Abomasal infusion of casein, starch and soybean oil differentially affect plasma concentrations of gut peptides and feed intake in lactating dairy cows

The effects of specific nutrients on secretion and plasma concentrations of gut peptides (glucagon-like peptide-1((7-36)) amide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin-8 (CCK)) differ across species, but are not reported for cattle. Our objective was to determine acute (hours) and chronic (1 week) effects of increased abomasal supply of protein, carbohydrate, or fat to the small intestine on dry matter intake (DMI) and plasma concentrations of GLP-1, GIP, CCK, and insulin. Four mid-lactation Holstein cows were used in a 4 x 4 Latin square design experiment. Treatments were 7-day abomasal infusions of water, soybean oil (500 g/d), corn starch (1100 g/d), or casein (800 g/d). Jugular vein plasma was obtained over 7 h at the end of the first and last day of infusions. Oil infusion decreased DMI on day 7, but total metabolizable energy (ME) supply (diet plus infusate) did not differ from water infusion. Casein and starch infusion had no effect on feed DMI; thus, ME supply increased. Decreased DMI on day 7 of oil infusion was accompanied by increased plasma GLP-1 concentration, but decreased plasma CCK concentration. Increased plasma GIP concentration was associated with increased ME supply on day 7 of casein and starch infusion. Casein infusion tended to increase plasma CCK concentration on both days of sampling, and increased plasma GLP-1 and insulin concentration on day 1 of infusion. The present data indicate a sustained elevation of plasma concentration of GLP-1, but not CCK, may contribute to the reduced DMI observed in dairy cows provided supplemental fat. (C) 2008 Elsevier Inc. All rights reserved.

Novel single chain antibodies to the prion protein identified by phage display

A well defined structure is available for the carboxyl half of the cellular prion protein (PrPc), while the structure of the amino terminal half of the molecule remains ill defined. The unstructured nature of the polypeptide has meant that relatively few of the many antibodies generated against PrPc recognise this region. To circumvent this problem, we have used a previously characterised and well expressed fragment derived from the amino terminus of PrPc as bait for panning a single chain antibody phage (scFv-P) library. Using this approach, we identified and characterised I predominant and 3 additional scFv-Ps that contained different V-H and V-L sequences and that bound specifically to the PrPc target. Epitope mapping revealed that all scFv-Ps recognised linear epitopes between PrPc residues 76 and 156. When compared with existing monoclonal antibodies (MAb), the binding of the scFvs was significantly different in that high level binding was evident on truncated forms of PrPc that reacted poorly or not at all with several pre-existing MAbs. These data suggest that the isolated scFv-Ps bind to novel epitopes within the aminocentral region of PrPc. In addition, the binding of MAbs to known linear epitopes within PrPc depends strongly on the endpoints of the target PrPc fragment used. (c) 2006 Elsevier Inc. All rights reserved.

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.

Polymorphisms in the apolipoprotein L1 gene and their effects on blood lipid and glucose levels in middle age males

Apolipoprotein L1 in plasma is associated with high- density lipoprotein. Novel APOL1 polymorphisms are investigated along with the association of two common haplotypes (Lys166Glu, Ile244Met, Lys271Arg) with circulating lipid and glucose levels. Although the amino acid substitutions occur in the amphipathic alpha helices region involved in lipid binding, these substitutions were found not to independently account for variability in circulating lipid and glucose levels in 149 middle age males.

A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

Use of a novel Hill-climbing genetic algorithm in protein folding simulations

We have developed a novel Hill-climbing genetic algorithm (GA) for simulation of protein folding. The program (written in C) builds a set of Cartesian points to represent an unfolded polypeptide's backbone. The dihedral angles determining the chain's configuration are stored in an array of chromosome structures that is copied and then mutated. The fitness of the mutated chain's configuration is determined by its radius of gyration. A four-helix bundle was used to optimise simulation conditions, and the program was compared with other, larger, genetic algorithms on a variety of structures. The program ran 50% faster than other GA programs. Overall, tests on 100 non-redundant structures gave comparable results to other genetic algorithms, with the Hill-climbing program running from between 20 and 50% faster. Examples including crambin, cytochrome c, cytochrome B and hemerythrin gave good secondary structure fits with overall alpha carbon atom rms deviations of between 5 and 5.6 Angstrom with an optimised hydrophobic term in the fitness function. (C) 2003 Elsevier Ltd. All rights reserved.

