Specific binding and uptake of apolipoprotein E-free high density lipoproteins by cultured liver parenchymal cells of copper-deficient rats

The influence of copper deficiency on the binding and uptake of apolipoprotein E-free high density lipoprotein (apo E-free HDL) in cultured rat hepatic parenchymal cells was examined in this study. Male weanling Sprague-Dawley rats were randomly divided into two treatments, a Cu-adequate (7.33 mg Cu/kg diet) or a Cu-deficient (1.04 mg Cu/kg diet) group. After 7 weeks, plasma apo E-free HDL were isolated by a combination of ultracentrifugation, gel filtration, and heparin-Sepharose affinity chromatography. Parenchymal cells were isolated from collagenase perfused liver of Cu-deficient and adequate rats and cultured for 16 hours at 37 degrees C prior to incubation with iodinated apo E-free HDL from the same treatment group. Cells were incubated with 5 microg/ml(125) I-apo E-free HDL for 2, 6, or 12 hours in the presence or absence of 200 microg/ml (40-fold) excess unlabeled apo E-free HDL. Increases in specific binding at 4 degrees C and specific cell-associated uptake at 37 degrees C as a function of time were observed with cells and HDL from Cu-deficient rats. Cells were also incubated for 6 hours with 8 concentrations of (125)I-apo E-free HDL in the presence or absence of excess unlabeled HDL. Although no significant increase in specific binding was detected at 4 degrees C as a function of ligand concentration, the response tended to be higher at 5 to 15 microg HDL/ml for the Cu-deficient treatment. However, at 37 degrees C the specific cell-associated uptake was increased markedly with cells and HDL from Cu-deficient rats. The observed increases in HDL binding and uptake indicate that these processes may be enhanced in Cu-deficient rats. These data are also consistent with recent in vivo results which indicate that plasma clearance and tissue uptake of HDL are increased in Cu-deficient rats.     

A group contribution method for the estimation of equilibrium constants for biochemical reactions

Using Gibbs Energies of compounds, as well as Gibbs Energy changes and equilibrium constants of biochemical reactions, the contributions of functional groups to the Gibbs Energy (in aqueous solution, temperature 25°C, and pH=7) have been estimated. These contributions allow the estimation of the Gibbs Free Energy and the equilibrium constant of a biochemical reaction, given the structure of the reactants and products.     

Presence of immunoreactive endothelin in human saliva and rat parotid gland.

The presence of immunoreactive endothelin (IR-ET) in human saliva and rat parotid gland was investigated by radioimmunoassay. The IR-ET concentration (mean +/- SEM) in saliva taken from normal volunteers was 2.0 +/- 0.2 pmol/l (n = 15). The IR-ET concentration in rat parotid gland was 19.2 +/- 2.2 fmol/g wet weight (n = 10). Fast protein liquid chromatography (FPLC) of human saliva extract revealed 6 peaks; one peak eluting in the void volume, one in a position between ET-1 and -3, and the other four in the positions of synthetic ET-1, -2, -3 and big ET(1-38), respectively. A similar pattern of rat parotid gland extract was noted with FPLC, except that there was no peak after the void volume. Presence of endothelin, a potent growth factor, in saliva and salivary gland points to a role in maintaining the integrity of the oral and gastrointestinal tract mucosa.     

Sequence and structural organization of the human gene encoding ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a potent polypeptide hormone whose actions appear to be restricted to the nervous system where it promotes survival, neurotransmitter synthesis and neurite outgrowth in certain neuronal populations. We have cloned the gene encoding human CNTF (hCNTF) and have characterized its structure and organization. The hCNTF gene appears to be a unique-copy gene with a simple genetic organization, since only a single intron interrupts the coding domain. The hCNTF gene is located on chromosome 11, as determined using human-hamster somatic cell hybrids. The CNTF protein is highly conserved in evolution. The amino acid (aa) sequences of rat and rabbit CNTF translated from cDNAs display approx. 85% homology with the deduced aa sequence encoding hCNTF.     

Occupancy of an adhesive glycoprotein receptor modulates expression of an antigenic site involved in cell adhesion

Binding of ligands that contain Arg-Gly-Asp to adhesion receptors induces cell spreading and aggregation and alters gene expression, possibly due to conformational changes within occupied adhesion receptors. PMI-1 is a monoclonal antibody which reacts with the platelet fibrinogen receptor, glycoprotein IIb-IIIa, and reports such a conformational change. ADP stimulation of platelets results in a fibrinogen-dependent increase in binding of the PMI-1 antibody. Peptides containing Arg-Gly-Asp also reversibly increase the binding of this antibody to cells and to purified glycoprotein IIb-IIIa. The PMI-1 antibody inhibits platelet adhesion and spreading on certain substrata (Shadle, P. J., Ginsberg, M. H., Plow, E. F., and Barondes, S. H. (1984) J. Cell Biol. 99, 2056-2060); thus this occupancy-modulated site may participate in adhesive function.     

