Nakao Ishida

The structure of neocarzinostatin chromophore possessing a novel bicyclo-[7,3,0]dodecadiyne system

Abstract The structure of the chromophore of an antitumor antibiotic neocarzinostatin has been elucidated as a bicycro[7,3,0]dodecadiyne system having naphthalenecarboxylic acid, aminosugar and ethylene carbonate units.

Interferon Induction by Glycyrrhizin and Glycyrrhetinic Acid in Mice

Licorice extract is an herbal drug which has long been used as a demulcent and elixir in Chinese medicine. One of the main active components of licorice extract is glycyrrhizin (GL), a kind of saponin. The biological effects of GL and its aglycone, glycyrrhetinic acid (GA), have been extensively studied. Their anti-inflammatory (5), anti-ulcerous (3), and antiviral (14) effects have been reported from 1948 (15) to 1979 (14). Moreover, a preparation of GL combined with glycine and cysteine (SNMC), has been widely and successfully used in Japan as an antihepatitis drug (6, II, 19), although its mechanism of pharmacological action remains unclear. Recently, interferon (IFN) with or without adenine arabinoside (Ara-A) has been used to treat hepatitis B patients (4, 7). Studies show that IFN consistently decreases the level of either DNA polymerase or hepatitis B surface antigen in hepatitis patients. There are also many reports which suggest that some irnmunopotentiators induce IFN (13) and explain their antiviral activity in vivo as interferon mediated (8, 16, 18). Therefore, in the light of GLs clinical effect on hepatitis patients and of the fact that its structure resembles that of hydrocortisone, the possibility that GL induces IFN was proposed. In this study we investigated the ability of both GL and GA to induce IFN in mice. Sixto 8-week-old male DDI mice obtained from the Institute for Experimental Animals, Tohoku University School of Medicine, were used in this experiment. Six-week-old male C3H, ddY, CDF-l, C57BL, BALB/c, and athymic nude mice (nujnu) of BALB/c background were obtained from the Funabashi Farm Co., Ltd., and were used to study the effect of the different mouse strains on interferon induction. GL and GA were supplied by Minophagen Pharmaceutical Co. Drugs were dissolved in 0.01 M phosphate buffered saline (PBS) and adjusted to pH 7.2 with I N sodium hydroxide. Pooled sera obtained from three mice were tested for anti-viral activity, which was determined by the 50% plaque reduction method on L-929 monolayer cell cultures with vesicular stomatitis virus (VSV) and was expressed in international reference units based on NIH reference mouse IFN (Catalog No. 002-904-511) (12).

Structural components of Sendai virus: Serological and physicochemical characterization of hemagglutinin subunit associated with neuraminidase activity

Sendai virions were disrupted with Triton X-100 at alkaline pH, and the neuraminidase preparation was isolated by gel filtration and density gradient centrifugations in sucrose and cesium chloride. The final neuraminidase preparation was homogeneous in antigenicity when tested by the agar gel immunodiffusion, and associated with the hemagglutination inhibition (HI) antibody blocking activity. Chicken antiserum against the neuraminidase preparation inhibited both the neuraminidase and hemagglutinating activities. Immunoelectrophoresis was used to precipitate neuraminidase extracted with Triton from [ 3 H]lencine-labelled virus. When peptide analysis of the precipitate was performed by the polyacrylamide gel electrophoresis, the neuraminidase and HI-antibody blocking activities were found to be on a single protein. This protein migrated at the identical position to a glycoprotein having a molecular weight of 67,000.

Induction of interferon-γ in mouse spleen cells by OK-432, a preparation of Streptococcus pyogenes

Abstract A bacterial antitumor and immunopotentiating agent, OK-432, induced Interferon in the spleen cell cultures but not in the thymus cell cultures of various inbred strains of mice. When 1 × 10 7 spleen cells were cultured in the presence of 5 μg/ml of OK-432, interferon activity was detected as early as 4 hr later and reached a maximum level of about 160 to 500 units/ ml 24 hr later. OK-432-induced interferon was mainly an IFN-γ of molecular weight approximately 40,000, but also contained IFN-α and IFN-β.

A Microplate Method for Isolation of Viruses from Infants and Children with Acute Respiratory Infections

Between December 1984 and December 1986, a microplate technique was adopted for isolation of viruses from infants and children with acute respiratory infections. By using two kinds of tissue culture microplates, i.e., the HHVM plate, containing human embryonic fibroblast (HEF), HEp‐2, Vero and MDCK cells, and the MK plate which contains secondary monkey kidney cells, 1,080 field viruses were isolated from 1,061 (24.9%) out of 4,254 throat swabs. Of these 1,080 isolates, 1,003 (92.9%) were recovered in the HHVM plates and the remaining 77 (7.1%) in the MK plates. With the HHVM plate, influenza A and B viruses were cultivated in MDCK, RS virus in HEp‐2, parainfluenza and mumps viruses in Vero, adenoviruses in both HEF and HEp‐2, polioviruses in HEF, HEp‐2 and Vero, coxsackie B viruses in both HEp‐2 and Vero, rhino and echo viruses in HEF, herpes simplex virus in both HEF and HEp‐2, and cytomegalovirus in HEF, although MK were more sensitive than Vero to parainfluenza and coxsackie B viruses. There was no difference in the rate of isolation of viruses between the microplate and ordinary tube methods. Cross contamination in the microplates was negligible for routine work.

