Isolation and characterization of microsatellites from the canine genome

Microsatellite sequences comprising (dC-dA)_n.(dG-dT)_n repeats have been isolated from canine libraries and sequenced. Oligonucleotide primers have been synthesized to the micro-satellite flanking sequences and used in the polymerase chain reaction to amplify those loci from genomic DNA. The degree of polymorphism of each microsatellite was estimated in a set of unrelated dogs. It is concluded that of the 10 loci studied, nine are sufficiently polymorphic to be useful in genetic studies.     

Isolation and characterization of immunopeptide from dialyzable leukocyte extract

The dialyzable leukocyte extract was fractionated using high-pressure liquid chromatography through an anion exchange column (preparative). Twenty-one fractions were obtained. Fraction 1, which gave maximum enhancement of E rosettes, was further purified by thin-layer chromatography on silica gel plates. Five ninhydrin-positive bands were seen. The area under each band was scraped from the plates and eluted. The fastest moving component was labeled S1 and the slowest moving S5. Fraction S3 had given maximum enhancement of E rosettes and was designated as immunopeptide. The immunopeptide was further characterized. It contained protein, ribose, and hexose. The molecular weight was determined to be 1870 by sedimentation equilibrium method. It contained 15 amino acid residues. The immunopeptide enhanced E-rosette formation in vivo when given in doses of 5 units/m², to four individuals with low E rosettes.     

Isolation and characterization of embryonic stem-like cells from canine blastocysts

Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES-like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11-16 of first estrus. A total of 80 normal embryos were obtained from 15 dogs. Of the embryos, 13 were at the morulae stage, 39 at the blastocyst stage, and 28 at the hatched blastocyst stage. The inside of morulae or inner cell masses (ICMs) of blastocysts were isolated mechanically, and cultured onto mouse embryonic fibroblasts (MEF) as feeder layers. Primary cell colonies were formed in 0% (0/13) of morulae, 25.6% (10/39) of blastocysts, and 67.9% (19/28) of hatched blastocysts. These colonies were separated either by enzymatic dissociation or by mechanical disaggregation. Dissociation with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remained undifferentiated, and two ES-like cell lines (cES1, cES2) continued to grow in culture after eight passages. These cells had typical stem cell-like morphology and expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4. These cells formed embryoid bodies (EBs) in a suspension culture; extended culture of EBs resulted in the formation of cystic EBs. When the simple EBs were cultured on tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells. These observations indicate that we successfully isolated and characterized canine ES-like cells.     

Isolation and characterization of a small intestinal surfactant-like particle containing alkaline phosphatase and other digestive enzymes

Digestive brush-border enzymes in particulate form have been reported in the intestinal lumen in vivo and in medium from organ explants in vitro. It has been suggested that these particles derive from membrane shedding of the apical brush border. This study describes the isolation and characterization of particles derived from the 105,000 x g supernatant fraction of intestinal luminal washings and from light scrapings of the mucosa itself after fat feeding of rats. These fractions were separated in a continuous NaBr gradient, producing a visible band of 1.07-1.08 g/liter density and resulting in a 15-fold enrichment of intestinal alkaline phosphatase in the band fraction. Other brush-border hydrolases were represented in the banded fraction, but at specific activities only 1/5th to 1/36th that of the brush border. The major phospholipid in the fraction was phosphatidylcholine (58 +/- 15%), containing 75% saturated fatty acids. In contrast, the major brush-border phospholipid was phosphatidyl-ethanolamine. These characteristics showed that the particles derived from the lumen and mucosal surface were not identical to fragments of the brush border. Electron microscopy of the banded fraction revealed partially coiled membrane fragments. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots, some proteins (e.g. surfactant protein B, collagenous protein 4) were found in common between the intestinal particles and rat pulmonary surfactant. These data suggest the production of a particle secreted by rat intestine that differs from brush-border membranes and that shares some morphological and biochemical similarities with pulmonary surfactant.     

