Chicken riboflavin-binding protein. cDNA sequence and homology with milk folate-binding protein

The Rd gene is expressed in the livers and oviducts of laying hens and codes for the riboflavin-binding protein (RfBP) of egg yolk and egg white. A lambda gt11 cDNA library derived from chicken oviduct poly(A)+ RNA was screened with polyclonal rabbit antiserum to chicken RfBP. Positive clones were isolated and rescreened with a mixed oligonucleotide probe corresponding to residues 20-25 of the mature protein. The largest cDNA clone (969 base pairs) was subcloned into plasmid pIBI21, and the nucleotide sequence was determined by the dideoxynucleotide method. This clone contained the entire coding region for RfBP. The published amino acid sequence of the mature protein was confirmed. In addition, the following 17-residue signal peptide was deduced: Met-Leu-Arg-Phe-Ala-Ile-Thr-Leu-Phe-Ala-Val-Ile-Thr-Ser-Ser-Thr-Cys. Unexpectedly, the nucleotide sequence codes for 2 adjacent arginine residues at the carboxyl terminus that are not observed in the mature protein. The amino acid sequence of RfBP is homologous with bovine milk folate-binding protein. Eight of the nine pairs of cysteines involved in disulfide bonds in RfBP are conserved in folate-binding protein, as are all of the tryptophan residues. Sequence identity between homologous regions of these two vitamin-binding proteins is more than 30%.     

Human Amnion Epithelial Cells Induced to Express Functional Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis, an autosomal recessive disorder caused by a mutation in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), remains a leading cause of childhood respiratory morbidity and mortality. The respiratory consequences of cystic fibrosis include the generation of thick, tenacious mucus that impairs lung clearance, predisposing the individual to repeated and persistent infections, progressive lung damage and shortened lifespan. Currently there is no cure for cystic fibrosis. With this in mind, we investigated the ability of human amnion epithelial cells (hAECs) to express functional CFTR. We found that hAECs formed 3-dimensional structures and expressed the CFTR gene and protein after culture in Small Airway Growth Medium (SAGM). We also observed a polarized CFTR distribution on the membrane of hAECs cultured in SAGM, similar to that observed in polarized airway cells in vivo. Further, hAECs induced to express CFTR possessed functional iodide/chloride (I^−/Cl⁻) ion channels that were inhibited by the CFTR-inhibitor CFTR-172, indicating the presence of functional CFTR ion channels. These data suggest that hAECs may be a promising source for the development of a cellular therapy for cystic fibrosis.     

Transferrin endocytosis and the mechanism of iron uptake by reticulocytes in the toad (Bufo marinus)

Reticulocytosis was induced in toads (Bufo marinus) by treatment with phenylhydrazine. Iron and transferrin uptake and transferrin endocytosis and exocytosis by these cells were measured. The mean number of transferrin receptors per cell was found to be 4.5 X 10(5) and the affinity constant of transferrin to receptors was 0.2 X 10(7) M-1. Iron and transferrin uptake were temp.-dependent processes. An inflection point occurred at 15-16 degrees C in the Arrhenius plots of endocytosis and iron uptake. The activation energies of these two processes above and below the inflection temperature were 31 and 71 kJ/mol. It is concluded that iron uptake by immature toad erythroid cells occurs by receptor-mediated endocytosis which still functions at temps as low as 5 degrees C.     

Quantitative secretome and glycome of primary human adipocytes during insulin resistance

Adipose tissue is both an energy storage depot and an endocrine organ. The impaired regulation of the secreted proteins of adipose tissue, known as adipocytokines, observed during obesity contributes to the onset of whole-body insulin resistance and the pathobiology of type 2 diabetes mellitus (T2DM). In addition, the global elevation of the intracellular glycosylation of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) via either genetic or pharmacological methods is sufficient to induce insulin resistance in both cultured cells and animal models. The elevation of global O-GlcNAc levels is associated with the altered expression of many adipocytokines. We have previously characterized the rodent adipocyte secretome during insulin sensitive and insulin resistant conditions. Here, we characterize and quantify the secretome and glycome of primary human adipocytes during insulin responsive and insulin resistant conditions generated by the classical method of hyperglycemia and hyperinsulinemia or by the pharmacological manipulation of O-GlcNAc levels. Using a proteomic approach, we identify 190 secreted proteins and report a total of 20 up-regulated and 6 down-regulated proteins that are detected in both insulin resistant conditions. Moreover, we apply glycomic techniques to examine (1) the sites of N-glycosylation on secreted proteins, (2) the structures of complex N- and O-glycans, and (3) the relative abundance of complex N- and O-glycans structures in insulin responsive and insulin resistant conditions. We identify 91 N-glycosylation sites derived from 51 secreted proteins, as well as 155 and 29 released N- and O-glycans respectively. We go on to quantify many of the N- and O-glycan structures between insulin responsive and insulin resistance conditions demonstrating no significant changes in complex glycosylation in the time frame for the induction of insulin resistance. Thus, our data support that the O-GlcNAc modification is involved in the regulation of adipocytokine secretion upon the induction of insulin resistance in human adipocytes.     

Iron-binding properties and amino acid composition of marsupial transferrins: comparison with eutherian mammals and other vertebrates

1. Some physicochemical properties of transferrin from three marsupials, viz a possum (Trachosurus vulpecula), a kangaroo (Macropus fuliginosus) and the quokka (Setonix brachyurus) were studied and compared with those of transferrins from mammalian and non-mammalian vertebrate species. 2. The molecular weight of the marsupial transferrins fell within the range of 76,000-79,000 daltons. 3. The marsupial transferrins were similar to the transferrins of eutherian mammals with respect to optical spectral properties, iron binding capacity and the pH-dependence of iron binding, and iron release mediated by 2,3-DPG. 4. The amino acid compositions of the marsupial transferrins were compared with each other and with the transferrins from the other vertebrate species. The compositions of the marsupial transferrin were closely related to each other, and also showed similarities with transferrins from eutherian mammals and chicken ovotransferrin.     

Isolation and characterization of nuclei from rice embryos.

A method has been developed to isolate pure preparations of nuclei in high yield from commercially available viable rice embryos (germ), employing extraction with buffer solution containing glycerol (without detergent) and polyamine, followed by centrifugation on a 30% Percoll cushion. The intactness of the isolated nuclei was confirmed by light microscopy as well as electron microscopy. The protein profiles of both whole nuclei and nuclear extracts obtained by SDS-PAGE, organellar marker enzyme activities, DNA and RNA analyses, and in vitro RNA synthesis, all indicate that the highly purified nuclei are isolated from rice embryos.     

Management of childhood urinary tract infection in family practice.

To find out to what extent doctors appreciate the potential damage that bacteriuria may cause, we sent questionnaires to 195 family practitioners and trainees in one health board in Northern Ireland. The results of the survey showed that, though doctors seemed to be aware of the risk of renal disease, this was not reflected in practice.