Sigeyasu Kobayashi

Structural Characterization of Recombinant Human Interferon-Gammas Derived from Two Different Mammalian Cells

Recombinant human interferon‐gammas (rHuIFN‐γs) were obtained from two different mammalian cells (mouse C127 cells and Chinese hamster ovary, CHO, cells) cultured in a microcarrier culture system. Both rHuIFN‐γs were purified using sequential chromatographies for their comparison of structural properties. The peptide maps of HuIFN‐γs digested with V8 protease and Western blot analysis demonstrated that C127 cells yielded mainly about 25kDa component and CHO cells produced about 25kDa and about 20kDa components. By the identification of glycosylated peptides, it was suggested that 20kDa and 25kDa components are glycosylated at one and at two sites, respectively. C‐terminal amino acid sequence analysis indicated that both rHuIFN‐γs consisted of at least six different species lacking 2 to 16 amino acid residues from C‐terminus, so that C‐termini of both rHuIFN‐γs were slightly different from each other.

[59] Assay of interferon by reduction of viral RNA synthesis: A convenient assay for tracer experiments with monolayer cultures

Publisher Summary This chapter presents the method using the special culture tubes, and the microtiter plate method. In special culture tubes, the dried tubes are put into counting vials containing a toluene-based scintillation fluor, and their radioactivity is determined. Since the two methods are quite similar to each other, the following comments apply to both. The host range of vesicular stomatitis virus (VSV) is known to be rather broad. Thus, both methods can be applied for assaying interferon of various species (including type II interferon) without any modification if suitable cells of the corresponding species are chosen. When mouse L cells, human FL cells, rabbit RK-13 cells, or other cells from established cell lines are employed, they can be seeded just prior to application of interferon samples. But, when human FS-7 cells or other diploid cells are employed, they take time to form good cell sheets. (For assaying human interferon, FS-7 cells are severalfold more sensitive than FL cells.) It is recommended to seed FS-7 cells a couple of days before use in order to minimize the variation in the incorporated radioactivity. In this case, the medium is replaced with fresh medium before use.

Translation of Human Interleukin 2 mRNA in Xenopus laevis Oocytes

Poly(A)‐positive mRNA extracted from tonsillar mononuclear cells stimulated with phytohemagglutinin‐M and 12‐o‐tetradecanoyl phorbol 13‐acetate was successfully translated into biologically active interleukin 2 (IL‐2) in Xenopus laevis oocytes, and secreted into the incubation medium. In control experiments, the extract of oocytes injected with either poly(A)‐negative RNA or buffer did not show any IL‐2 activity. By sucrose density gradient centrifugation analysis, IL‐2 mRNA was found as a single peak corresponding to a sedimentation coefficient of 10‐11S.

Topical Administration of Human Fibroblast Interferon in Herpes Simplex Keratitis of the Rabbit

The action of interferon (IFN) is generally considered to be species specific. However, IFN produced by human cells is known to block viral replication not only in infected human cells but also in infected rabbit or simian cells (2, 3). There are two antigenically distinct human IFNs, one produced by leukocytes (Hu IFN-a) and the other by diploid fibroblasts (Hu IFN-,B). McGill et al (6) described the effect of Hu IFN-a on herpes simplex keratilis of the rabbit. As yet, the effect of Hu IFN-,B on this keratilis of rabbit has not been documented. This study was conducted to evaluate Hu IFN-,B eye drops on keratitis caused by herpes simplex virus in albino rabbits. Partially purified Hu IFN-,B was prepared in our laboratory (Basic Research Laboratories, Toray Industries, Inc.), as described previously (5). IFN was titrated by a semi-micromethod based on the inhibition of the cytopathic effect of vesicular stomatitis virus (VSV) (CPEso method) (1) or a method based on the inhibition of VSV-RNA synthesis (INASso method) (8,9). The titer was corrected to the value of the international reference Hu IFN-,B (G-023-902-527). The preparation had a titer of 2.8 X 106 international reference units (IU) Iml with a specific activity of about 5 X 107 IUImg of protein. The species specificity of the antiviral activity of the IFN-,B preparation used in this study was examined in in vitro experiments using human FL cells and rabbit RK-13 cells which are very sensitive to IFN. In the assay systems using both the INASsoand CPEso methods, the IFN-,B showed fairly high activity in the rabbit RK-13 cells (about 10% of the titer in the human cell system). Similar results were obtained in the assay of antiviral activity of Hu IFN-a (kindly furnished by Dr. Kari Cantell of Finland) by the same methods. These results suggest that Hu IFN-,B, like Hu IFN-a, might exert an antiviral effect on herpes simplex keratitis of rabbit. An in vivo masked study was conducted with eight rabbits. Herpes simplex virus type 1 was used. A purified solution of human plasma albumin (1 mgjrnl of normal saline) was used as a placebo, since the same concentration of albumin was added to the final IFN-,B preparation as a stabilizer. A viral suspension containing

Constitutive Long-Term Production of Recombinant Human Interferon-Gamma by Transformed Mouse C127 Cells Cultured in Serum Free Medium

Transformed mouse C127 cells in which human interferon-gamma cDNA was inserted were grown well in a microcarrier culture system. The cells were maintained for more than two months even in a serum free condition without cell proliferation and continued to produce a high level of recombinant human interferon-gamma (rHuIFN-r). The maintenance of the cells grown on the microcarriers was fairly prolonged by adding 0, 1% bovine serum albumin to the serum free medium. The flow-cytometrical analysis for cell cycle showed more than 85% of these maintained cells remained in G0/G1 phase, differing from the case of the growing cells. However, we confirmed the copy-numbers of rHuIFN-r cDNA were safely retained even in the maintained cells for a long period. The rHuIFN-r preparation obtained in the serum free condition was also glycosylated protein similar to natural HuIFN-r derived from human periferal blood lymphocytes.