Bioavailability of manganese from manganese proteinate, manganese sulfate and manganese oxide in broilers reared under heat distress

The relative bioavailabilities of manganese from manganese monoxide/ manganese sulfate and Mn-proteinate were compared in two different environmental conditions. Birds were raised at a cycling temperature of 18.3 to 23.9 °C or at a cycling temperature of 23 to 35 °C. Experimental animals used were day-old male commercial broiler chicks (Arbor Acre X Arbor Acre). Treatments were prepared from a starter and a grower diet (26 ppm Mn dry matter basis) which were supplemented with 0, 1000, 2000, and 3000 ppm Mn as Mnproteinate, manganese sulfate or manganese monoxide. Birds had ad libitum access to feed and water. Bone was the most sensitive tissue followed by kidney to manganese supplementation from all sources. Tibia manganese concentration increased linearly (P \u3c 0.05). Based on ratios of slopes from multiple linear regression analysis of bone manganese on manganese intake from various sources, the relative bioavailabilities were 120 and 91% from manganese proteinate and manganese oxide, respectively, compared with 100% from manganese sulfate in three-week old chicks and 125 and 83% from manganese proteinate and manganese oxide, respectively, compared with 100% from manganese sulfate in chicks older than three weeks. All values were significantly different from 100% (P \u3c 0.05). Heat distress was observed to increase the manganese bioavailabilities of the various manganese sources (P \u3c 0.05) . Manganese from Mn-proteinate was more available than manganese from manganese sulfate followed by manganese oxide under each environmental temperature regimen

Doctor of Philosophy

dissertationMost proteins assemble into oligomeric complexes. These supramolecular associations may confer many advantages to the substituents. Polyhedral capsids represent a common, highly symmetric nanoscale architecture in which multiple subunits self-assemble to form a hollow three-dimensional surface which often serve as molecular containers or platforms for multivalent display of ligands. Capsids can be tailored to serve in applications such as drug delivery, biocatalysis, and materials synthesis. In this dissertation, I present a body of work undertaken on the Aquifex aeolicus lumazine synthase (AaLS) capsid to expand our knowledge of supramolecular protein associations and to generate capsids with novel functions. First, the construction and characterization of a novel nanoreactor is described. Using a previously established tagging system, an esterase was encapsulated in a laboratory-evolved variant of AaLS. Characterization of the purified complex shows an average loading of two esterases per capsid and an approximately 20-fold decrease in efficiency compared to the free esterase. This decrease is larger than most of the previously reported capsid-based nanoreactor systems which suggests that both the confinement molarity and the electrostatic environment of the capsid interior may significantly influence the kinetic parameters of guest enzymes. Second, I utilize charge complementarity to decorate the exterior of an AaLS capsid variant with green fluorescent protein (GFP). A new interface was engineered by negatively supercharging the five-fold symmetric capsid pores and appending a deca-arginine tag to the C-terminus of GFP. This interaction requires the engineered features of both binding partners and shows steep dependence on the buffer ionic strength, although it retains high affinity at physiological ionic strength. Thus, charge complementarity can provide a simple, powerful, and general method for designing protein associations de novo. Finally, I expand upon previous work in which a redox switch was developed to control capsid assembly. The original switch relies on the formation of a disulfide-bonded adduct between a pentameric variant of AaLS and thiophenol. I explore alterations to the prosthetic group structure which reveal that the three-fold symmetric interface of the assembled capsid is highly plastic and can tolerate a range of different adduct sizes and shapes. These studies also identified two new disassembly switches, providing greater control over the supramolecular chemistry of the AaLS capsid

Automatic sorting of point pattern sets using Minkowski Functionals

Point pattern sets arise in many different areas of physical, biological, and applied research, representing many random realizations of underlying pattern formation mechanisms. These pattern sets can be heterogeneous with respect to underlying spatial processes, which may not be visually distinguishable. This heterogeneity can be elucidated by looking at statistical measures of the patterns sets and using these measures to divide the pattern set into distinct groups representing like spatial processes. We introduce here a numerical procedure for sorting point pattern sets into spatially homogeneous groups using Functional Principal Component Analysis (FPCA) applied to the approximated Minkowski functionals of each pattern. We demonstrate that this procedure correctly sorts pattern sets into similar groups both when the patterns are drawn from similar processes and when the 2nd-order characteristics of the pattern are identical. We highlight this routine for distinguishing the molecular patterning of fluorescently labeled cell membrane proteins, a subject of much interest in studies investigating complex spatial signaling patterns involved in the human immune response.Comment: 11 pages, 6 figures, submitted to Physical Review E (05 March 2013

Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs

AbstractStructural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41 000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519–541), and also FP analogs (FP520V/E with Val→Glu at residue 520; FP527L/R with Leu→Arg at 527; FP529F/Y with Phe→Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection

Advances in Hyaluronan Biology: Signaling, Regulation, and Disease Mechanisms

Hyaluronan is an extracellular glycosaminoglycan polymer consisting of linear disaccharide units containing alternating glucuronate and N-acetylglucosamine.Many cell types make hyaluronan, which unlike most other macromolecules is assembled at the plasmamembrane and concurrently translocated through the hyaluronan synthase enzyme. The normal function of large hyaluronan polymers (\u3e1MDa) in tissue cushioning, hydration, and lubrication is well established. The aberrant accumulation and degradation of hyaluronan and the receptor-mediated signaling of smaller hyaluronan fragments have also been extensively implicated in a variety of pathological states including inflammation and cancer. More recently, the discovery that hyaluronan can either be a structural matrix component or appear as smaller processed polymers and oligomers that differentially engage a diverse range of signaling receptors has created an exciting paradigm shift and reenergized hyaluronan research in a broad range of fields. In this special issue, eight review articles focus on summarizing the latest contributions to understanding hyaluronan synthesis and catabolism and the regulation of hyaluronan functions. Seven novel primary research articles also investigate multiple roles of hyaluronan in disease progression and targeting

An organotypic slice culture model of chronic white matter injury with maturation arrest of oligodendrocyte progenitors

<p>Abstract</p> <p>Background</p> <p>CNS myelination disturbances commonly occur in chronic white matter lesions in neurodevelopmental and adult neurological disorders. Recent studies support that myelination failure can involve a disrupted cellular repair mechanism where oligodendrocyte (OL) progenitor cells (OPCs) proliferate in lesions with diffuse astrogliosis, but fail to fully differentiate to mature myelinating OLs. There are no <it>in vitro </it>models that reproduce these features of myelination failure.</p> <p>Results</p> <p>Forebrain coronal slices from postnatal day (P) 0.5/1 rat pups were cultured for 1, 5, or 9 days <it>in vitro </it>(DIV). Slices rapidly exhibited diffuse astrogliosis and accumulation of the extracellular matrix glycosaminoglycan hyaluronan (HA), an inhibitor of OPC differentiation and re-myelination. At 1 DIV ~1.5% of Olig2⁺OLs displayed caspase-3 activation, which increased to ~11.5% by 9 DIV. At 1 DIV the density of PDGFRα⁺and PDGFRα⁺/Ki67⁺OPCs were significantly elevated compared to 0 DIV (<it>P </it>< 0.01). Despite this proliferative response, at 9 DIV ~60% of white matter OLs were late progenitors (preOLs), compared to ~7% in the postnatal day 10 rat (<it>P </it>< 0.0001), consistent with preOL maturation arrest. Addition of HA to slices significantly decreased the density of MBP⁺OLs at 9 DIV compared to controls (217 ± 16 <it>vs. </it>328 ± 17 cells/mm², respectively; <it>P </it>= 0.0003), supporting an inhibitory role of HA in OL lineage progression in chronic lesions.</p> <p>Conclusions</p> <p>Diffuse white matter astrogliosis and early OPC proliferation with impaired OL maturation were reproduced in this model of myelination failure. This system may be used to define mechanisms of OPC maturation arrest and myelination failure related to astrogliosis and HA accumulation.</p

Single Cell Ras-GTP Analysis Reveals Altered Ras Activity in a Subpopulation of Neurofibroma Schwann Cells but Not Fibroblasts

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouse Nf1−/− Schwann cells but never in Nf1−/− mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts

Hyaluronan Synthesis, Catabolism, and Signaling in Neurodegenerative Diseases

The glycosaminoglycan hyaluronan (HA), a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS). HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair