Impact of Dietary Organic Mineral Supplementation on Reproductive Performance,Egg Quality Characteristics,Lipid Oxidation,Ovarian Follicular Development,and Immune Response in Laying Hens Under High Ambient Temperature

Biological Trace Element Research - The objectives of this study were to investigate the impact of dietary organic mineral mixture (manganese, zinc, and copper) supplementation on reproductive...     

Effect of oxidation on the properties of apolipoproteins A-I and A-II

Purified apolipoprotein A-I has been separated by reversed-phase high performance liquid chromatography (HPLC) into multiple peaks and these peaks have been characterized. One peak, apoA-Ib had a relatively longer retention time on HPLC but its retention time could be shortened by treatment by hydrogen peroxide. CNBr cleavage studies indicated that the differences in apoA-Ib and in its oxidation product, apoA-Ia, were due to the different oxidation states of methionine. This phenomenon was also observed in apoA-II, where methionine oxidation produced two more forms of this apolipoprotein in addition to the native form. These isomers were found to have different secondary structures and affinities for lipid. Model peptide analogs of the amphipathic helix with the same sequence but with methionine and methionine sulfoxide at the nonpolar face of the amphipathic helix were synthesized and studied. It was found that the lipid affinities of these synthetic peptide isomers were very different. They also differed in their secondary structures as studied by circular dichroism (CD). We propose that methionine oxidation introduces hydrophilic residues at the nonpolar face of the amphipathic helical domains of these apolipoproteins and, therefore, alters their secondary structure and lipid affinity.     

Myeloid Cells Restrict MCMV and Drive Stress-Induced Extramedullary Hematopoiesis through STAT1

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The Triggering Receptor Expressed on Myeloid Cells 2 Inhibits Complement Component 1q Effector Mechanisms and Exerts Detrimental Effects during Pneumococcal Pneumonia

Phagocytosis and inflammation within the lungs is crucial for host defense during bacterial pneumonia. Triggering receptor expressed on myeloid cells (TREM)-2 was proposed to negatively regulate TLR-mediated responses and enhance phagocytosis by macrophages, but the role of TREM-2 in respiratory tract infections is unknown. Here, we established the presence of TREM-2 on alveolar macrophages (AM) and explored the function of TREM-2 in the innate immune response to pneumococcal infection in vivo. Unexpectedly, we found Trem-2 ^−/− AM to display augmented bacterial phagocytosis in vitro and in vivo compared to WT AM. Mechanistically, we detected that in the absence of TREM-2, pulmonary macrophages selectively produced elevated complement component 1q (C1q) levels. We found that these increased C1q levels depended on peroxisome proliferator-activated receptor-δ (PPAR-δ) activity and were responsible for the enhanced phagocytosis of bacteria. Upon infection with S. pneumoniae, Trem-2 ^−/− mice exhibited an augmented bacterial clearance from lungs, decreased bacteremia and improved survival compared to their WT counterparts. This work is the first to disclose a role for TREM-2 in clinically relevant respiratory tract infections and demonstrates a previously unknown link between TREM-2 and opsonin production within the lungs.     

Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C

We have synthesized four oligopeptides that are structural analogues of a low-affinity Ca2+-specific binding site (site II) of rabbit skeletal troponin C. One analogue (peptide 3) was a dodecapeptide with a sequence corresponding to the 12-residue Ca2+-binding loop (residues 63-74 in troponin C), two (peptides 4 and 5) were 23-residue in length, corresponding to residues 52-74 of the protein, and the fourth (peptide 6) was a 25-residue peptide corresponding to residues 50-74. All four peptides had one amino acid substitution within the 12-residue binding loop in which phenylalanine at position 10 was replaced by tyrosine to provide a marker for spectroscopic studies. In addition, peptides 3 and 4 each had a second substitution within the binding loop where glycine at position 6 was replaced by alanine. The second substitution was motivated by the conservation of glycine at the position in the Ca2+-binding loops of all four Ca2+-binding sites in troponin C. The peptides were characterized by their intrinsic fluorescence, ability to enhance the emission of bound Tb3+, affinity for Ca2+ and Tb3+, and circular dichroism. The affinity for Ca2+ was in the range 10-10(2) M-1, and the affinity for Tb3+ was in the range 10(4)-10(5) M-1. The binding constants of the longer peptides were several-fold larger than that of the dodecapeptide. With peptides 4 and 5, substitution of glycine by alanine at position 6 within the 12-residue loop decreased the affinity for Ca2+ by a factor of four, but had little effect on the affinity for Tb3+. However, the mean residue ellipticity of peptide 4 was substantially higher than that of peptide 5. Since peptide 4 differs from peptide 5 only in the substitution of glycine at position 6 in the loop segment, the conservation of glycine at that position may serve a role in providing a suitable secondary structure of the binding sites for interaction with troponin I. Peptides 4 and 6, when present in a large excess, mimic troponin C in regulating fully reconstituted actomyosin ATPase by showing partial calcium sensitivity and activation of the ATPase. Since these peptides are the smallest peptides containing the Ca2+-binding loop of site II, their biological activity suggests that a Ca2+-dependent binding site of troponin C for troponin I could be as short as the segment comprising residues 52-62.     

