Plasma amino acids of lean and obese Zucker rats subjected to a cafeteria diet after weaning.

Plasma amino acids of Zucker obese (fa/fa) and lean (Fa/?) rats fed either a reference nonpurified pellet or a cafeteria diet have been studied from 30 to 60 days after birth. Obese rats showed higher plasma branched chain amino acid levels but similar total amino acids, urea and glucose concentrations. The ingestion of a cafeteria diet induced higher levels in many amino acids, as well as in the composite figure in lean rats, but failed to alter total 2-amino nitrogen concentrations in obese rats, despite high levels in several non-essential amino acids and lower values in essential amino acids; urea levels were much lower in rats fed the cafeteria diet. The results are consistent with an impairment of amino acid nitrogen elimination via urea cycle in cafeteria diet-fed rats. This is independent of the hyperinsulinemia-driven plasma accumulation of several essential amino acids induced by genetic obesity. The effects were, then additive.     

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Nuclease hypersensitivity in the beta-globin gene region of K562 cells

We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.     

Protonmotive Q cycle pathway of electron transfer and energy transduction in the three-subunit ubiquinol-cytochrome c oxidoreductase complex of Paracoccus denitrificans

Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans consists of only three polypeptide subunits (Yang, X., and Trumpower, B. L. (1986) J. Biol. Chem. 261, 12282-12289), whereas the analogous complexes of eukaryotic mitochondria consist of nine or more polypeptides (Schagger, H., Link, T. A., Engel, W. D., and von Jagow, G. (1986) Methods Enzymol. 126, 224-237). Using the purified three-subunit Paracoccus complex we have tested whether this simple cytochrome bc1 complex has the same electron transfer pathway and proton translocation activity as the bc1 complexes of mitochondria. Under presteady state conditions, the effects of inhibitors on reduction of cytochromes b and c1 by quinol and oxidant-induced reduction of cytochrome b indicate a cyclic electron transfer pathway and two routes of cytochrome b reduction in the three-subunit Paracoccus cytochrome bc1 complex. A novel method was developed to incorporate the cytochrome bc1 complex into liposomes with the detergent dodecyl maltoside. The enzyme reconstituted into liposomes translocated protons with an H+/2e value of 3.9. Carbonyl cyanide m-chlorophenylhydrazone eliminated proton translocation, while permitting the scalar release of protons from quinol, and thus reduced the H+/2e ratio to 2. These values agree with the predicted stoichiometries for proton translocation by a protonmotive Q cycle pathway. No inhibition of proton translocation by N',N'-dicyclohexylcarbodiimide was detected when the Paracoccus cytochrome bc1 complex was incubated with N',N'-dicyclohexylcarbodiimide before or after reconstitution into liposomes. Electron transfer in the three-subunit complex thus appears to occur by a protonmotive Q cycle pathway identical to that in mitochondrial cytochrome bc1 complexes. Only three polypeptides, cytochromes b, c1, and the Rieske iron-sulfur protein, are required for respiration and energy transduction in the cytochrome bc1 complex. The function of the supernumerary polypeptides in mitochondrial bc1 complexes is thus unclear.     

Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles

MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl[3H]tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl[3H]tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface.