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At least 19 recordsLinked to original sources

Macronutrient selection through post-ingestive signals in sharpsnout seabream fed gelatine capsules and challenged with protein dilution.

Sharpsnout seabream ability for macronutrient self-selection was studied using gelatine capsules containing pure macronutrients. In particular, the existence of non-oropharyngeal chemosensory pathways involved on protein (P), fat (F) and carbohydrate (CH) selection, as well as sharpsnout seabream response to dietary protein dilution were investigated. In a sequence of experimental phases, sharpsnout seabream were fed a pelleted complete diet, an encapsulated complete diet or a combination of separately encapsulated pure macronutrients. In order to induce associative learning, capsules containing a given macronutrient were paired with a particular colour. The animals composed a diet containing 62.7% P, 21.3% CH and 16.0% F, in terms of macronutrient percentage intake, and this selection pattern was maintained throughout all experimental phases. In a second experiment, individually kept sharpsnout seabream were challenged with protein dilution. After protein capsules were diluted (from 91.9% P to 56.3% P) with cellulose, the animals increased their protein intake to compensate for dilution in such a way that their energy intake was not significantly modified (17.4 kJ/100 g BW vs. 17.6 kJ/100 g BW after dilution). These results show that sharpsnout seabream feeding on encapsulated diets are able to select and maintain a particular diet composition, as well as sustain their energy intake, without using the diet's oropharyngeal chemosensory properties. Moreover, they were also able to maintain their protein intake after dilution, which highlights the importance of this macronutrient in this omnivorous species.

Animals↗

An apparatus for the concentration of large volumes of dilute protein solutions to a predetermined volume.

An apparatus was designed and manufactured that can be used for the concentration of large quantities of dilute protein solutions to a predetermined volume. The method is based on the osmotic transfer of water to a concentrated hydrophilic polymer (poly ethylene glycol, PEG) through a protein-stopping dialysis membrane and is a refinement of a method previously reported by van Oss. The apparatus is made of perspex. Concentration takes place through a commercially available dialysis tube and is aided by a 40% polyethylene glycol solution. Large volumes (5l) of dilute protein solution could be reduced to 50 ml at a rate of 30 ml per hour with no significant loss in biological activity.

Animals↗

Inverse correlation between pink-eyed dilution protein expression and induction of melanogenesis by bafilomycin A1.

The pink-eyed dilution protein (p) plays a pivotal role in the synthesis of eumelanin. In its absence, critical melanosomal proteins fail to traffic to the melanosome. Pink-eyed dilution gene (P) mutations are the most common cause of tyrosinase-positive oculocutaneous albinism worldwide. Thus, reports that bafilomycin A1 was able to induce synthesis of melanin in tyrosinase-positive melanomas led us to test the drug on p-null murine melanocytes. We found that in melanocytes lacking p, bafilomycin A1 was able to induce melanin synthesis. These cells, once transfected with an expression vector encoding an epitope-tagged p transcript, failed to respond to the drug. The increase in melanin synthesis is accompanied by a reduction in tyrosinase protein cleavage and secretion with subsequent accumulation within the melanocyte. Bafilomycin A1 has also been reported to induce pigmentation of normal Caucasian melanocytes. Based on these data we hypothesize that p may serve as a key control point at which ethnic skin color variation is determined.

Animals↗

A statistical-mechanical theory of fibril formation in dilute protein solutions.

We outline a theoretical treatment that describes fibril formation in dilute protein solutions. For this, we combine a theory describing self-assembly and conformational transition with a description of the lateral association of linear chains. Our statistical-mechanical model is able to predict the mean degree of polymerization and the length of the fibrils and their precursors, as well as the weight fractions of the different aggregated species in solution. We find that there appear to exist two regimes as a function of concentration, and as a function of the free energies of protein association: one in which low-molecular weight compounds dominate and one in which the fibrils do. The transition between these regimes can be quite sharp, and becomes sharper as more filaments are allowed to associate into a single fibril. The fraction of fibrils consisting of less than the maximum allowed number of filaments turns out to be negligible, in agreement with experimental studies, where the fibril thickness is found to be practically monodisperse. In addition, we find that the description of the fibril ends has a large effect on the predicted fibril length.

Amyloid↗

Pink-eyed dilution protein modulates arsenic sensitivity and intracellular glutathione metabolism.

Mutations in the mouse p (pink-eyed dilution) and human P genes lead to melanosomal defects and ocular developmental abnormalities. Despite the critical role played by the p gene product in controlling tyrosinase processing and melanosome biogenesis, its precise biological function is still not defined. We have expressed p heterologously in the yeast Saccharomyces cerevisiae to study its function in greater detail. Immunofluorescence studies revealed that p reaches the yeast vacuolar membrane via the prevacuolar compartment. Yeast cells expressing p exhibited increased sensitivity to a number of toxic compounds, including arsenicals. Similarly, cultured murine melanocytes expressing a functional p gene were also found to be more sensitive to arsenical compounds compared with p-null cell lines. Intracellular glutathione, known to play a role in detoxification of arsenicals, was diminished by 50% in p-expressing yeast. By using the glutathione-conjugating dye monochlorobimane, in combination with acivicin, an inhibitor of vacuolar gamma-glutamyl cysteine transpeptidase, involved in the breakdown of glutathione, we found that p facilitates the vacuolar accumulation of glutathione. Our data demonstrate that the pink-eyed dilution protein increases cellular sensitivity to arsenicals and other metalloids and can modulate intracellular glutathione metabolism.

Acid Phosphatase↗

Experimental and numerical studies of the chromatofocusing of dilute proteins using retained pH gradients formed on a strong-base anion-exchange column.

The separation of dilute protein mixtures was achieved using simple monovalent buffering species to form retained, internally produced pH gradients on a strong-basic anion-exchange column. Highly focused proteins bands localized on stepwise pH transitions were produced experimentally under trace and volume overloaded feed conditions. Numerical simulations were performed that accurately predict the pH profile and protein band shapes in the column effluent. Experimental results were combined with numerical investigations to explore strategies for designing efficient preparative-scale chromatofocusing systems using simple, inexpensive buffers and adsorbents.

Buffers↗

Broiler response to energy or energy and protein dilution in the finisher diet.

Two experiments were carried out with male broiler chickens in which diets from 35 to 49 d of age were diluted by oat hulls and sand. In one experiment only energy was diluted; in the second experiment, both energy and protein (amino acid) levels were affected. Each diet was tested with four replicate floor pens each containing 25 1-d-old birds. All birds received a standard starter diet to 16 d and then grower diet to 35 d. In Experiment 1, dilution of only energy resulted in a significant linear (P < 0.01) reduction in body weight at 42 d, although there was growth compensation after this time such that all birds weighed the same at 49 d. Diet energy dilution resulted in increased feed intake, although energy intake was not maintained (P < 0.01). Diet energy dilution generally had little effect on carcass weight or yield of breast meat, although there was less abdominal fat (P < 0.01). In Experiment 2, dilution of both energy and protein had a comparable effect on growth rate as described in Experiment 1; however, in this study there was a linear decrease in carcass weight and breast meat yield as the diet was diluted. These results suggest that the male broiler chicken can grow quite well on very low energy diets but that a period of at least 7 d is required for adjustment to feed intake. Even with compensatory increase in feed intake, however, the bird is unable to maintain its energy intake when fed such diluted diets.

Amino Acids↗

Effect of type III antifreeze protein dilution and mutation on the growth inhibition of ice.

Mutation of residues at the ice-binding site of type III antifreeze protein (AFP) not only reduced antifreeze activity as indicated by the failure to halt ice crystal growth, but also altered ice crystal morphology to produce elongated hexagonal bipyramids. In general, the c axis to a axis ratio of the ice crystal increased from approximately 2 to over 10 with the severity of the mutation. It also increased during ice crystal growth upon serial dilution of the wild-type AFP. This is in marked contrast to the behavior of the alpha-helical type I AFPs, where neither dilution nor mutation of ice-binding residues increases the c:a axial ratio of the ice crystal above the standard 3.3. We suggest that the ice crystal morphology produced by type III AFP and its mutants can be accounted for by the protein binding to the prism faces of ice and operating by step growth inhibition. In this model a decrease in the affinity of the AFP for ice leads to filling in of individual steps at the prism surfaces, causing the ice crystals to grow with a longer c:a axial ratio.

