κB-activity in human cell lines U373 and HeLa upon stimulation using lipopolysaccharides (LPS). Immunocytochemistry further revealed a reduced nuclear translocation of NF-κB p65, while qPCR and western blot analyses showed strongly attenuated expression of NF-κB target genes. Treatment with 1,8-cineol further led to increased protein levels of IκBα in an IKK-independent matter, while FRET-analyses showed restoring of LPS-associated loss of interaction between NF-κB p65 and IκBα. We likewise observed reduced amounts of phosphorylated c-Jun N-terminal kinase 1/2 protein in U373 cells after exposure to 1,8-cineol. In addition, 1,8-cineol led to decreased amount of nuclear NF-κB p65 and reduction of its target gene IκBα at protein level in human peripheral blood mononuclear cells. Our findings suggest a novel mode of action of 1,8-cineol through inhibition of nuclear NF-κB p65 translocation via IκBα resulting in decreased levels of proinflammatory NF-κB target genes and may therefore broaden the field of clinical application of this natural drug for treating inflammatory diseases.