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)     

Isolation and characterization of pteroylpolyglutamate hydrolase from rat intestinal mucosa

Pteroylpolyglutamate hydrolase was isolated from rat intestinal mucosa and purified with the aid of affinity chromatography. The affinity ligand was poly-gamma-glutamic acid (Mr approximately 12,000) derived from Bacillus subtilis. The specific enzymatic activity was increased 2,000-fold over the 100,000 X g supernatant of the mucosal homogenate with a yield of 20%. Sephadex G-200 gel filtration yielded an estimated molecular mass of 80,000 daltons. The isoelectric point was pH 8.2. The pH optimum in acetate buffer containing 1 mM zinc was 4.5. The KM values for pteroylheptaglutamate and pteroyltriglutamate were 0.21 and 0.67 microM, respectively. Polyanionic compounds, poly-gamma-glutamic acid, dextran sulfate, and heparin were noncompetitive inhibitors. Studies of the time course of hydrolysis of synthetic [3H]pteroylheptaglutamate by three separate techniques demonstrated the appearance of [3H]pteroylmonoglutamate, synchronous with substrate cleavage. Intermediate pteroyloligoglutamates were not detected. An endopeptidase-like mode of hydrolysis was further established by identification of a hexaglutamyl peptide as the other reaction product.     

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.     

Isolation and characterization of glycoproteins from canine tracheal pouch secretions.

Canine tracheal pouch secretions were solubilized with 1% sodium dodecyl sulfate and visualized by sodium dodecyl sulfate-agarose-acrylamide gel electrophoresis. Intact mucus, and water-soluble and insoluble fractions of mucus were shown to be composed of high molecular weight glycoproteins (Mr greater than or equal to 3 . 10(6)) and three major classes of proteins of lower molecular weight (Mr approximately 4 . 10(5), 2 . 10(5), and 6 . 10(4)). When the mucus secretions were further treated with a reducing agent, the glycoproteins were dissociated into subunits which appeared on the gel as three discrete bands. Separation of the high molecular weight glycoproteins from the other proteins was achieved by gel filtration on Biogel A-15m in the presence of 1% dodecyl sulfate following reduction and alkylation of mucus. These glycoproteins were further resolved, using DEAE cellulose chromatography in the presence of 6 M urea, into two protein fractions. Both fractions contained approximately 87% carbohydrate, high amounts of serine and threonine but differed significantly in contents of N-acetyl glucosamine and sialic acid; their mobility on gel electrophoresis was also different. Significant contents of cysteine were noted in both fractions. Results of this study indicate that the canine tracheal pouch preparations provide normal tracheal secretions which bear similarity in structure to the tracheobronchial secretions obtained from human patients.     

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border

Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-order fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1.5 M KCl) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of Mr 55,000, 48,000, and 40,000 was noted. On two- dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (Mr 48,000, and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the Mr 55,000 protein which focused at pH values higher than 6.4. The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins. Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell. The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue- specific patterns of their protein subunits.     

Isolation and characterization of canine distemper virus-specific RNA

Ten species of virus-specific RNA were detected in Vero cells infected with the FXNO strain of canine distemper virus (CDV). The largest RNA was the genome-sized RNA and the nine smaller species were polyadenylated RNAs. Similar results were obtained for nine other strains of CDV. The molecular weights of these ten RNAs were determined to be 4.61 X 10(6), 2.46 X 10(6), 1.52 X 10(6), 1.32 X 10(6), 1.19 X 10(6), 1.07 X 10(6), 0.77 X 10(6), 0.65 X 10(6), 0.58 X 10(6), and 0.48 X 10(6). By in vitro translation of the polyadenylated RNAs in a rabbit reticulocyte lysate system, three different proteins which probably correspond to H, NP, and M were synthesized from the fraction containing RNAs 7, 8, 9, and 10.     

Isolation and characterization of the apolipoprotein E receptor from canine and human liver

Previous results have demonstrated that liver membranes possess two distinct lipoprotein receptors: a low density lipoprotein (LDL) receptor that binds lipoproteins containing either apolipoprotein (apo-) B or apo-E, and an apo-E-specific receptor that binds apo-E-containing lipoproteins, but not the apo-B-containing LDL. This study reports the isolation and purification of apo-B,E(LDL) and apo-E receptors from canine and human liver membranes. The receptors were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and were partially purified by DEAE-cellulose chromatography. The apo-B,E(LDL) receptor was isolated by affinity chromatography on LDL-Sepharose. The apo-E receptor, which did not bind to the LDL-Sepharose column, was then purified by using an HDLc (cholesterol-induced high density lipoprotein)-Sepharose affinity column and an immunoaffinity column. Characterization of the receptors revealed that the hepatic apo-B,E(LDL) receptor is similar to the extrahepatic LDL receptor with an apparent Mr = 130,000 on non-reducing sodium dodecyl sulfate-polyacrylamide gels. The apo-E receptor was found to be distinct from the apo-B,E(LDL) receptor, with an apparent Mr = 56,000. The purified apo-E receptor displayed Ca2+-dependent binding to apo-E-containing lipoproteins and did not bind to LDL or chemically modified apo-E HDLc. Antibodies raised against the apo-B,E(LDL) receptor cross-reacted with the apo-E receptor. However, an antibody prepared against the apo-E receptor did not react with the apo-B,E(LDL) receptor. The apo-E receptor also differed from the apo-B,E(LDL) receptor in amino acid composition, indicating that the apo-E receptor and the apo-B,E(LDL) receptor are two distinct proteins. Immunoblot characterization with anti-apo-E receptor immunoglobulin G indicated that the apo-E receptor is present in the hepatic membranes of man, dogs, rats, and mice and is localized to the rat liver parenchymal cells.     