Repair of closely opposed cyclobutyl pyrimidine dimers in UV-sensitive human diploid fibroblasts

An enzyme-sensitive site assay has been used to examine the fate of closely opposed pyrimidine dimers (bifilar enzyme-sensitive sites) in fibroblasts from individuals afflicted with various genetic disorders that confer increased cellular sensitivity to UV radiation. The disappearance of bifilar enzyme-sensitive sites was found to be normal in cells from individuals with Fanconi's anemia, Cockayne's syndrome, dyskeratosis congenita and the variant form of xeroderma pigmentosum. The rate of bifilar enzyme-sensitive site removal in XP cells assigned to complementation group C was reduced by an amount similar to that observed for the repair of isolated dimers. Our results indicate that the initiation of repair at closely opposed dimers is slow in XP-C cells but normal in all other cells examined.     

Genomic DNA sequence for human C-reactive protein

The gene for the prototype acute phase reactant, C-reactive protein, has been isolated from two lambda phage libraries containing inserted human DNA fragments using synthetic oligonucleotide probes. Nucleotide sequence analysis indicates that after coding for a signal peptide of 18 amino acids and the first two amino acids of the mature protein, there is an intron of 278 base pairs followed by the nucleotide sequence for the remaining 204 amino acids. The intron is unusual in that it contains on the positive strand a poly(A) stretch 16 nucleotides long and a poly(GT) region 30 nucleotides long which could adopt the Z-form of DNA. The nucleotide sequence reported here confirms the amino acid sequence of mature C-reactive protein as originally reported except that it codes for an additional 19 amino acids beginning at position 62. Thus DNA sequence analysis predicts that the mature protein consists of 206 amino acids rather than 187 as originally reported. The mRNA cap site is located 104 nucleotides from the start of the signal peptide and there is a 3' noncoding region 1.2 kilobase pairs in length. The gene has a typical promoter containing the sequences TATAAAT and CAAT 29 and 81 base pairs upstream, respectively, of the cap site.     

An improved DNA sequencing strategy

A modification of Hong's systematic DNA sequencing strategy is described. The original procedure has been simplified and transfectant yield increased. After DNase I limited cleavage in the presence of Mn2+, the single-cut linear DNA does not have to be separated from supercoiled or open circular DNA on an agarose gel. After ligation, the DNA is digested with a second restriction endonuclease for which a unique cleavage site resides between the insert and the first restriction endonuclease cutting site. The original intact DNA is linearized whereas the deleted subclone is not. The background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb DNA fragment containing the araC regulatory gene from Erwinia carotovora. A set of subclones sufficient to sequence the fragment on both strands was produced in 2 days and the yield was at least 60-fold higher than in the original protocol.     

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane : Studies of Photosystem II using Pronase Digestion and Chemical Labeling

The accessibility of various Photosystem II (PSII)-associated polypeptides to the protease pronase and the chemical modifier trinitrobenzene-sulfonic acid (TNBS) has been investigated. Three polypeptides with apparent molecular weight of 32, 21, and 16 kilodaltons, known to be associated with O₂ evolution, are all resistant to pronase digestion and TNBS labeling in intact thylakoids. All the polypeptides in the isolated PSII preparation were labeled with TNBS while a different pattern of labeling was observed when the PSII complex was isolated from TNBS-modified thylakoids. Attempts to prepare PSII particles from pronase-treated thylakoids using the Triton X-100 solubilization method were unsuccessful. Pronase-treated thylakoids were probed with antisera against the chlorophyll proteins of PSII using immunoblotting techniques. This allowed for a positive identification of proteolytic fragments from the respective proteins. The results are discussed in relation to the transmembrane organization of PSII in spinach thylakoids.     

A vehicle for the introduction of transposons into plant-associated pseudomonads

A recombinant plasmid with wide-host-range transfer functions, narrow-host-range replication functions, and carrying a kanamycin-resistant transposon transferred kanamycin resistance to a number of plant-associated pseudomonads. Southern hybridization studies suggest that only a small portion of the plasmid, coinciding with the location of the transposon, is present in the kanamycin-resistant Pseudomonas derivatives. The plasmid sequences appear to be inserted at a number of different sites in the recipient genome. This plasmid can thus be used as a vehicle for the introduction of transposons into some plant-associated pseudomonads and should be useful in both genetic and ecological studies of these bacteria.