Isolation and characterization of two novel rat ovarian lactogen receptor cDNA species

Two novel lactogen receptor cDNA clones (2.1 and 1.2 kb) were isolated from a rat ovarian cDNA library. Nucleotide sequence of the 2.1 kb clone codes for a 610 aa receptor (nonglycosylated mol. wgt. 66,000 D) with an extracellular domain, a transmembrane region and an intracellular domain, and exhibited significant overall similarity with the rat liver receptor (310 aa) and both rabbit mammary and human hepatoma receptors (616 and 622 aa). However, the ovarian lactogen receptor sequence contains a unique cytoplasmic domain of 110 aa and consensus sequences for both a tyrosine phosphorylation site and an ATP/GTP type A binding site, and thus has potential for signal transduction and mitogenic activity. The 1.2 kb clone codes for a truncated binding form of 150 aa that is identical with the ovarian long form over only the first 130 residues, and lacks the transmembrane region. Differences between long and short forms of the ovarian lactogen receptors and the truncated liver species may result from alternative splicing. The prolactin holoreceptor gene(s) has the potential for producing several receptor subtypes that differ in tissue-specific expression, size, compartmentalization and mode of signal transduction, and may subserve the divergent functions of prolactin in its several target cells.     

Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density

Influenza virus gains access to the cytoplasm of its host cell by means of a fusion event between viral and host cell membrane. Fusion is mediated by the envelope glycoprotein hemagglutinin (HA) and is triggered by low pH. To learn how many hemagglutinin trimers are necessary to cause membrane fusion, we have used two NIH 3T3 fibroblast cell lines that express HA protein at different surface densities. On the basis of quantitations of the number of HA trimers per cell and the relative surface areas of the two cell lines, the HAb-2 cells have a 1.9-fold higher plasma membrane surface density than the GP4F cells. The membrane lateral diffusion coefficient and the mobile fraction for HA is the same for both cell lines. A Scatchard analysis of the binding of glycophorin-bearing liposomes to the cells showed 1700 binding sites for the GP4F cells and 3750 binding sites for the HAb-2 cells, with effectively the same liposome-cell binding constant, about 7 x 10(10) M-1. Binding was specific for glycophorin on the liposomes and HA expressed on the cells. A competition experiment employing toxin-containing and empty liposomes allowed us to quantitate the number of liposomes that fused per cell, which was a small constant fraction of the number of bound liposomes. For the HAb-2 cells, about 1 in every 70 bound liposomes fused and for the GP4F cells about 1 in every 300 bound liposomes fused. Hence, the HAb-2 cells showed 4.4 times more fusion per bound liposome, even though the surface density of HA was only 1.9 times greater. We conclude the following: (i) One HA trimer is not sufficient to induce fusion. (ii) The HA bound to glycophorin is not the HA that induces fusion. That is, even though each HA has a binding and a fusion function, those functions are not performed by the same HA trimer.     

Isolation and partial characterization of a lactotransferrin receptor from mouse intestinal brush border

Several lines of evidence have recently suggested the occurrence of a specific lactotransferrin receptor in the small intestinal brush-border membrane in several animal species, which is thought to be involved in lactotransferrin-mediated intestinal iron absorption. We report here for the first time the isolation and partial characterization of this receptor from mouse intestinal brush border. The receptor has been purified to homogeneity by affinity chromatography on an immobilized human lactotransferrin column. The purified receptor was found to be active in that it binds iron-free and iron-saturated lactotransferrin with a Kd of 0.1 microM. Anti-receptor antibodies were prepared, and the receptor was further isolated by immunoaffinity chromatography in higher yield but in a denatured form. The purified receptor was revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis to be a protein of about Mr = 130,000, consisting of a single polypeptide chain. The isoelectric point was determined to be 5.8. The receptor was further shown to bear concanavalin A and phytohemagglutinin L binding glycans. Digestion by N-glycanase and endo-N-acetyl-beta-D-glucosaminidase B led to a decrease of Mr = 25,000, while the endo-N-acetyl-beta-D-glucosaminidase H was uneffective, suggesting that the lactotransferrin receptor is mainly glycosylated by bi- and triantennary glycans. To gain further insight into the interaction of the receptor with lactotransferrin, namely, the number of ligand molecules bound per molecule of receptor, mouse lactotransferrin was cross-linked to its membrane-bound enterocyte receptor by use of radiolabeled sulfosuccinimidyl 3-[[2-(p-azidosalicylamido)ethyl]dithio]propionate (SASD).(ABSTRACT TRUNCATED AT 250 WORDS)     

Enumeration and identification of IS3 elements in Escherichia coli strains.

Escherichia coli K-12 strains ordinarily contain five IS3 elements. Three of these correspond to previously mapped IS3 elements (R. C. Deonier, G. R. Oh, and M. Hu, J. Bacteriol. 129:1129--1140, 1977; S. Hu, E. Ohtsubo, and N. Davidson, J. Bacteriol. 122:749--763, 1975), and two additional IS3 elements are identified. The distribution of IS3 elements among deoxyribonucleic acid fragments generated by digestion with EcoRI indicates a basic pattern from which deviation is detected.     

Kinetics of glycogen phosphorylase a with a series of semisynthetic, branched saccharides. A model for binding of polysaccharide substrates.

The requirement of muscle phosphorylase for branched polysaccharide substrates was investigated by kinetic studies on semisynthetic branched saccharides. One series of saccharides was prepared from maltoheptose by oxidizing the reducing group to a carboxyl group and coupling this with an amino group of ethylenediamine. The resulting aminooligosaccharide was coupled with p-nitrophenyl esters of mono-, di-, tetra-, and polycarboxylic aicds to produce saccharides containing one, two, four, and approximately 52 maltodextrin chains per molecule. A similar series of saccharides was prepared from a heterogeneous maltodextrin of average chain length 11.7. Kinetic constants were determined for the reaction with phoshorylase a in the direction of chain elongation. Michaelis constants are equilibrium constants for dissociation of saccharide from the enzyme-AMP-glucose-1P-saccharide complex. The Michaelis constants, expressed in terms of the concentration of nonreducing end groups, are independent of maltodextrin chain length but decrease considerably as the number of chains per molecule increases. Maximum velocities do not differ greatly from that for glycogen. Among the synthetic saccharides, only the polymer behaves similarly to glycogen in exhiiting a decreasing reaction rate as the chains are elongated. The kinetic constants are quantitatively consistent with a model in which two chain termini from the same saccharide molecule bind to the phosphorylase molecule simultaniously, Differences in binding between saccharides having different numbers of equally accessible chains are caused solely by statistical factors in the equilibrium. Highly branched substrates bind better because of their greater multiplicity of two end-group pairs.     

Single-cell transcriptomic atlas of primate cardiopulmonary aging

Aging is a major risk factor for many diseases,especially in highly prevalent cardiopulmonary comorbidities and infectious diseases including Coronavirus Diseas...     

Genetic Polymorphisms of Pneumocystis Jirovecii in HIV-Positive and HIV-Negative Patients in Northern China

Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and β-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes (dhfr, dhps and cyb) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China. Open in a separate window