The role of lipid composition on the interaction between a tryptophan-rich protein and model bacterial membranes

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

In vitro batch cultures of gut microbiota from healthy and ulcerative colitis (UC) subjects suggest that sulphate-reducing bacteria levels are raised in UC and by a protein-rich diet.

Imbalances in gut microbiota composition during ulcerative colitis (UC) indicate a role for the microbiota in propagating the disorder. Such effects were investigated using in vitro batch cultures (with/without mucin, peptone or starch) inoculated with faecal slurries from healthy or UC patients; the growth of five bacterial groups was monitored along with short-chain fatty acid (SCFA) production. Healthy cultures gave two-fold higher growth and SCFA levels with up to ten-fold higher butyrate production. Starch gave the highest growth and SCFA production (particularly butyrate), indicating starch-enhanced saccharolytic activity. Sulphate-reducing bacteria (SRB) were the predominant bacterial group (of five examined) for UC inocula whereas they were the minority group for the healthy inocula. Furthermore, SRB growth was stimulated by peptone presumably due to the presence of sulphur-rich amino acids. The results suggest raised SRB levels in UC, which could contribute to the condition through release of toxic sulphide.

Probing protein−tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein−tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein−tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein−tannin interactions.

Probing protein-tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein-tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein-tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein-tannin interactions.

Chemical composition and in vitro fermentation of tannin-rich tree fruits

Dry and mature tree fruits are a potential source of protein for goats in the semi-arid areas of southern Africa, but their chemical composition and feeding value is largely unknown. This study presents the chemical composition and in vitro fermentation of indehiscent whole fruits and separated seed and hull fractions from Acacia nilotica, Acacia erubescens, Acacia sieberiana, Acacia erioloba, Piliostigma thonningii and Dichrostachys cinerea trees. Results indicate that the N contents of whole fruits ranged between 13.5 g/kg DM (A. nilotica) and 27.1 g/kg DM (A. erubescens). Seeds had a higher N content than hulls for all tree species. A. nilotica, D. cinerea and P thonningii fruits had high levels of extractable phenolics (758, 458 and 299 g/kg DM, respectively). Soluble phenolics (SPh) and ytterbium precipitable phenolics (YbPh) levels were negatively correlated to in vitro gas production but positively correlated to in vitro organic matter degradability (iOMD). Partition factors for whole fruits at 48 h ranged between 3.6 mg/ml for A. erioloba and 7.8 mg/ml for A. nilotica. Seeds of A. erioloba, A. erubescens and P thonningii were consistently fermented more efficiently throughout the incubation period compared to their whole fruits or hulls. Estimating in vitro degradability of phenolic-rich substrates through filtration procedures can give erroneous results due to the loss of soluble phenolics, which are not necessarily degradable. The feeding value of fruits from D. cinerea and A. nilotica tree species may be reduced due to the presence of high levels of phenolics. (C) 2007 Elsevier B.V. All rights reserved.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

A novel method for production of lipid hydroperoxide- or oxysterol-rich low-density lipoprotein

LDL oxidation may be important in atherosclerosis. Extensive oxidation of LDL by copper induces increased uptake by macrophages, but results in decomposition of hydroperoxides, making it more difficult to investigate the effects of hydroperoxides in oxidised LDL on cell function. We describe here a simple method of oxidising LDL by dialysis against copper ions at 4 degrees C, which inhibits the decomposition of hydroperoxides, and allows the production of LDL rich in hydroperoxides (626 +/- 98 nmol/mg LDL protein) but low in oxysterols (3 +/- 1 nmol 7-ketocholesterol/mg LDL protein), whilst allowing sufficient modification (2.6 +/- 0.5 relative electrophoretic mobility) for rapid uptake by macrophages (5.49 +/- 0.75 mu g I-125-labelled hydroperoxide-rich LDL vs. 0.46 +/- 0.04 mu g protein/mg cell protein in 18 h for native LDL). By dialysing under the same conditions, but at 37 degrees C, the hydroperoxides are decomposed extensively and the LDL becomes rich in oxysterols. This novel method of oxidising LDL with high yield to either a hydroperoxide- or oxysterol-rich form by simply altering the temperature of dialysis may provide a useful tool for determining the effects of these different oxidation products on cell function. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cepsilon

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.

Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four beta-strands and two alpha-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.

An unusual choanoflagellate protein released by hedgehog autocatalytic processing

Hedgehog proteins are important cell-cell signalling proteins utilized during the development of multicellular animals. Members of the hedgehog gene family have not been detected outside the Metazoa, raising unanswered questions about their evolutionary origin. Here we report a highly unusual hedgehog-related gene from a choanoflagellate, a close unicellular relative of the animals. The deduced C-terminal domain, Hoglet-C, is homologous to the autocatalytic domain of Hedgehog proteins and is predicted to function in autocatalytic cleavage of the precursor peptide. In contrast, the N-terminal Hoglet-N peptide has no similarity to the signalling peptide of Hedgehog (Hh-N). Instead, Hoglet-N is deduced to be a secreted protein with an enormous threonine-rich domain of unprecedented size and purity (over 200 threonine residues) and two polysaccharide-binding domains. Structural modelling reveals that these domains have a novel combination of features found in cellulose-binding domains (CBD) of types IIa and IIb, and are expected to bind cellulose. We propose that the two CBD domains enable Hoglet-N to bind to plant matter, tethering an amorphous nucleophilic anchor, facilitating transient adhesion of the choanoflagellate cell. Since HhC and Hoglet-C are homologous, but Hh-N and Hoglet-N are not, we argue that metazoan hedgehog genes evolved by fusion of two distinct genes.

Comparison of heat-treated rapeseed expeller and solvent-extracted soya-bean meal as protein supplements for dairy cows given grass silage-based diets

Sixteen early to mid lactation Finnish Ayrshire dairy cows were used in a cyclic change-over experiment with four 21-day experimental periods and a 4 5 2 factorial arrangement of treatments to evaluate the effects of heat-treated rapeseed expeller and solvent-extracted soya-bean meal protein supplements on animal performance. Dietary treatments consisted of grass silage offered ad libitum supplemented with a fixed amount of a cereal based concentrate (10 kg/day on a fresh weight basis) containing 120, 150, 180 or 210 g crude protein (CP) per kg dry matter (DM). Concentrate CP content was manipulated by replacement of basal ingredients (g/kg) with either rapeseed expeller (R; 120, 240 and 360) or soya-bean meal (S; 80, 160 and 240). Increases in concentrate CP stimulated linear increases (P < 0.05) in silage intake (mean 22.5 and 23.8 g DM per g/kg increase in dietary CP content, for R and S, respectively) and milk production. Concentrate inclusion of rapeseed expeller elicited higher (P < 0.01) milk yield and milk protein output responses (mean 108 and 3.71 g/day per g/kg DM increase in dietary CP content) than soya-bean meal (corresponding values 62 and 2.57). Improvements in the apparent utilization of dietary nitrogen for milk protein synthesis (mean 0.282 and 0.274, for R and S, respectively) were associated with higher (P < 0.05) plasma concentrations of histidine, branched-chain, essential and total amino acids (35, 482, 902 and 2240 and 26, 410, 800 and 2119 mu mol/l, respectively) and lower (P < 0.01) concentrations of urea (corresponding values 4.11 and 4.52 mmol/l). Heat-treated rapeseed expeller proved to be a more effective protein supplement than solvent-extracted soya-bean meal for cows offered grass silage-based diets.

Differential uptake of subfractions of triglyceride-rich lipoproteins by THP-1 macrophages

It is well known that raised plasma triglycerides (TG) are positively linked to the development of coronary heart disease. However, triglycerides circulate in a range of distinct lipoprotein subtractions and the relative atherogenicity of these subtractions is not clear. In this study, three fractions of triglyceride rich lipoprotein (TRL) were isolated from normolipidaemic males according to their differing Svedberg flotation (S-f) rates: chylomicron (CM, S-f > 400), very low-density lipoprotein (VLDL)-1 (S-f 60-400) and VLDL-2 (S-f 20-60). These fractions were incubated with THP-1 monocyte-derived macrophages for determination of cholesterol and TG accumulation, in the presence and absence of the lipoprotein lipase (LPL) inhibitor orlistat. Expression of LDL receptor related protein (LRP) and apolipoprotein B48 receptor (apoB48R) was also examined in both differentiating monocytes, and monocyte-derived macrophages, incubated with TRL. VLDL-I caused a significantly greater accumulation of TG within macrophages compared to VLDL-2. Binding studies also tended to show a greater preference for VLDL-1. No change in expression of LRP or apoB48R was observed in fully differentiated macrophages incubated with VLDL-1, VLDL-2 or CM, although a greater expression of LRP mRNA was observed in differentiating monocytes exposed to VLDL-1, compared to those incubated with CM or VLDL-2. TG loading in response to all three TRL fractions was blocked by orlistat, suggesting that it is likely that the major pathway for uptake of TG was hydrolysis by LPL. Calculations suggested that direct uptake of particles accounts for between 12 and 25% of total TAG uptake. In conclusion, THP monocyte-derived macrophages demonstrate a preference for VLDL-1, both through the LPL pathway and by direct uptake of whole particles. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Influence of apolipoprotein E genotype and dietary alpha-tocopherol on redox status and C-reactive protein levels in apolipoprotein E3 and E4 targeted replacement mice

The molecular basis of the positive association between apoE4 genotype and CVD remains unclear. There is direct in vitro evidence indicating that apoE4 is a poorer antioxidant relative to the apoE3 isoform, with some indirect in vivo evidence also available. Therefore it was hypothesised that apoE4 carriers may benefit from alpha-tocopherol (alpha-Toc) supplementation. Targeted replacement mice expressing the human apoE3 and apoE4 were fed with a diet poor (0 mg/kg diet) or rich (200 mg/kg diet) in alpha-Toc for 12 weeks. Neither apoE genotype nor dietary alpha-Toc exerted any effects on the antioxidant defence system, including glutathione, catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities. In addition, no differences were observed in mitogen-induced lymphocyte proliferation. alpha-Toc concentrations were modestly higher in plasma and lower in tissues of apoE4 compared with apoE3 mice, with the greatest differences evident in the lung, suggesting that an apoE4 genotype may reduce alpha-Toc delivery to tissues. A tendency towards increased plasma F-2-isoprostanes in apoE4 mice was observed, while liver thiobarbituric acid-reactive substances did not differ between apoE3 and apoE4 mice. In addition, C-reactive protein (CRP) concentrations were reduced in apoE4 mice indicating that this positive effect on CRP may in part negate the increased CVD risk associated with an apoE4 genotype.

Probing protein-tannin interactions by isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) has been applied to investigate protein-tannin interactions. Two hydrolyzable tannins were studied, namely myrabolan and tara tannins, for their interaction with bovine serum albumin (BSA), a model globular protein, and gelatin, a model proline-rich random coil protein. Calorimetry data indicate that protein-tannin interaction mechanisms are dependent upon the nature of the protein involved. Tannins apparently interact nonspecifically with the globular BSA, leading to binding saturation at estimated tannin/BSA molar ratios of 48:1 for tara- and 178:1 for myrabolan tannins. Tannins bind to the random coil protein gelatin by a two-stage mechanism. The energetics of the first stage show evidence for cooperative binding of tannins to the protein, while the second stage indicates gradual saturation of binding sites as observed for interaction with BSA. The structure and flexibility of the tannins themselves alters the stoichiometry of the interaction, but does not appear to have any significant affect on the overall binding mechanism observed. This study demonstrates the potential of ITC for providing an insight into the nature of protein-tannin interactions.

The role of 80K/MARCKS, a specific substrate of protein kinase C, in cell growth and tumour progression

Since its discovery more than a decade ago [Wu et al., 1982; Rozengurt et al., 1983], the 80-87 kDa myristoylated a lanine-rich C-kinase substrate (80K/MARCKS) protein has attracted a great deal of attention from researchers interested in cell growth and tumour progression. However, despite its ubiquitous distribution, a definitive functional role for 80K/MARCKS has not been found. The purpose of this review is to describe the properties, distribution and regulation of 80K/MARCKS and to discuss some of the most recent findings, both from our laboratory and from others, that have suggested a functional role for this protein in modulating cell growth and tumour progression. Furthermore, I will present data from our laboratory that implicates 80K/MARCKS as a novel tumour suppressor in cells of melanocyte origin.