James A. North

In vitro virucidal activity of selected anthraquinones and anthraquinone derivatives

Anthraquinones and anthraquinone derivatives were characterized for their antiviral and virucidal activities against viruses representing several taxonomic groups. One of these compounds, hypericin, had activity against vesicular stomatitis virus, herpes simplex virus types 1 and 2, parainfluenza virus, and vaccinia virus (from 0.5 to 3.8 log10 reductions in infectivity) at concentrations of less than 1 microgram/ml as determined by a direct pre-infection incubation assay. Human rhinovirus was not sensitive to hypericin at concentrations up to 10 micrograms/ml. Addition of small amounts of Tween-80 to solutions containing hypericin enhanced, by up to 2.6 log10, hypericins virucidal activity. Anthraquinones and anthraquinone derivatives with the hydroxyl and alkyl substitution pattern of emodin (i.e. emodin, emodin anthrone, emodin bianthrone and hypericin) were active against the enveloped viruses tested. The following general pattern of activity was found: hypericin greater than emodin bianthrone greater than emodin anthrone greater than emodin. Chrysophanic acid, aloe-emodin, and sennosides A and B did not possess activity against any of the viruses tested.

The antiviral agent hypericin has in vitro activity against HSV-1 through non-specific association with viral and cellular membranes

The antiviral and virucidal compound, hypericin, was studied regarding its activity and possible mechanism against herpes simplex virus (HSV-1). It was determined that hypericin caused slight inhibition of viral adsorption to and penetration of Vero cells. Additionally, yield reduction assays suggested that hypericin was most effective against HSV-1 as a virucidal agent rather than as an intracellular antiviral agent. Fluorescence microscopy revealed that hypericin initially associated with cytoplasmic membranes and that over the course of time it became concentrated in intracellular membranous regions, probably the Golgi apparatus or endoplasmic reticulum (ER). These concentration events failed to inhibit glycosylation of either viral or cellular proteins and were effectively blocked by compounds which inhibit endocytosis or membrane cycling between the ER and Golgi. Based on fluorescence studies, it was determined that hypericin had non-specific affinity for protein and higher affinity for detergent and lipid. The evidence suggested that strong, non-specific association with membranes, both viral and cellular, are probably the basis of hypericins virucidal and antiviral activity.

The isolation of N5-methyltetrahydrofolate-homocysteine transmethylase from bovine brain☆

Abstract The enzyme ( N 5 -methyltetrahydrofolate-homocysteine transmethylase) that catalyzes the transfer of a methyl group from N 5 -methyltetrahydrofolate to homocysteine to form methionine has been found in a number of bovine tissues. The level of transmethylase activity was higher in brain than in any other tissue examined including liver and kidney. The brain enzyme has been purified approximately 370-fold. An initial fractionation with protamine sulfate and ammonium sulfate was followed by column chromatography on DEAE-Sephadex and Sephadex G-200. As the specific activity of the transmethylase increased the enzyme showed a greater dependency upon S -adenosyl methionine and a reducing system.

The isolation of serine transhydroxymethylase from bovine brain.

Abstract Serine transhydroxymethylase has been purified 150-fold from bovine brain. The initial fractionation with portamine sulfate and ammonium sulfate was followed by chromatography on DEAE-Cellulose and Sephadex G-200. The cofactor requirements for bovine brain serine transhydroxymethylase included pyridoxal phosphate and tetrahydrofolate. The maximum catalytic activity occurred at pH 7.6 and the Michaelis constant for serine was 6.7 × 10 −4 m .

5-methyltetrahydrofolate: synthesis and utilization in normal and SV40-transformed BHK-21 cells.

Abstract BHK-21 cells and SV40-transformed BHK-21 cells (A-8) grew rapidly in a medium containing methionine (I). BHK-21 cells also proliferated in a methionine-deficient medium (II) supplemented with homocysteine and vitamin B 12 ; however, the A-8 cells did not proliferate and ultimately died in this medium. The 5,10-methylenetetrahydrofolate reductase activity in transformed cells was only 10% of that found in BHK-21 cells. While A-8 cells in medium II could not be maintained on folate or 5-formyltetrahydrofolate, they survived and even proliferated slowly when provided 5-methyltetrahdyrofolate. The low level of the reductase in A-8 cells and the inability of these cells to utilize folate or 5-formyltetrahydrofolate suggests that A-8 cells are unable to synthesize 5-methyltetrahydrofolate at a rate that will satisfy the cellular demands for methionine.

The purification of a soluble cytochrome from pig kidney with spectral properties similar to those of microsomal cytochrome b5

Abstract A soluble cytochrome has been partially purified from pig kidney. The absorption spectra of this cytochrome in both the oxidized and reduced state resembles microsomal cytochrome b5. However, this cytochrome is present in crude homogenates in a soluble form and, therefore, the purification scheme does not involve the use of proteases, lipase or detergents.

Effect of ribavirin on HeLa cells expressing E. coli xanthine-guanine phosphoribosyl transferase

HeLa cells expressing the E. coli gene Ecogpt and cultured with xanthine were extremely resistant to antiproliferation by mycophenolic acid, but were only partially resistant to the effects of ribavirin. Nucleotide analyses of these transfected cells, and the parental HeLa cells, demonstrated an association between the GTP/ribavirin triphosphate ratio and virus yield reductions.

In vitro antiviral activity of ribavirin against picornaviruses

The in vitro antiviral activity of ribavirin (Rby; 1-β-D-ribof uranosyl-1,2,4-triazole-3-carboxamide) against selected members of the Picornaviridae is described. When antiviral activity was determined by reduction of infectious virus progeny, Rbv was active against human rhinovirus type 2 (HRV-2) and poliovirus type 1 (Polio-1) in both a drug concentration and multiplicity of infection-(MOI) dependent response. However, an antiviral activity rating assay based on the reductions of cellular cytopathic effects (CPE) indicated that Rbv was active against human rhinoviruses, but was less active against polioviruses. Prophylactic administration of Rbv significantly improved virus yield reduction, especially in Polio-1. SDS Polyacrylamide gel electrophoresis (SDS-PAGE) of HRV-2- and Polio-1-infected cell lysates demonstrated that Rbv inhibited the synthesis of viral-specific proteins. Although actinomycin D (Act D) did not significantly influence Picornavirus yields, when added concomitantly with Rbv, Act D reversed Rbvs antiviral effect.

Isolation of N 5 ,N 10 -methylene tetrahydrofolate reductase from bovine brain.

Abstract N5,N10 -methylene tetrahydrofolate reductase has been purified 100-fold from bovine brain. The initial fractionation with protamine sulfate and ammonium sulfate was followed by chromatography on DEAE-polyacrylamide gel (Bio Gel DM-30) and Sephadex G-200 as well as the selective adsorption and elution of the enzyme on calcium phosphate gel. The purified enzyme required FADH2 and catalyzed the reduction of the methylene group of N5,N10 -methylene tetrahydrofolate to the methyl group of N5 -methyl tetrahydrofolate. The pH optimum of the bovine brain reductase occurred at a pK of 6.5. The enzyme proved quite unstable. Both air oxidation and prolonged periods of storage at -20° inactivated the enzyme.