Akira Kuninaka

A sensitive radioimmunoassay for adenosine in biological samples.

Abstract A simple, sensitive, specific, and reproducible radioimmunoassay for the measurement of adenosine in biological materials has been developed. Adenosine antibody was obtained by immunizing rabbits with an immunogen prepared by conjugating 2′,3′-disuccinyladenosine to human serum albumin. By succinylating adenosine in samples at the 2′- and 3′-O positions with a premixed reagent consisting of succinic anhydride, triethylamine, and dioxane, the assay became sensitive enough to detect less than picomole amounts of adenosine in minute quantities of tissues. The corss-reactivity of structurally related compounds with the antibody was mostly negligible except for 2′-deoxyadenosine, whose usual concentration was very low. The use of this method made it possible to measure adenosine without any prior purification procedure. The immunoreactive materials in various biological samples disappeared during incubation of the samples with adenosine deaminase.

Antitumor Activity and Pharmacology of 1‐β‐D‐Arabinofuranosylcytosine‐5′‐stearylphosphate: An Orally Active Derivative of 1‐β‐D‐Arabinofuranosylcytosine

The antitumor activity of l‐β‐D‐arabinofuranosylcytosine‐5′‐alkylphosphates (CnPCAs) against L1210 leukemia in mice after oral administration was demonstrated. The optimum length of the alkyl group on the phosphate moiety of CnPCA for exhibiting a high antitumor activity was found to be between tetradecyl (C14) and tricosyl (C23). The most active alkyl derivative in this system was found to be l‐β‐D‐arabinofuranosylcytosine‐5′‐stearylphosphate (C18PCA). The optimum and minimum effective doses of C18PCA were 100 and 6,25 mg/kg/day (q1d, day 1 to day 5), respectively. The maximum T/C% of C18PCA was approximately 220. The antitumor activity of C18PCA was not greatly dependent on the treatment schedule and route. Plasma concentration of 1‐β‐D‐arabinofurano‐sylcytosine (ara‐C) remained in the range of 0.4 to 0.75 μmol/ml for 24 h after oral administration of 100 mgAg (170 μmol/kg) of C18PCA. These results indicate that C18PCA administered per orally is absorbed intact through the gastrointestinal tract and ara‐C is released for a long period of time. C18PCA is regarded as an orally active depot form of ara‐C

In Vitro Antiherpesviral Activity of 5-Alkyl Derivatives of 1-β-d-Arabinofuranosyluracil

Several 5-alkyl derivatives of 1-β-d-arabinofuranosyluracil (araU) were tested for antiherpesviral activity and inhibitory action on cell growth in human embryonic lung fibroblasts. 1-β-d-Arabinofuranosylcytosine, 9-β-d-arabinofuranosyladenine, and 5-iododeoxyuridine (IUdR) were included as reference materials. Among the 5-alkyl derivatives of araU, arabinosylthymine was the most active, followed by 5-ethyl- and 5-propyl-araU. 5-Ethyl-araU was as active as IUdR and more active than 9-β-d-arabinofuranosyladenine against herpes simplex virus (HSV) type 1 and did not inhibit cell growth at a concentration as high as 1,000 μg/ml. 5-Butyl- and 5-methoxymethyl-araU, as well as araU, exhibited relatively low activity. The araU derivatives tested were as active against HSV WT-34, an isolate from a patient with keratitis, as against HSV type 1. Against an IUdR-resistant isolate, HSV WT-20, arabinosylthymine was less inhibitory than IUdR. Deoxyribonucleic acid synthesis in HSV type 1-infected cells was markedly inhibited by arabinosylthymine, IUdR, and 5-ethyl-araU, whereas cellular deoxyribonucleic acid synthesis in uninfected cells was significantly inhibited by IUdR but not by arabinosylthymine or 5-ethyl-araU.

Antiherpesviral activity and inhibitory action on cell growth of 5-alkenyl derivatives of 1-beta D-arabinofuranosyluracil.

Antiherpesviral activity of 5-vinyl-1-beta-D-arabinofuranosyluracil was as high as that of 1-beta-D-arabinofuranosylthymine, whereas the former was less inhibitory to cell growth than the latter. 5-Propenyl- and 5-butenyl-1-beta-D-arabinofuranosyluracil were less active than 5-vinyl-1-beta-D-arabinofuranosyluracil.

Studies on 5′-Phosphodiesterases in Microorganisms: Part II. Properties and Application of Penicillium citrinum 5′-Phosphodiesterase

5′-Phosphodiesterase, which degrades RNA into nucleoside-5′-monophosphates but does not attack DNA, is present not only in mycelium but also in culture filtrate of Penicillium citrinum Thorn 1131. For the formation of this enzyme pH of the culture medium must be kept below 7.0 during culture, as this enzyme is inactivated rapidly in alkaline solution. The pH optimum of this enzyme is in the region of pH 5. Cysteine, Mg++, sodium fluoride, and inorganic ortho- or pyrophosphate are without appreciable effect on this enzyme. Nucleoside-5′-monophosphates, which have been regarded as new chemical seasonings, can be produced economically in a large scale by using the microbial 5′-phosphodiesterase.

