Purification of Transferrin and Albumin from Mouse Ascites Fluid

Mouse ascites fluid, which is readily obtained when cell lines and hybridomas are maintained in host mice, is a convenient source of several plasma proteins. This paper describes procedures for the purification of albumin and transferrin from mouse ascites fluid. Mouse transferrin was prepared from a 50–75% ammonium sulfate fraction of mouse ascites fluid by CM- and DEAE-cellulose chromatography. Mouse albumin was obtained by the same purification route, but required an additional chromatography step on Cibacron Blue F3GA-agarose. Both proteins were shown to be homogeneous by polyacrylamide gel electrophoresis and Immunoelectrophoresis. Characterization, which included a determination of amino acid composition, partial N-terminal sequence, molecular weight and extinction coefficient, correlated well with known values reported for human transferrin and albumin. The purified mouse proteins may be useful for biochemical studies, antibody preparation, and as growth factors for hybridomas or other mouse cell lines maintained in culture.     

Contact Heterogeneity,Rather Than Transmission Efficiency,Limits the Emergence and Spread of Canine Influenza Virus

Host-range shifts in influenza virus are a major risk factor for pandemics. A key question in the study of emerging zoonoses is how the evolution of transmission efficiency interacts with heterogeneity in contact patterns in the new host species, as this interplay influences disease dynamics and prospects for control. Here we use a synergistic mixture of models and data to tease apart the evolutionary and demographic processes controlling a host-range shift in equine H3N8-derived canine influenza virus (CIV). CIV has experienced 15 years of continuous transfer among dogs in the United States, but maintains a patchy distribution, characterized by sporadic short-lived outbreaks coupled with endemic hotspots in large animal shelters. We show that CIV has a high reproductive potential in these facilities (mean R₀ = 3.9) and that these hotspots act as refugia from the sparsely connected majority of the dog population. Intriguingly, CIV has evolved a transmission efficiency that closely matches the minimum required to persist in these refugia, leaving it poised on the extinction/invasion threshold of the host contact network. Corresponding phylogenetic analyses show strong geographic clustering in three US regions, and that the effective reproductive number of the virus (R_e) in the general dog population is close to 1.0. Our results highlight the critical role of host contact structure in CIV dynamics, and show how host contact networks could shape the evolution of pathogen transmission efficiency. Importantly, efficient control measures could eradicate the virus, in turn minimizing the risk of future sustained transmission among companion dogs that could represent a potential new axis to the human-animal interface for influenza.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Hydrolysis of peptides by carboxypeptidase A: equilibrium trapping of the ES2 intermediate

The cobalt absorption and electron paramagnetic resonance (EPR) spectra of cobalt carboxypeptidase undergo unique variations on formation of catalytic peptide and ester intermediates as previously recorded in cryoenzymologic experiments employing rapid-scanning spectroscopy and cryotrapping [Geoghegan, K. F., Galdes, A., Martinelli, R. A., Holmquist, B., Auld, D.S., & Vallee, B. L. (1983) Biochemistry 22, 2255-2262]. We here describe a means of stabilizing these intermediates, which we have termed "equilibrium trapping". It allows peptide intermediates to be observed for longer periods (much greater than 1 min) at ambient as well as subzero temperatures. The reaction intermediate with the rapidly turned over peptide substrate Dns-Ala-Ala-Phe is trapped when the cobalt enzyme (greater than 10 microM) has catalyzed the attainment of chemical equilibrium between high concentrations of the hydrolysis products Dns-Ala-Ala, 10 mM, and L-phenylalanine, 50 mM, and the product of their coupling Dns-Ala-Ala-Phe. Under these conditions, Dns-Ala-Ala-Phe is present in the equilibrated substrate-product reaction mixture at a level that exceeds the one predicted on the basis of K'eq for hydrolysis of this substrate and is close to the enzyme concentration. Other pairs of peptide hydrolysis products yield similar results. Visible absorption and EPR spectra of the cobalt enzyme show that the synthesized peptide binds to the active site in the mode previously recognized as the ES2 catalytic intermediate in peptide hydrolysis. Equilibrium trapping of the ES2 intermediate allows analysis of its physicochemical properties by methods that could not be employed readily under cryoenzymological conditions, e.g., circular dichroic and magnetic circular dichroic spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

