Byron K. Murray

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.

Thymidine kinase: diagnostic and prognostic potential

Thymidine kinase is a cell cycle-dependent marker that can be detected in the serum of patients diagnosed with many different types of cancer. Serum levels of thymidine kinase have also been shown to reflect the progression of cancer as well as an indication of the efficacy of chemotherapeutic intervention. A new monoclonal antibody assay for thymidine kinase has been developed, which is capable of detecting thymidine kinase in both serum and tumor tissue. Thymidine kinase assay kits should be available at low cost and could serve as an effective low cost test for the detection and progression of many types of human cancer.

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.

High resolution separation and quantitation of ribonucleotides using capillary electrophoresis

A method for the analysis of ribonucleotides using capillary electrophoresis has been developed. A cross-linked polyacrylamide coated column, Tris-HCl, and phosphate-mixed buffer were used, which produced reproducible separations (solute migration time average RSD% of 1.2) of 14 ribonucleotides within 50 min. Linear relationships between peak areas and sample concentrations, an average minimum detectable concentration of 5.4 microM, and an average minimum detectable quantity of 0.08 pmol were obtained. The described method allowed reproducible and reliable qualitative and quantitative analysis of intracellular ribonucleotide pools in HeLa cells.

Cell cycle arrest and differential gene expression in HT-29 cells exposed to an aqueous garlic extract

Epidemiological data show an inverse correlation between garlic consumption and the risk for colon cancer. To examine this relationship, HT-29 human adenocarcinoma cells were cultured in the presence and absence of an aqueous garlic extract. Garlic treatment resulted in a fraction of cells detaching from the culture flasks. These cells remained viable. Flow cell cytometry showed that untreated cells exhibited a normal distribution among phases of the cell cycle, with 12% of cells at the G2/M boundary. Of the garlic-treated cells remaining attached to the flask, 27% were present at the G2/M boundary. Treated cells that detached from the flask were found almost exclusively (89%) at the G2/M boundary. RNA fingerprinting and microarray analysis showed that expression of the gene for menin was twice as high in control cells as in detached treated cells. In contrast, expression of genes for epidermal growth factor receptor and integrin-α6 was nearly twice as high in detached treated cells as in control cells. These changes in gene expression were consistent with an arrest of the cell cycle at the G2/M boundary. Garlic=s arrest of the cell cycle in human adenocarcinoma cells may explain in part its anticarcinogenic properties.

Virion disruption by ozone-mediated reactive oxygen species

It is well documented in the scientific literature that ozone-oxygen mixtures inactivate microorganisms including bacteria, fungi and viruses (Hoff, J.C., 1986. Inactivation of microbial agents by chemical disinfectants. EPA 600 S2-86 067. Office of Water, U.S. Environmental Protection Agency, Washington, DC; Khadre, M.A., Yousef, A.E., Kim, J.-G., 2001. Microbiological aspects of ozone applications in food: a review. J. Food Sci. 66, 1242-1252). In the current study, delivery and absorption of precisely known concentrations of ozone (in liquid media) were used to inactivate virus infectivity. An ozone-oxygen delivery system capable of monitoring and recording ozone concentrations in real time was used to inactivate a series of enveloped and non-enveloped viruses including herpes simplex virus type-1 (HHV-1, strain McIntyre), vesicular stomatitis Indiana virus (VSIV), vaccinia virus (VACV, strain Elstree), adenovirus type-2 (HAdV-2), and the PR8 strain of influenza A virus (FLUAVA/PR/8/34/H1N1; FLUAV). The results of the study showed that ozone exposure reduced viral infectivity by lipid peroxidation and subsequent lipid envelope and protein shell damage. These data suggest that a wide range of virus types can be inactivated in an environment of known ozone exposure.

Over-pressure suppresses ultrasonic-induced drug uptake.

Ultrasound (US) is used to enhance and target delivery of drugs and genes to cancer tissues. The present study further examines the role of acoustic cavitation in US-induced permeabilization of cell membranes and subsequent drug or gene uptake by the cell. Rat colon cancer cells were exposed to ultrasound at various static pressures to examine the hypothesis that oscillating bubbles, also known as cavitating bubbles, permeabilize cells. Increasing pressure suppresses bubble cavitation activity; thus, if applied pressure were to reduce drug uptake, cell permeabilization would be strongly linked to bubble cavitation activity. Cells were exposed to 476 kHz pulsed ultrasound at average intensities of 2.75 W/cm(2) and 5.5 W/cm(2) at various pressures and times in an isothermal chamber. Cell fractions with reversible membrane damage (calcein uptake) and irreversible damage (propidium iodide uptake) were analyzed by flow cytometry. Pressurization to 3 atm nearly eliminated the biological effect of US in promoting calcein uptake. Data also showed a linear increase in membrane permeability with respect to insonation time and intensity. This research shows that US-mediated cell membrane permeability is likely linked to cavitation bubble activity.

Conversion of Herpetic Lesions to Malignancy by Ultraviolet Exposure and Promoter Application

Many lines of evidence exist associating herpes simplex virus (HSV) with the development of carcinoma, but much of this evidence is anecdotal or associative in nature and does not prove a cause and effect. The purpose of this research was to investigate the oncogenic potential of HSV type 2 (HSV-2) in vivo. A mouse model for lip carcinogenesis was designed to combine HSV-2 infection, u.v. exposure and tetradecanoyl-phorbol-acetate (TPA) application. HSV-2 inoculation on to abraded mouse lips was capable of causing vesicular ulcerative lesions which healed completely after 10 to 14 days. Ultraviolet irradiation of the HSV lesion site daily for 6 min at 42 ergs/mm2/s on days 3 to 6 post-infection caused hyperkeratosis, acanthosis and dysplasia to develop in several lips; while the same u.v. exposure by itself failed to alter the histologic appearance. The addition to repeated TPA application to the HSV+ u.v. regimen promoted tumour emergence. Thirty-two of 156 Balb/c mice developed tumours. Although the majority were papillomas, six were squamous cell carcinomas. These tumour-bearing mice had increased HSV-specific antibody titres. HSV antigens were shown to be present in outgrowths from explanted tumours as well as in tumour biopsies by immunoperoxidase staining with HSV-specific antisera. It is suggested that the in vivo u.v.-irradiated HSV acted as the inducer and TPA as the promoter, analogous to the classical two-stage carcinogenesis model.

Bovine parvovirus uses clathrin-mediated endocytosis for cell entry.

Entry events of bovine parvovirus (BPV) were studied. Transmission electron micrographs of infected cells showed virus particles in cytoplasmic vesicles. Chemical inhibitors that block certain aspects of the cellular machinery were employed to assess viral dependency upon those cellular processes. Chlorpromazine, ammonium chloride, chloroquine and bafilamicin A1 were used to inhibit acidification of endosomes and clathrin-associated endocytosis. Nystatin was used as an inhibitor of the caveolae pathway. Cytochalasin D and ML-7 were used to inhibit actin and myosin functions, respectively. Nocodazole and colchicine were employed to inhibit microtubule activity. Virus entry was assessed by measuring viral transcription using real-time PCR, synthesis of capsid protein and assembly of infectious progeny virus in the presence of inhibitor blockage. The results indicated that BPV entry into embryonic bovine trachael cells utilizes endocytosis in clathrin-coated vesicles, is dependent upon acidification, and appears to be associated with actin and microtubule dependency. Evidence for viral entry through caveolae was not obtained. These findings provide a fuller understanding of the early cell-entry events of the replication cycle for members of the genus Bocavirus.

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.