Isolation and characterization of a variant form of vasoactive intestinal polypeptide

A variant form of the vasoactive intestinal polypeptide (VIP) has been isolated. It was found to consist of a molecule which instead of the C-terminal asparagine amide of VIP has a C-terminal extension of Gly-Lys-Arg. This VIP variant displaces VIP in a VIP receptor assay, reacts with N-terminally-directed antisera in a VIP radioimmunoassay and possesses VIP-like bioactivity in an assay measuring pancreatic juice secretion in the cat.     

Isolation and characterization of two isozymes of myosin heavy chain from canine atrium

To clarify the characteristics of myosin isozymes in the atrium, we fractionated two isoforms of myosin heavy chain (HC), atrial HC alpha (A-HC alpha) and HC beta (A-HC beta), from the canine heart by affinity chromatography, using monoclonal antibodies specific for HC alpha (CMA19) and HC beta (HMC50), respectively, and then compared their peptide composition and enzymatic properties with those of ventricular HC alpha (V-HC alpha) and HC beta (V-HC beta). The reactivity of these isozymes with three monoclonal antibodies revealed that there are at least three different epitopes between A-HC alpha and A-HC beta. Differences in the primary structure of A-HC alpha and A-HC beta were confirmed by one- and two-dimensional gel electrophoretic analyses of these peptides, produced by digestion with alpha-chymotrypsin and cyanogen bromide (CNBr). A-HC alpha and V-HC alpha were indistinguishable proteins, and A-HC beta was also very similar to V-HC beta. Furthermore, there were differences between A-HC alpha and A-HC beta in their Ca2+-activated ATPase activities. The ATPase activity of A-HC beta was lower than that of A-HC alpha and was similar to that of V-HC beta. We concluded that there are two different isozymes of myosin heavy chain in the atrium (A-HC alpha and A-HC beta), as well as in the ventricle (V-HC alpha and V-HC beta), and that A-HC beta is very similar to V-HC beta, the predominant form of ventricular myosin, in its molecular structure and enzymatic activity.     

Immunochemical characterization of neurotensin-like peptides in chicken

Using radioimmunoassay and 3 region specific antisera toward bovine neurotensin (NT), the NT-like peptides in chicken have been shown to differ from NT but to strongly resemble its COOH-terminal region. Three substances were identified, one of which resembled NT biologically and appeared to share 7 or 8 of its COOH-terminal residues. The two other peptides were smaller than NT but seemed to possess 4–6 residue homologies with it. Tissue distribution studies indicated that the chicken pancreas and thymus had unusually high levels of this material (>200 fold that in rat) and that the 3 substances were distributed differently in tissues. Chromatographic studies showed that the peptides obtained from brain, intestine, thymus, and pancreas were similar. These results, demonstrating evolutionary conservation of the COOH-terminal region of NT, are in keeping with the known importance of this region for biological activity. These findings also suggest the existence of an NT-family of peptides serving multiple biological roles.     

Isolation and structural characterization of porcine coupling factor 6 from intestinal tissues

A polypeptide purified from an extract of thermostable, porcine intestinal peptides was found to correspond to coupling factor 6, previously known as a component of the mitochondrial oxidative phosphorylation system. The intestinal presence of this peptide offers a new source for preparation of the component in large quantities, and possibly suggests further functions of the polypeptide. Amino acid sequence analysis of this porcine form reveals it to be identical to the bovine form, except for two replacements, at position 62 (Thr in the porcine, Phe/Thr in the bovine form), and position 70 (Ala/Val). The extensive conservation suggests strict structural constraints on the functional properties of the polypeptide.