The composition of peptidochitodextrins from sarcophagid puparial cases

1. N-Bromosuccinimide cleaved proteins and pigments from fly puparia, increasing the chitin:protein ratio from 0.5 to 1.5. The product afforded subfractions (ratio 5:1) of molecular weights of 1200 and 1600 devoid of aromatic residues and N-terminal beta-alanine, direct aryl links between polysaccharide chains being discounted. 2. The chitin-protein complex decreased in molecular weight when treated with Pronase, which suggested polypeptide bridges within the native chitin micelle. The limit dextrins generated by chitinase were mixtures of unsubstituted dextrins and peptidylated oligosaccharides, with the former predominating. 3. Peptidochitodextrins of similar molecular weight but markedly different solubility were prepared, which were indistinguishable with respect to amino acid, glucosamine, acetyl, X-ray or infrared characteristics. It is suggested that physical interactions contribute to the stability of the integument in addition to the covalent bonds that form during sclerotization.     

Molecular Characterization of the Interaction of Staphylococcal Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMM) ClfA and Fbl with Fibrinogen

The ligand-binding domain of Fbl (the fibrinogen binding protein from Staphylococcus lugdunensis) shares 60% sequence identity with ClfA (clumping factor A) of Staphylococcus aureus. Recombinant Fbl corresponding to the minimum fibrinogen-binding region (subdomains N2N3) was compared with ClfA for binding to fibrinogen. Fbl and ClfA had very similar affinities for fibrinogen by surface plasmon resonance. The binding site for Fbl in fibrinogen was localized to the extreme C terminus of the fibrinogen γ-chain at the same site recognized by ClfA. Isothermal titration calorimetry showed that Fbl and ClfA had very similar affinities for a peptide mimicking the C-terminal segment of the fibrinogen γ-chain. The peptide also inhibited binding of Fbl and ClfA to fibrinogen. A series of substituted γ-chain variant peptides behaved very similarly when used to inhibit ClfA and Fbl binding to immobilized fibrinogen. Both ClfA and Fbl bound to bovine fibrinogen with a lower affinity compared with human fibrinogen and did not bind detectably to ovine fibrinogen. The structure of the N2N3 subdomains of Fbl in complex with the fibrinogen γ-chain peptide was modeled based on the crystal structure of the N2N3 subdomains of the ClfA-γ-chain peptide complex. Residues in the putative binding trench likely to be involved in fibrinogen binding were identified. Fbl variant proteins with alanine substitutions in key residues had reduced affinities for fibrinogen. Thus Fbl and ClfA bind the same site in fibrinogen by similar mechanisms.     

Nonneutral evolution of tandem repeats in the mitochondrial DNA control region of lagomorphs

The mitochondrial DNA of the European rabbit (Oryctolagus cuniculus) contains a tandem array of 153-bp repeats in the vicinity of the replication origin of the H-stand. Variation among molecules in the number of these repeats results in inter- and intraindividual length polymorphism (heteroplasmy). Generally, in an individual, one predominant molecular type is observed, the others representing a low percentage of the mtDNA content. At the tissue level, we observe a particular distribution of this polymorphism in the gonads compared with liver, kidneys, or brain, implying a relationship between the differentiation status of the cells and the types of new mtDNA molecules which appear and accumulate during lifetime. Similar tandem repeats were also found in the mtDNA noncoding region of European hares (Lepus europaeus), a cottontail (Sylvilagus floridanus), and a pika (Ochotona rufescens). The lengths and the sequences of these units evolve rapidly and in a concerted way, but the number of repeats is maintained in a narrow range, and an internal 20-bp segment is highly conserved. Constraints restrict the evolution of the primary sequence of these repeated units, the number of which is probably controlled by a stabilizing selection.