Isolation and properties of a soluble sialidase from the culture fluid of Chinese hamster ovary cells

A Soluble sialidase that can degrade recombinant glycoproteinsexpressed in Chinese hamster ovary (CHO) cells has been isolatedand purified to near homogeneity from the cell culture fluidof this host. Purification of     

Autoradiographic localization of ornithine decarboxylase in mouse kidney by use of radiolabeled α-difluoromethylornithine

Summary Ornithine decarboxylase, a key enzyme in polyamine biosynthesis and cell growth, has been localized in mouse kidney by autoradiography after administration of radiolabeled -difluoromethylornithine. This drug is an enzyme-activated irreversible inhibitor of ornithine decarboxylase and forms a covalent bond with the enzyme. It was found that ornithine decarboxylase is present in all cell types studied but that the highest content occurs in the proximal convoluted tubules followed by the distal convoluted tubules and the collecting tubules. The majority of the enzyme is located in the cytoplasm but about 10–15% is present in the nuclei (often associated with nucleolus-like components) of the cells of the proximal and distal convoluted tubules. The labeled ornithine decarboxylase was lost rapidly from both nucleus and cytoplasm of all the cell types examined, and labeling by radioactive -difluoromethylornithine was greatly reduced if the mice were pretreated for 5 h with cycloheximide to block protein synthesis. These results indicate that ornithine decarboxylase turns over rapidly in all of the cells.     

Structure of bovine beta-lactoglobulin at 6A resolution.

Bovine β-lactoglobulin is a dimer with a molecular weight of 2 × 18,400. In solution it undergoes a pH-dependent transition at pH 7.0 between two alternative structures, named N and R. The structures of four different crystal forms have been determined by multiple isomorphous replacement with heavy-atoms. Two of them, lattices K and X, were crystallised at pH 6.5, corresponding to the N state in solution; and the other two, lattices Y and Z, were crystallised at pH 7.5, corresponding to the R state in solution. The figures of merit of the phase angles determined for these lattices were 0.76, 0.77, 0.80 and 0.80, respectively. The four structures that emerged are similar and show certain features suggestive of α-helices and pleated sheets, but the resolution is insufficient to trace the entire course of the polypeptide chain. No clear distinction can yet be made between the structures above or below pH 7.0, nor between the native molecule and the molecule from which the C-terminal leucine and histidine residues have been cleaved. Analyses at higher resolution are in progress.     

The Interactions of Superoxide Ion(O-2) with Metallo-Porphyrins [(C18TPP)M, M = FE,MN,CO,ZN]; Models for Biological Systems and Superoxide Dismutases

In dimethylformamide superoxide ion forms a l:l adduct with tctrakis (2.6-dichlorophenyl) porphinatoiron, (Cl₈ TPP)FeOO^-, as well as with its manganese analogue, (Cl₈ TPP)MnOO^-. On the basis of their electrochemical, spectroscopic, and magnetic properties these adducts have a metal-oxygen covalent bond (PorM-OO^-), oxygen-centered redox chemistry. and reactivities that are similar to the hydroperoxide ion (HOO^-). Addition of ^-OH to a solution of PorFe and O₂ results in the formation of PorFe(OH)(OO^-), which can be electrochemically oxidized to PorFeOH plus O₂ (-0.2 V vs SCE). Addition of protons to the PorM-OO^- adducts promotes their rapid decomposition to PorM, HOOH. and O₂. This chemistry provides insight to the reactions of biological superoxide and superoxide dismutases.     

Induction of erythropoietin responsiveness in murine hematopoietic cells by the gag-myb-ets-containing ME26 virus.

ME26 virus, which was generated by inserting the coding region of the acute avian leukemia-inducing virus E26 into a murine retrovirus vector, encodes a 135-kDa gag-myb-ets fusion protein. Amphotropic murine leukemia virus pseudotypes of ME26 virus induce a high incidence of erythroleukemia 2 to 4 months after injection into newborn NFS/N mice. Spleen cells from the majority of these mice proliferate to high levels in the presence of the erythroid hormone erythropoietin (Epo) and can easily be established as permanent Epo-dependent cell lines. The cell lines contain multiple copies of ME26 viral DNA and express viral message and protein. An Epo receptor mRNA of normal size can be detected in these cells, and binding studies reveal a single class of lower-affinity Epo receptor with an affinity for Epo that is in the range of that previously reported for erythroid cells. The ME26 virus-induced Epo-dependent cell lines, however, appear more immature than previously described erythroid cell lines and more closely resemble early hematopoietic precursor cells, suggesting that the virus may be activating the Epo receptor in hematopoietic cells that do not normally express it. Consistent with this idea, we are able to infect an interleukin-3-dependent myeloid cell line, FDC-P2, with ME26 virus and convert it to Epo dependence. The ME26 virus-infected FDC-P2 cells, even before growth on Epo, showed a large increase in the amount of Epo receptor mRNA. However, no ME26 viral integrations can be detected adjacent to the Epo receptor gene, indicating that the virus is not activating the Epo receptor gene by promoter/enhancer insertion. Our results are more consistent with the hypothesis that the gag-myb-ets-encoded viral fusion protein, which is known to bind DNA, is directly or indirectly activating the expression of the Epo receptor gene in these cells.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20