Mapping the immune response to the outer domain of a human immunodeficiency virus-1 clade C gp120

The outer domain (OD) of human immunodeficiency virus (HIV)-1 gp120 represents an attractive, if difficult, target for a beneficial immune response to HIV infection. Unlike the entire gp120, the OD is structurally stable and contains the surfaces that interact with both the primary and secondary cellular receptors. The primary strain-specific neutralizing target, the V3 loop, lies within the OD, as do epitopes for two cross-reactive neutralizing monoclonal antibodies (mAbs), b12 and 2G12, and the contact sites for a number of inhibitory lectins. The OD is poorly immunogenic, at least in the context of complete gp120, but purposeful OD immunization can lead to a substantial antibody response. Here, we map the antibody generated following immunization with a clade C OD. In contrast to published data for the clade B OD, the majority of the polyclonal response to the complete clade C OD is to the V3 loop; deletion of the loop substantially reduces immunogenicity. When the loop sequence was substituted for the epitope for 2F5, a well-characterized human cross-neutralizing mAb, a polyclonal response to the epitope was generated. A panel of mAbs against the clade C OD identified two mAbs that reacted with the loop and were neutralizing for clade C but not B isolates. Other mAbs recognized both linear and conformational epitopes in the OD. We conclude that, as for complete gp120, V3 immunodominance is a property of OD immunogens, that the responses can be neutralizing and that it could be exploited for the presentation of other epitopes.

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.

Nitrogen management is essential to prevent tropical oil palm plantations from causing ground-level ozone pollution

More than half the world's rainforest has been lost to agriculture since the Industrial Revolution. Among the most widespread tropical crops is oil palm (Elaeis guineensis): global production now exceeds 35 million tonnes per year. In Malaysia, for example, 13% of land area is now oil palm plantation, compared with 1% in 1974. There are enormous pressures to increase palm oil production for food, domestic products, and, especially, biofuels. Greater use of palm oil for biofuel production is predicated on the assumption that palm oil is an "environmentally friendly'' fuel feedstock. Here we show, using measurements and models, that oil palm plantations in Malaysia directly emit more oxides of nitrogen and volatile organic compounds than rainforest. These compounds lead to the production of ground-level ozone (O-3), an air pollutant that damages human health, plants, and materials, reduces crop productivity, and has effects on the Earth's climate. Our measurements show that, at present, O-3 concentrations do not differ significantly over rainforest and adjacent oil palm plantation landscapes. However, our model calculations predict that if concentrations of oxides of nitrogen in Borneo are allowed to reach those currently seen over rural North America and Europe, ground-level O-3 concentrations will reach 100 parts per billion (10(9)) volume (ppbv) and exceed levels known to be harmful to human health. Our study provides an early warning of the urgent need to develop policies that manage nitrogen emissions if the detrimental effects of palm oil production on air quality and climate are to be avoided.

A new route to furanoeremophilane sesquiterpenoids. Synthesis of Senecio metabolites (+/-)-6-hydroxyeuryopsin, (+/-)-1,10-epoxy-6-hydroxyeuryopsin, (+/-)-toluccanolide A and (+/-)-toluccanolide C

A new strategy for the synthesis of sesquiterpenoids of the furanoeremophilane family was developed in which the tricyclic nucleus was assembled in an A + C -> A - C -> A - B - C sequence. The A - C connection was made via coupling of a cyclohexenylmethyl bromide with a stannylfuran under "ligandless" Stille conditions, and the key cyclization which closed ring B was accomplished with complete stereocontrol by intramolecular formylation of a 2-silylfuran in the presence of trimethylsilyl triflate. This route was used to complete the first total syntheses of the furanoeremophilane 6-hydroxyeuryopsin and the eremophilenolides toluccanolide A and toluccanolide C, as well as a formal synthesis of 1,10-epoxy-6-hydroxyeuryopsin.

Apolipoprotein B-48: comparison of fasting concentrations measured in normolipidaemic individuals using SDS-PAGE, immunoblotting and ELISA

Raised levels of chylomicrons and chylomicron remnants, which circulate following a meal, have been implicated in the development of atherosclerosis. Apolipoprotein (apo) B-48 is exclusively associated with chylomicron particles and provides a specific direct measurement of the number of intestinally derived lipoproteins in the circulation. The quantification of apo B-48 in biological samples is difficult due to the very low concentration in plasma, structural similarity to the N-terminal 48% of apo B-100 and lack of an appropriate standard for apo B-48. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by coomassie blue staining, has been used for many years to measure apo B-48 levels in triacylglycerol (TAG)-rich lipoprotein samples. The raising of antiserum to apo B-48 has led to development of more sensitive and specific methods including immunoblotting and enzyme-linked immunosorbant assays (ELISAs). This has enabled direct measurement of apo B-48 in plasma without the need for separation into TAG-rich lipoproteins. A high degree of variability was observed in the apo B-48 concentrations reported in the literature both within and between the SDS-PAGE, immunoblotting and ELISA methods. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

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.