Molecular Characterization of Factor V Leiden G1691A and Prothrombin G20210A Mutations in Saudi Newborns with Stroke

This study examined a possible association between the mutations related to Factor V Leiden and Factor II (prothrombin) and stroke in Saudi neonates. A multiplex PCR was established to detect Factor V Leiden G1691A and prothrombin G20210A mutations in 72 neonatal stroke subjects and 70 healthy adult controls with no family history of thromboembolic diseases. The frequency of the homozygous normal genotype (GG) of both genes was found to be significantly lower in the stroke subjects than in the controls (P < 0.0001). The stroke cases also had higher frequencies of the combined Factor II heterozygous mutant form (GA) and the homozygous normal Factor V (GG) (P < 0.0001) and of the combined heterozygous Factor V and the homozygous normal Factor II genotypes (GG) (P = 0.0) than controls. The study concluded that prothrombin and Factor V Leiden may be important risk factors for neonatal stroke in Saudi children.     

Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I

We have isolated five Chinese hamster ovary cell mutants defective in galactosyltransferase I (UDP-D-galactose:xylose beta-1,4-D-galactosyltransferase) and studied the effect of p-nitrophenyl-beta-D-xyloside supplementation on glycosaminoglycan biosynthesis in the mutant cells. Assays of galactosyltransferase I showed that the mutants contained less than 2% of the enzyme activity present in wild-type cells, and enzyme activity was additive in mixtures of mutant and wild-type cell extracts, suggesting that the mutations most likely defined the structural gene encoding the enzyme. Cell hybridization studies showed that the mutations in all five strains were recessive and that the mutants belonged to the same complementation group. The mutants contained wild-type levels of xylosyltransferase (UDP-D-xylose:core protein (serine) beta-D-xylosyltransferase), lactose synthase (UDP-D-galactose:N-acetyl-glucosaminide beta-1,4-D-galactosyltransferase), and lactosylceramide synthase (UDP-D-galactose:glucosylceramide beta-1,4-D-galactosyltransferase). Their sensitivity to lectin-mediated cytotoxicity was virtually identical to that of the wild-type, indicating that there were no gross alterations in glycoprotein or glycolipid compositions. Anion-exchange high performance liquid chromatography of 35S-glycosaminoglycans from one of the galactosyltransferase I-deficient mutants showed a dramatic reduction in both heparan sulfate and chondroitin sulfate, demonstrating that galactosyltransferase I is responsible for the formation of both glycosaminoglycans in intact cells. Surprisingly, the addition of 1 mM-p-nitrophenyl-beta-D-xyloside, a substrate for galactosyltransferase I, restored glycosaminoglycan synthesis in mutant cells. This finding suggested that another galactosyltransferase, possibly lactose synthase, can transfer galactose to xylose in intact cells.     

Studies of synthetic peptide analogs of the amphipathic helix. Effect of charge distribution, hydrophobicity, and secondary structure on lipid association and lecithin:cholesterol acyltransferase activation

Four peptides capable of forming an amphipathic alpha-helix have been synthesized and their conformational and lipid-binding properties studied. These peptides have been designed to vary the alpha-helix-forming potential as well as the charge distribution of the model peptide. The resulting peptide analogs and their complexes with dimyristoyl phosphatidylcholine were studied by using right angle light scattering, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, circular dichroism, intrinsic tryptophan fluorescence, and differential scanning calorimetry techniques. The four analogs, [Glu4,9, Leu11,17] (reverse-18A, [Glu4,9, Leu5,11,17] reverse-18A, [Glu1,8, Leu11,17] 18A, and [Glu1,8, Leu5,11,17] 18A were derived from a model amphipathic peptide Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) whose lipid-associating properties strongly mimic apolipoprotein A-I or derived from Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala-Phe (reverse-18A), a peptide with little affinity for lipid and having a reversed charge distribution compared to the 18A peptide. We have shown that by substituting glutamic acid and leucine for aspartic acid and alanine, respectively, in a weak lipid-associating amphipathic helix peptide, the lipid-associating ability can be increased. Thus, peptides with both kinds of charge distribution can associate with the lipid. The ability of the peptide to disrupt phospholipid bilayers, however, is higher for 18A analogs compared to the reverse-18A analogs even after increasing the helix-forming potential and hydrophobicity. In addition to forming smaller lipoprotein particles, the modified 18A analogs were much superior to the modified reverse-18A analogs in their ability to activate the enzyme lecithin:cholesterol acyltransferase. This demonstrates that the positions of charged residues in the amphipathic helix play an important role in lecithin:cholesterol acyltransferase activation.