Ligand bridging of the DNA Holliday junction: molecular recognition of a stacked-X four-way junction by a small molecule

Planning a Holliday: A new mode of binding to a stacked-X, four-way Holliday junction is described in which a chromophore molecule binds across the center of the junction and two adenine residues are replaced by the acridine chromophores at either side of the crossover. This binding mode is specific for the Holliday junction and does not cause unwinding of the DNA helices.

Can a consecutive double turn conformation be considered as a peptide based molecular scaffold for supramolecular helix in the solid state?

Helices and sheets are ubiquitous in nature. However, there are also some examples of self-assembling molecules forming supramolecular helices and sheets in unnatural systems. Unlike supramolecular sheets there are a very few examples of peptide sub-units that can be used to construct supramolecular helical architectures using the backbone hydrogen bonding functionalities of peptides. In this report we describe the design and synthesis of two single turn/bend forming peptides (Boc-Phe-Aib-Ile-OMe 1 and Boc-Ala-Leu-Aib-OMe 2) (Aib: alpha-aminoisobutyric acid) and a series of double-turn forming peptides (Boc-Phe-Aib-IIe-Aib-OMe 3, Boc-Leu-Aib-Gly-Aib-OMe 4 and Boc-gamma-Abu-Aib-Leu-Aib-OMe 5) (gamma-Abu: gamma-aminobutyric acid). It has been found that, in crystals, on self-assembly, single turn/bend forming peptides form either a supramolecular sheet (peptide 1) or a supramolecular helix (peptide 2). unlike self-associating double turn forming peptides, which have only the option of forming supramolecular helical assemblages. (c) 2005 Elsevier Ltd. All rights reserved.

Conformational and self-assembly studies of helix forming hexapeptides containing two alpha-amino isobutyric acids

Single crystal X-ray diffraction studies reveal that three hexapeptides with general formula Boc-Ile-Aib-Xx-Ile-Aib-Yy-OMe, where Xx and Yy are Leu in peptide I, Len and Phe in peptide II, and Phe and Leu in peptide III, respectively, adopt equivalent conformations that can be described as mixed 3(10)/alpha-helice with two 4 -> 1 and two 5 -> 1 intramolecular N-H center dot center dot center dot O=C H-bonds. The peptides do not generate any helixterminating Schellman motif despite having Aib at the penultimate position from C-terminus. In the crystalline state, the helices are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. The CD Studies of the three hexapeptides in acetonitrile indicate that they are folded in well-developed 3(10)-helical structures. NMR studies of peptide I in CDCl3 also suggest the formation of a homogeneous 3 m-helical structure. The field emission scanning electron microscopic (FE-SEM) images of peptide 11 in the solid state reveal a non-twisted ribbon-like morphology, which is formed through lateral association of non-twisted filaments. (c) 2007 Elsevier Ltd. All rights reserved.

Overlapping double turn conformations adopted by tetrapeptides containing non-coded alpha-Amino Isobutyric Acid (AIB) and formation of tape-like structures through supramolecular helix mediated self-assembly

Single crystal X-ray diffraction studies and solvent dependent H-1 NMR titrations reveal that a set of four tetrapeptides with general formula Boc-Xx(1)-Aib(2)-Yy(3)-Zz(4)-OMe, where Xx, Yy and Zz are coded L- amino acids, adopt equivalent conformations that can be described as overlapping double turn conformations stabilized by two 4 -> 1 intramolecular hydrogen bonds between Yy(3)-NH and Boc C=O and Zz(4)-NH and Xx(1)C=O. In the crystalline state, the double turn structures are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. Field emission scanning electron microscopic (FE-SEM) images of the tetrapeptides in the solid state reveal that they can form flat tape-like structures. The results establish that synthetic Aib containing supramolecular helices can form highly ordered self-aggregated amyloid plaque like human amylin.

Polymorphisms in the apolipoprotein L1 gene and their effects on blood lipid and glucose levels in middle age males

Apolipoprotein L1 in plasma is associated with high- density lipoprotein. Novel APOL1 polymorphisms are investigated along with the association of two common haplotypes (Lys166Glu, Ile244Met, Lys271Arg) with circulating lipid and glucose levels. Although the amino acid substitutions occur in the amphipathic alpha helices region involved in lipid binding, these substitutions were found not to independently account for variability in circulating lipid and glucose levels in 149 middle age males.