'Your secret language': classics in the British colonies of West Africa

What were the cultural politics of classics in British colonies? Did classical education operate as a sign of oppression, or as a tool for forging an anti-colonial politics? Why did Europeans bring the classics to West Africa, and how did they manage the developing dynamic when West Africans laid independent claim to the classical heritage? This ground-breaking study examines the ways in which European colonisers and West African nationalists clashed, or collaborated, over the uses of Latin, Greek and the classics.

Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.

Corpus approaches to language in the media

The main aim of this chapter is to offer an overview of research that has adopted the methodology of Corpus Linguistics to study aspects of language use in the media. The overview begins by introducing the key principles and analytical tools adopted in corpus research. To demonstrate the contribution of corpus approaches to media linguistics, a selection of recent corpus studies is subsequently discussed. The final section summarises the strengths and limitations of corpus approaches and discusses avenues for further research.

Legacy effects of grassland management on soil carbon to depth

The importance of managing land to optimise carbon sequestration for climate change mitigation is widely recognised, with grasslands being identified as having the potential to sequester additional carbon. However, most soil carbon inventories only consider surface soils, and most large scale surveys group ecosystems into broad habitats without considering management intensity. Consequently, little is known about the quantity of deep soil carbon and its sensitivity to management. From a nationwide survey of grassland soils to 1 m depth, we show that carbon in grasslands soils is vulnerable to management and that these management effects can be detected to considerable depth down the soil profile, albeit at decreasing significance with depth. Carbon concentrations in soil decreased as management intensity increased, but greatest soil carbon stocks (accounting for bulk density differences), were at intermediate levels of management. Our study also highlights the considerable amounts of carbon in sub-surface soil below 30cm, which is missed by standard carbon inventories. We estimate grassland soil carbon in Great Britain to be 2097 Tg C to a depth of 1 m, with ~60% of this carbon being below 30cm. Total stocks of soil carbon (t ha-1) to 1 m depth were 10.7% greater at intermediate relative to intensive management, which equates to 10.1 t ha-1 in surface soils (0-30 cm), and 13.7 t ha-1 in soils from 30-100 cm depth. Our findings highlight the existence of substantial carbon stocks at depth in grassland soils that are sensitive to management. This is of high relevance globally, given the extent of land cover and large stocks of carbon held in temperate managed grasslands. Our findings have implications for the future management of grasslands for carbon storage and climate mitigation, and for global carbon models which do not currently account for changes in soil carbon to depth with management.

Effects of dietary urea levels on milk protein fractions of Holstein cows

The aim of this study was to evaluate the effects of substituting soybean meal for urea on milk protein fractions (casein, whey protein and non-protein nitrogen) of dairy cows in three dietary levels. Nine mid-lactation Holstein cows were used in a 3 x 3 Latin square arrangement, composed of 3 treatments, 3 periods of 21 days each, and 3 squares. The treatments consisted of three different diets fed to lactating cows, which were randomly assigned to three groups of three animals: (A) no urea inclusion, providing 100% of crude protein (CP), rumen undegradable protein (RUP) and rumen degradable protein (RDP) requirements, using soybean meal and sugarcane as roughage; (B) urea inclusion at 7.5 g/kg DM in partial substitution of soybean meal CP equivalent; (C) urea inclusion at 15 g/kg DM in partial substitution of soybean meal CP equivalent. Rations were isoenergetic and isonitrogenous-1 60 g/kg DM of crude protein and 6.40 MJ/kg DM of net energy for lactation. When the data were analyzed by simple polynomial regression, no differences were observed among treatments in relation to milk CP content, true protein, casein, whey protein, non-casein and non-protein nitrogen, or urea. The milk true protein:crude protein and casein:true protein ratios were not influenced by substituting soybean meal for urea in the diet. Based on the results it can be concluded that the addition of urea up to 15 g/kg of diet dry matter in substitution of soybean meal did not alter milk protein concentration casein, whey protein and its non-protein fractions, when fed to lactating dairy cows. (c) 2007 Elsevier B.V. All rights reserved.