A practically sensitive radioimmunoassay for cyclic CMP by 2′-O-acetylation

Abstract An improved procedure has been developed for the measurement of low levels of cyclic CMP in biological materials. Cyclic CMP antibody was obtained by immunizing rabbits with an antigen prepared by conjugating 2′- O -succinyl cyclic CMP to human albumin. Based on the remarkable affinity of the cyclic CMP antibody thus prepared for 2′- O -acyl cyclic CMP, the acetylation of cyclic CMP in samples at the 2′- O -position was adopted to enhance the assay sensitivity. Acetylation of cyclic CMP was performed simply with a premixed reagent consisting of acetic anhydride and triethylamine. Although the acetic acid and triethylamine in the sample solution originating from the acetylating reagent interfered with the subsequent binding to the antibody, they were quickly evaporated. Thus, the dilution of samples to reduce interference is not needed at all throughout the procedure. By the acetylation technique described, the total sensitivity of the assay was increased so markedly that cyclic CMP in small quantities of urine and tissue extracts was readily determined.

Interferon Production in Human Diploid Cell Strains Derived from Embryonic Lungs

Twenty‐three strains of human diploid cells derived from embryonic lungs were tested for production of interferon by “superinduction.” Strain HAIN‐55 produced a relatively high level of interferon. The optimal concentration of cycloheximide for superinduction was essentially equal to that reported with foreskin fibroblasts. On the other hand, actinomycin D at a concentration of 4 to 16 μg/ml enhanced the production of interferon more strikingly than at a concentration of 1 μg/ml, which was usually employed for superinduction in the foreskin fibroblasts. Inhibition of interferon production was observed when fetal bovine serum was added to the medium during treatment with metabolic inhibitors for superinduction. Minimal essential medium was superior to Eagles basal medium as growth medium for interferon production, and serum added after removal of metabolic inhibitors could be replaced by bovine serum albumin. The yield of interferon produced under the best conditions in this study, with strain HAIN‐55, was more than 10,000 reference units/ml.

Secondary Structure of Nuclease P1

The molecular conformation of nuclease P1 in aqueous solution was investigated by measuring the optical rotatory dispersion (ORD) and circular dichroism (CD). The optical rotatory dispersion constant, λ was 281 nm. The Moffit-Yang parameters, a0 and b0, were −2 and −195, respectively. The ORD spectrum showed a minimum at 234 nm and the reduced mean residue rotation at 233 nm, [m]233, was −5880. The CD spectrum showed a double minimum at 213 and 226 nm and the molecular ellipticity at 222 nm, [θ]22, was -11,900. From these data, the α-helix content was calculated to be 29 to 31 %. The computer fit of CD suggests that the α-structure is about 6% and the random coil is about 63%. The helical structure was found to be quite stable to denaturing reagents such as urea and guanidine hydrochloride. However, removal of zinc atoms from the enzyme resulted in disruption of the helical structure with inactivation.

Susceptibility of Influenza Viruses to Interferon and to Poly (I) · Poly (C) Determined by the Plaque Reduction Method

Susceptibility of eight strains of influenza A and B viruses to interferon and to poly(I) · poly(C) were determined by the plaque reduction method. All strains tested were slightly less susceptible than vesicular stomatitis virus (VSV) in an established line of canine kidney (MDCK) cells. The 50% plaque depression doses (PD50) of poly(I) · poly(C) for influenza A and B viruses were as high as 3.0‐ to 4.5‐fold and 6‐ to 18‐fold that for VSV, respectively. The amounts of interferon required to inhibit plaque formation of influenza A and B viruses by 50% were 3.0–6.2 and 7.3–15.2 units/ml, respectively. The ratio of PD50 of poly(I) · poly(C) for each strain of influenza viruses tested to that for VSV in chick embryo cells was almost the same as in MDCK cells. Furthermore, in chick embryo cells, the strains of influenza virus tested were demonstrated to be much more susceptible to poly (I) · poly(C) than both Newcastle disease virus and vaccinia virus. It is suggested that influenza viruses may be relatively susceptible to interferon and to poly(I) · poly(C).

Effect of Nucleosides on Interferon Production and Development of Antiviral State Induced by Poly I·Poly C

Effect of nucleosides both on induction of antiviral state in chick embryo cells (CEC) or rabbit kidney cells (RK13) and on interferon production in RK13 or mouse fibroblast cells (L cells) by polyriboinosinic‐polyribocytidylic acid (poly I·poly C) was studied. Addition of inosine or a fifty‐fifty mixture of inosine and uridine at a final concentration of 0.1 mM to 10 mM to a growth medium enhanced development of antiviral state in CEC. The nucleoside effect was also observed in RK13 at 0.1 mM but not at a concentration higher than 1 mM. Interferon production in RK13 by superinduction (sequential treatment with metabolic inhibitors after exposure to poly I·poly C) was enhanced 1.5‐ to 4.0‐fold by addition of the nucleoside mixture to the growth medium. When RK13 was pretreated with 10 units per ml of interferon and then superinduced by inhibitors, the enhancing effect of nucleosides on interferon production was not observed. Interferon production in L cells was potentiated a little by addition of 1 mM of the nucleoside mixture to the growth medium. The effect of nucleoside was not observed when the nucleosides were added after exposure to poly I·poly C. The nucleoside effect may be applicable for production of high titered interferon.