Estrogen receptor-alpha populations change with age in commercial laying hens.

Older hens in production lay larger but fewer eggs than younger birds, and the incidence of soft and broken shells is greater in older hens than younger. These changes are attributable at least in part to changing hormone profiles and diminished ability of the hen to transport calcium at the duodenum. In further exploration of this relationship, a study was conducted with three ages of Hy-Line W-36 birds: prelay pullets (PL; 19 wk, 0% production), peak-production hens (PP; 29 wk, approximately 93% production), and late-stage hens (LS; 71 wk, approximately 80% production). Hens from the PP and LS groups were palpated for presence of an egg in the shell gland; hens were then euthanized and tissues (kidney, shell gland, hypothalamus) were removed for quantification of estrogen receptor-alpha (ERalpha) populations via immunocytochemical and Western blot analyses. Localization of ERalpha by immunostaining in the shell gland showed differences among age groups; however, no differences were noted in localization of ERalpha between age groups in the kidney and hypothalamus. In both the kidney and the shell gland there was a decrease in the amount of ERalpha, as detected by immunoblotting, in the LS hens compared to PL and PP birds (P < 0.05). The results suggest that failure of calcium regulating mechanisms with age may be mediated at least in part through the reduced populations of estrogen receptors in certain critical tissues.

Identification of IgE-binding proteins in soy lecithin.

BACKGROUND: Soy lecithin is widely used as an emulsifier in processed foods, pharmaceuticals and cosmetics. Soy lecithin is composed principally of phospholipids; however, it has also been shown to contain IgE-binding proteins, albeit at a low level. A few clinical cases involving allergic reactions to soy lecithin have been reported. The purpose of this investigation is to better characterize the IgE-binding proteins typically found in lecithin. METHODS: Soy lecithin proteins were isolated following solvent extraction of lipid components and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated lecithin proteins were immunoblotted with sera from soy-sensitive individuals to determine the pattern of IgE-binding proteins. The identity of IgE-reactive bands was determined from their N-terminal sequence. RESULTS: The level of protein in six lecithin samples obtained from commercial suppliers ranged from 100 to 1,400 ppm. Lecithin samples showed similar protein patterns when examined by SDS-PAGE. Immunoblotting with sera from soy-sensitive individuals showed IgE binding to bands corresponding to 7, 12, 20, 39 and 57 kD. N-terminal analysis of these IgE-binding bands resulted in sequences for 3 components. The 12-kD band was identified as a methionine-rich protein (MRP) and a member of the 2S albumin class of soy proteins. The 20-kD band was found to be soybean Kunitz trypsin inhibitor. The 39-kD band was matched to a soy protein with unknown function. CONCLUSIONS: Soy lecithin contains a number of IgE-binding proteins; thus, it might represent a source of hidden allergens. These allergens are a more significant concern for soy-allergic individuals consuming lecithin products as a health supplement. In addition, the MRP and the 39-kD protein identified in this study represent newly identified IgE-binding proteins.

Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein.

Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII, and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin.

Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants.

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.

Phosphopeptides as substrates for thylakoid protein phosphatase activity.

Lack of a suitable substrate has been a major obstacle in studying the chloroplastic thylakoid membrane protein phosphatase activity. In this study, the suitability of synthetic phosphopeptides for this purpose was investigated. Phosphothreonine-containing phosphopeptides mimicking the N-terminal phosphorylation site of the major thylakoid phosphoprotein, the light-harvesting chlorophyll a/b-binding protein (LHCP-II), were dephosphorylated by isolated peak thylakoid membranes. Phosphopeptides representing unrelated sequences or in which the target phosphothreonine had been changed to a phosphoserine were not dephosphorylated. The dephosphorylation of phosphopeptides by thylakoid membranes was similar to the dephosphorylation of endogenous LHCP-II in its pH-dependence profile, sensitivity to inhibitors, and bivalent cation requirement. The same phosphopeptide analogs of the LHCP-II phosphorylation site inhibited endogenous LHCP-II dephosphorylation in isolated thylakoids, whereas the dephospho-analogs and nonsubstrate phosphopeptides had no effect. Collectively, these results suggest that phosphopeptides mimicking a thylakoid phosphoprotein dephosphorylation site can be exploited for further study of the thylakoid protein phosphatase activity.