Physiological mechanisms mediating aspartame-induced satiety

Aspartame has been previously shown to increase satiety. This study aimed to investigate a possible role for the satiety hormones cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) in this effect. The effects of the constituents of aspartame, phenylalanine and aspartic acid, were also examined. Six subjects consumed an encapsulated preload consisting of either 400 mg aspartame, 176 mg aspartic acid + 224 mg phenylalanine, or 400 mg corn flour (control), with 1.5 g paracetamol dissolved in 450 ml water to measure gastric emptying. A 1983-kJ liquid meal was consumed 60 min later. Plasma CCK, GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucose, and insulin were measured over 0-120 min. Gastric emptying was measured from 0 to 60 min. Plasma GLP-1 concentrations decreased following the liquid meal (60-120 min) after both the aspartame and amino acids preloads (control, 2096.9 pmol/l min; aspartame, 536.6 pmol/l min; amino acids, 861.8 pmol/l min; incremental area under the curve [AUC] 60-120 min, P<.05). Desire to cat was reduced from 60 to 120 min following the amino acids preload (control, -337.1 mm min; aspartame, -505.4 mm min; amino acids, -1497.1 mm min; incremental AUC 60-120 min, P<.05). However, gastric emptying rates, plasma CCK, GIP, insulin, and glucose concentrations were unaffected. There was a correlation between the increase in plasma phenylalanine and decrease in desire to eat after the liquid meal following the constituent amino acids (r = -.9774, P=.004). In conclusion, it is unlikely that aspartame increases satiety via CCK- or GLP-1-mediated mechanisms, but small changes in circulating phenylalanine concentrations may influence appetite. (C) 2003 Elsevier Science Inc. All rights reserved.

Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite

Protein, generally agreed to be the most satiating macronutrient, may differ in its effects on appetite depending on the protein source and variation in digestion and absorption. We investigated the effects of two milk protein types, casein and whey, on food intake and subjective ratings of hunger and fullness, and on postprandial metabolite and gastrointestinal hormone responses. Two studies were undertaken. The first study showed that energy intake from a buffet meal ad libitum was significantly less 90 min after a 1700 kJ liquid preload containing 48 g whey, compared with an equivalent casein preload (P<0.05). In the second study, the same whey preload led to a 28 % increase in postprandial plasma amino acid concentrations over 3 h compared with casein (incremental area under the curve (iAUC), P<0.05). Plasma cholecystokinin (CCK) was increased by 60 % (iAUC, P<0.005), glucagon-like peptide (GLP)-1 by 65 % (iAUC, P<0.05) and glucose-dependent insulinotropic polypeptide by 36 % (iAUC, P<0.01) following the whey preload compared with the casein. Gastric emptying was influenced by protein type as evidenced by differing plasma paracetamol profiles with the two preloads. Greater subjective satiety followed the whey test meal (P<0.05). These results implicate post-absorptive increases in plasma amino acids together with both CCK and GLP-1 as potential mediators of the increased satiety response to whey and emphasise the importance of considering the impact of protein type on the appetite response to a mixed meal.

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillits phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5. a K-m of 14 mM with alpha-D-Manp-(1 -> 6)-D-Manp as substrate. It was strongly inhibited by Mn2+ and did not need Ca2+ or any other metal cofactor of those tested. The enzyme cleaves specifically (1 -> 6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocus sing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1 -> 3)-D-Manp 10 times faster than alpha-D-Manp-(1 -> 6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1 -> 2)-D-Manp. The activity towards (1 -> 3)-linked mannobiose is strongly activated by 1 mM Ca2+ and inhibited by 10 mM EDTA, while (1 -> 6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities. (c) 2005 Elsevier Ltd. All rights reserved.

Singularities of optimal control problems on some 6-D lie groups

This paper considers the motion planning problem for oriented vehicles travelling at unit speed in a 3-D space. A Lie group formulation arises naturally and the vehicles are modeled as kinematic control systems with drift defined on the orthonormal frame bundles of particular Riemannian manifolds, specifically, the 3-D space forms Euclidean space E-3, the sphere S-3, and the hyperboloid H'. The corresponding frame bundles are equal to the Euclidean group of motions SE(3), the rotation group SO(4), and the Lorentz group SO (1, 3). The maximum principle of optimal control shifts the emphasis for these systems to the associated Hamiltonian formalism. For an integrable case, the extremal curves are explicitly expressed in terms of elliptic functions. In this paper, a study at the singularities of the extremal curves are given, which correspond to critical points of these elliptic functions. The extremal curves are characterized as the intersections of invariant surfaces and are illustrated graphically at the singular points. It. is then shown that the projections, of the extremals onto the base space, called elastica, at these singular points, are curves of constant curvature and torsion, which in turn implies that the oriented vehicles trace helices.