A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the α2,6-sialyltransferase (α2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both α2,6- and α2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing α2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the α2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-γ and the tissue inhibitor of metalloproteinases-1. Interferon-γ purified from the universal host carried 40.4% α2,6- and 59.6% α2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-γ produced by normal CHO cells.     

Molecular analysis of the cystinuria disease gene: identification of four new mutations,one large deletion,and one polymorphism

A cystinuria disease gene (rBAT) has recently been identified, but evidence strongly suggests that only Type-I cystinuria is due to mutations in this gene. Sixteen point mutations and a large deletion causing the disease have so far been described in the rBAT gene sequence. To identify new mutated alleles, genomic DNA was analyzed, after the determination of the entire genomic structure of the rBAT gene, by RNA-single strand conformation polymorphism analysis, an accurate and sensitive method able to detect nucleotide changes. Four new point mutations, a large deletion, and a common intragenic polymorphism were detected. These new mutations increase to 22 the number of mutated alleles so far characterized in rBAT. In addition, the frequency of 21 mutations was assessed in a sample of accurately defined Type-I cystinuria choromosomes. They account for about 58% of all Type-I chromosomes, mutation M467T being the most common (0.26). Received: 15 March 1996 / Revised: 17 May 1996     

Oxygen consumption as a measure yeast starter culture vitality for the wine industry

Summary A simple technique for rapid determination of fermentation starters vitality which eliminates the need for determination of viable cells counts is described. The mathematical relationship between cell number and oxygen consumption of eight strains of Saccharomyces cerevisiae was studied. Results confirmed the possibility of utilizing a pO2 probe as an indicator of cell viability for fermentation starter.     

Structural features of the pore formed by Staphylococcus aureusα-toxin inferred from chemical modification and primary structure analysis

Abstract Staphylococcus aureus α-toxin makes cells and model membranes permeable to ions and uncharged molecules by opening oligomeric pores of uniform size. Its primary sequence reveals peculiar features which give some hints on the structure of the pore. A flexible region separating the toxin into two halves, several amphiphilic β-strands and two amphiphilic α-helices long enough to span the hydrophobic core of the lipid bilayer are predicted. In analogy to bacterial porins, we propose that the inner walls of the pore are, at least in part, built by an amphiphilic β-barrel. The model is consistent with circular dichroism data and with the electrophysiological properties of the pore. Functional information on this toxin were obtained by chemical modification of its four histidine residues. Specific carbethoxylation suggested they have different roles: one is required for specific receptor binding, one for oligomerisation and two for unspecific lipid binding. A tentative assignment of each histidine to its specific role is done on the basis of the structural predictions. A functionally related hemolysin, Aeromonas hydrophyla aerolysin, reveals remarkably similar features including the presence and location of histidines involved in receptor binding and oligomerisation.     

Helicobacter pylori and gastric autoimmunity

Host specific T-cell response is critical for the outcome of Helicobacter pylori infection. In genetically susceptible individuals, H. pylori can activate gastric CD4⁺ Th1 cells that recognize cross-reactive epitopes shared by H. pylori proteins and self H⁺, K⁺-ATPase, leading to gastric autoimmunity via molecular mimicry.     

Linear oligopeptides: c.d. and n.m.r. study of Dnp-pNA derivatives of Aib-containing tetrapeptides in a β-bend conformation

A circular dichroism investigation of two β-bend forming, Aib-containing tetrapeptides, with the -Aib-l-Ala- and -l-Ala-Aib- central sequences, occurring in peptaibol antibiotics, blocked at the N-terminal end with the 2,4-dinitrophenyl group and at the C-terminal end with the p-nitroanilino group, is described. A comparison is made with a tetrapeptide containing the -l-Ala-l-Ala- central sequence, also observed in peptaibol antibiotics. The amount of β-bend conformers, determined from the intensities of the exciton couplets arising from the intramolecular interaction of the p-nitroanilino chromophores, has been assessed as a function of the hydrogen-bonding properties of the solvent. The conformational analysis in dimethylsulphoxide has been extended to ¹H nuclear magnetic resonance. Some information on the type of β-bend formed has additionally been obtained. The preparation and characterization of the three chromophoric tetrapeptides, along with some analogues and synthetic precursors, are also reported.