The smallest protein of Sendai virus: Its candidate function of binding nucleocapsid to envelope

The smallest nonglycosylated protein, VP5 (MW, 35,000), of Sendai virus was investigated for its location and function. Virion-associated VP5 resisted digestion by trypsin, fungal semi-alkali protease, or chymotrypsin, suggesting that VP5 is not an external protein. A close association of VP5 with both envelope and nucleocapsid was first established by polypeptide analyses and electron microscopic examinations of fractions obtained by a rate zonal centrifugation of alkali-Tween 20-treated virions. Second, by CsCl equilibrium centrifugation of the same alkali-Tween 20-disrupted virions, nucleocapsids free of VP5 (rho = 1.323) and those associated with VP5 (rho = 1.297) were separated. The former had a width of 18 nm, but the latter, a broad width ranging from 18 to 40 nm (mean, 30 nm). Third, Triton X-100 treatment of the virions without KCl left VP5 in association with nucleocapsid, and, by electron microscopy, globular structures consisting of folded nucleocapsid were seen, while the same treatment with 1 M KCl completely removed VP5 from nucleocapsid and straightened nucleocapsids were observed. These three results suggest that VP5 bound to nucleocapsid is functioning to associate it to envelope and to sustain the spherical shape of the virion.

On the study of Sendai virus hemolysis: I. Complete Sendai virus lacking in hemolytic activity

Abstract The hemolytic activity of Sendai virus was examined with samples collected at various times after infection of the embryonated eggs. The virus obtained just after the end of the one-step growth cycle (early harvest) was shown to exhibit no hemolytic activity, whereas it expressed full activity of cell fusion and infectivity. Incubation at 36°, freezing and thawing, or sonic treatment enabled the early harvests to be hemolytic. Conversion of the virus from the nonhemolytic state to a hemolytic one was always followed by the alteration of the virus envelope so that it became permeable to the uranyl acetate stain used for the negative staining (UA-particle). The degree of hemolysis and the number of UA-particles of the early harvest increased as freezing and thawing was repeated while the degree of cell fusion and infectivity was decreasing. On the basis of these findings, it was concluded that Sendai virus with intact envelope does not exhibit hemolysis, while it has the potential activity to do so.

Prevention of rotavirus infection by oral administration of cow colostrum containing antihumanrotavirus antibody

After immunizing 8-month pregnant Holstein cows with human rotavirus, Wa strain, cow colostrum containing neutralizing antibody to human rotavirus, designated as Rota colostrum, was obtained. After randomly grouping 13 infants from a single orphanage, 6 infants received 20 ml of Rota colostrum every morning and 7 control infants received 20 ml of market milk. One month later, rotavirus associated diarrhea was observed in 6 of the 7 infants given milk and 1 out of the 6 infants given Rota colostrum. Orally administered Rota colostrum significantly protected infants from diarrhea caused by rotavirus (P < 0.05). Two out of 5 Rota colostrum recipients who were free from diarrhea showed rises in complement fixation (CF) antibody titer after the rotavirus infection epidemic. Thus, Rota colostrum prevented the outbreak of diarrhea but did not prevent immunological responses to natural rotavirus infection. In the therapeutic trial Rota colostrum had no effect on duration of diarrhea, bowel movements or virus shedding in stool. However, there were no side-effects of Rota colostrum.

Mode of action of neocarzinostatin: inhibition of DNA synthesis and degradation of DNA in Sarcina lutea.

Neocarzinostatin, a polypeptide anti-tumour substance produced by Streptomyces carzinostaticus, was investigated for its bactericidal action on Sarcina lutea. The primary action of the antibiotic on Sarcina lutea was the selective inhibition of DNA synthesis. Incorporation of [14C]thymidine into DNA was inhibited immediately after the addition of neocarzinostatin at a concentration as low as 0.005 μg/ml. Incorporation of [14C]uridine into RNA and of [14C]leucine into protein were not inhibited at a concentration of 0.5 μg/ml which caused almost complete inhibition of DNA synthesis. Moreover, DNA was markedly degraded into free bases at concentrations over 0.5 μg/ml. Despite the considerable DNA degradation, the DNA extracted from the cells treated with neocarzinostatin was able to serve as the template for DNA synthesis in vitro and did not differ from normal DNA in thermal denaturation profile. A relation between the mechanism of inhibition of DNA synthesis and degradation of DNA is discussed.

Isolation and characterization of two distinct types of HVJ (Sendai virus) spikes

Abstract Solubilization of the envelope of HVJ (Sendai virus) with alkali-Tween 20 and subsequent fractionation by sucrose density electrofocusing in the presence of Tween 20 resulted in the separation of two biologically distinct glycoproteins: one protein banded at pH 4.9 without hemagglutinin and neuraminidase activities (HANA − ) and the other at pH 6.5 with both hemagglutinin and neuraminidase activities (HANA + ). Morphologically, both fractions consisted of dumbbell-shaped spikes, with HANA − spikes exhibiting more definite contour in or shape of knob and shaft than HANA + spikes. Serologically, HANA − and HANA + spikes were distinct from each other in micro-Ouchterlony test. Moreover, SDS-polyacrylamide gel electrophoresis revealed that HANA − spikes were composed of two species of glycoproteins, VP4, (MW, 51,000) and SGP (MW, 15,000), while HANA + spikes contained single glycoprotein, VP2 (MW, 67,000). Radioactivity of HANA − spike fraction did not adsorb onto chicken erythrocyte ghosts, whereas radioactivity, neuraminidase, and hemagglutinin activity of HANA + spike fraction adsorbed onto the ghosts. No hemolytic activity was detected in either HANA − or HANA + spike fractions.