Ca(2+)-activated transbilayer movement of plasma membrane phospholipids in Leishmania donovani during ionomycin or thapsigargin stimulation

The protozoan parasite Leishmania causes serious infections in humans all over the world. After being inoculated into the skin through the bite of an infected sandfly, Leishmania promastigotes must gain entry into macrophages to initiate a successful infection. Specific, surface exposed phospholipids have been implicated in Leishmania-macrophage interaction but the mechanisms controlling and regulating the plasma membrane lipid distribution remains to be elucidated. Here, we provide evidence for Ca(2+)-induced phospholipid scrambling in the plasma membrane of Leishmania donovani. Stimulation of parasites with ionomycin increases intracellular Ca(2+) levels and triggers exposure of phosphatidylethanolamine at the cell surface. We found that increasing intracellular Ca(2+) levels with ionomycin or thapsigargin induces rapid transbilayer movement of NBD-labelled phospholipids in the parasite plasma membrane that is bidirectional, independent of cellular ATP and not specific to the polar lipid head group. The findings suggest the presence of a Ca(2+)-dependent lipid scramblase activity in Leishmania parasites. Our studies further show that lipid scrambling is not activated by rapid exposure of promastigotes to higher physiological temperature that increases intracellular Ca(2+) levels. (C) 2011 Elsevier B.V. All rights reserved.

Insulin suppresses LPS-induced iNOS and COX-2 expression and NF-kappa B activation in alveolar macrophages

The development of septic shock is a common and frequently lethal consequence of gram-negative infection. Mediators released by lung macrophages activated by bacterial products such as lipopolysaccharide (LPS) contribute to shock symptoms. We have shown that insulin downregulates LPS-induced TNF production by alveolar macrophages (AMs). In the present study, we investigated the effect of insulin on the LPS-induced production of nitric oxide (NO) and prostaglandin (PG)-E(2), on the expression of inducible nitric oxide synthase ( iNOS) and cyclooxygenase (COX)-2, and on nuclear factor kappa B (NF-kappa B) activation in AMs. Resident AMs from male Wistar rats were stimulated with LPS (100 ng/mL) for 30 minutes. Insulin (1 mU/mL) was added 10 min before LPS. Enzymes expression, NF-kappa B p65 activation and inhibitor of kappa B (I-kappa B) a phosphorylation were assessed by immunobloting; NO by Griess reaction and PGE(2) by enzyme immunoassay (EIA). LPS induced in AMs the expression of iNOS and COX-2 proteins and production of NO and PGE(2), and, in parallel, NF-kappa B p65 activation and cytoplasmic I-kappa B alpha phosphorylation. Administration of insulin before LPS suppressed the expression of iNOS and COX-2, of NO and PGE(2) production and Nuclear NF-kappa B p65 activation. Insulin also prevented cytoplasmic I-kappa Ba phosphorylation. These results show that in AMs stimulated by LPS, insulin prevents nuclear translocation of NF-kappa B, possibly by blocking I-kappa Ba degradation, and supresses the production of NO and PGE(2), two molecules that contribute to septic shock. Copyright (C) 2008 S. Karger AG, Basel.

Efeito de diferentes fontes de carboidrato e da privação de alimento sobre aspectos biológicos de Telenomus remus Nixon (Hymenoptera, Scelionidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do corante Vermelho de sudan B na marcaçao e no desenvolvimento de Diatraea saccharalis ( Fabricius, 1794) (Lepidoptera: Crambidae) e de Cotesia flavipes (Cameron, 1891) ( Hymenoptera: Braconidae)

The objective of this work was to evaluate biological aspects of Diatraea saccharalis fed on artificial diet containing different concentrations of the Sudan B Red dye and the possibility to mark the parasitoid Cotesia flavipes, when submitted to the parasitism of dyed caterpillars. For that, were added to the artificial diet four concentrations of Sudan Red B dye (100, 200, 300 and 400 ppm) and control (no dye addition). It was evaluated larval and pupal period, larval and pupal viability, longevity, sex rate, pupal weigh, eggs per female, eggs per day, number of eggs per egg mass, egg viability and embrionary period; besides same were accomplished measurements in the caterpillars (bioassay I). Caterpillars of 17 days old (30) of each treatment were removed from the tubes and exposed to the parasitism of C. flavipes (bioassay 2). The egg-pupae period, sex rate, pupal period and viability, number of females, males, total of emerged adults and longevity were evaluated. The data were submitted to the multivariate analisys methods: cluster analysis, two-way and principal component analysis. Based on analysis, it was observed that the treatment of 100 ppm was the least harmful to the biology of the sugar cane borer larvae by groping to the control and did not influence negatively its biological aspects. The concentration of 400 ppm affected negatively the biology of C. flavipes. The Sudan Red B it is ended doses marked the caterpillars and the adults, however the concentration of 100 ppm is the most suitable to dye D. saccharalis. None of the tested concentration marked adults of C. flavipes, despite to affect negatively its biology. It is unviable to increase the concentration seeking futures tests, for that dye to be harmful to the biological aspects of D. saccharalis and C. flavipes.