Animals↗

Protein dilution effect on thiouracil-seroalbumin interactions.

As the spectra and binding parameters calculated for the thiouracil-albumin interaction change with the protein concentration, a human seroalbumin conformational change depending on protein concentration has been suggested. This protein-conformational change is tested by dilatometry and viscosimetry. At low concentrations, albumin showed a greater thiouracil binding capacity and a second positive peak in its interaction with the drug, detected by difference spectroscopy. Both effects are due to a monomerisation of protein dimers and not to a conformational change depending on protein concentration. This monomerisation would imply a major accessibility of thiouracil and propylthiouracil to other binding sites on HSA.

Binding Sites↗

The pinkeyed-dilution protein and the eumelanin/pheomelanin switch: in support of a unifying hypothesis.

The two major types of mammalian melanin are pheomelanin (yellow or red pigment) and eumelanin (black or brown). The agouti (A) and extension (E) loci determine whether follicular melanocytes will deposit pheomelanin or eumelanin within their melanosomes. Mutations at the murine pinkeyed-dilution (P) locus cause a striking reduction in deposition of eumelanic, but not pheomelanic, pigment. The mRNA encoded at the P locus is not expressed in skin that exclusively produces pheomelanic pigment as a result of mutation at the agouti locus. We have suggested, based upon both genetic and biochemical evidence, that three key melanogenic proteins--tyrosinase, tyrosinase-related-protein-1 (TRP-1), and TRP-2, encoded at the albino (C), brown (B), and slaty (Slt) loci, respectively--form a high-molecular-weight "melanogenic complex" within the melanosome. High-molecular-weight forms of tyrosinase, TRP-1 and TRP-2, are absent from eumelanic ocular tissues of p(un)/p(un) mice that fail to produce normal P-locus transcript, even though these mice are genetically normal at the loci that regulate production of the three melanogenic proteins. We have hypothesized that the presence of the p-locus protein is important for the integrity of the melanogenic complex and for the levels of members of the TRP family. We show here that the yellow skins of mice mutant at the agouti or extension loci, as well as the nonyellow skins of pinkeyed-unstable (p(un)/p(un)) mice, demonstrate greatly diminished levels of tyrosinase, TRP-1 and TRP-2, and an absence or markedly decreased proportion of high-molecular-weight forms of melanogenic proteins. We conclude that normal levels of wild-type P-locus protein are necessary for eumelanogenesis and that the absence of this protein may be necessary, but is not sufficient to cause the melanosome to switch to the production of pheomelanin. We discuss the implications of our results in relation to the interacting genetic controls regulating melanogenesis.

Agouti Signaling Protein↗

Direct observation of the self-association of dilute proteins in the presence of inert macromolecules at high concentration via tracer sedimentation equilibrium: theory, experiment, and biological significance.

The technique of tracer sedimentation equilibrium [Rivas, G., et al. (1994) Biochemistry, 2341-2348 (1); Rivas, G., et al. (1996) J. Mol. Recognit. 9, 31-38 (2)] is utilized, together with an extension of the theory of sedimentation equilibrium of highly nonideal solutions [Chatelier and Minton, (1987) Biopolymers 26, 1097-1113 (3)], to characterize the thermodynamic activity and/or the state of association of a dilute, labeled macromolecular solute in the presence of an arbitary concentration of a second, unlabeled macromolecular solute. Experiments are performed on solutions of labeled fibrinogen (0.25-1 g/L) in bovine serum albumin (0-100 g/L) in the presence and absence of divalent cations (Ca(2+), Mg(2+)), and on solutions of labeled tubulin (0.2-0.6 g/L) in dextran (0-100 g/L). It is found that in the absence of the divalent cations, the large dependence of the thermodynamic activity of fibrinogen on BSA concentration is well accounted for by a simple model for steric repulsion. In the presence of the cations and sufficiently large concentrations of BSA (>30 g/L), fibrinogen appears to self-associate to a weight-average molar mass approximately twice that of monomeric fibrinogen. Tubulin appears to self-associate to an extent that increases monotonically with increasing dextran concentration, reaching a weight-average molar mass almost 3 times that of the alphabeta dimer in the presence of 100 g/L dextran. Possible biological ramifications are discussed.

Buffers↗