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.

Measuring antioxidant capacity using the ORAC and TOSC assays.

Recent epidemiological studies have shown that there may be a link between oxidative stress and the development of several types of chronic diseases. Studies have also shown that diets rich in fruits and vegetables may decrease the incidence of cancer and other chronic diseases. The antioxidant activity of the phytochemicals these foods contain may be partially responsible for the decreased incidence of these diseases in people who regularly consume them. While there are several assays currently used to assess the antioxidant activity of phytochemicals and other antioxidant compounds, two are reviewed here in detail. The first is the oxygen radical absorbance capacity (ORAC) assay, which measures the decrease in fluorescence decay caused by antioxidants, and the second is the total oxyradical scavenging capacity (TOSC) assay, which measures the decrease in ethylene gas production caused by the inhibition of the thermal hydrolysis of ABAP (2,2'-Azobis(2-methyl-(propionamidine) dihydrochloride) by KMBA (alpha-keto-gamma-(methylthio)butyric acid sodium salt) in the presence of antioxidant compounds. These two assays are discussed here, with an in depth review of their methodology and correlation.

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.

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.

Comparison of the total oxyradical scavenging capacity and oxygen radical absorbance capacity antioxidant assays.

Epidemiological studies have shown that phytochemicals in fruits and vegetables may decrease the incidence of cancer. The antioxidant activity of phytochemicals may be partially responsible for the reduced cancer risk. In this study, the antioxidant activity of several phytochemicals was compared using two different antioxidant assays: the oxygen radical absorbance capacity (ORAC) assay, which measures the decrease in fluorescence decay caused by antioxidants, and the total oxyradical scavenging capacity (TOSC) assay, which measures the decrease in ethylene production caused by antioxidants. TOSC and ORAC values were measured for 11 different phytochemicals, and values were expressed as micromol of Trolox equivalents/mg. As expected, a correlation was seen between the TOSC values and the ORAC values (R2 = 0.60). Quercitin, maritime pine bark extract (Pycnogenol, Horphag Research Ltd., Geneva, Switzerland), grape skin extract, and green tea polyphenols had the highest overall antioxidant activity of the 11 phytochemicals measured. Lemon fruit and citrus bioflavonoids had the lowest overall antioxidant activity. Rutin and alpha-lipoic acid had low ORAC values but high TOSC values when compared to the other phytochemicals. The correlation between the in vitro TOSC and ORAC antioxidant assays suggests that both assays may be useful in identifying phytochemicals with high antioxidant activity.

Cell death in bovine parvovirus-infected embryonic bovine tracheal cells is mediated by necrosis rather than apoptosis.

The helper-independent bovine parvovirus (BPV) was studied to determine its effect on host embryonic bovine tracheal (EBTr) cells: whether the ultimate outcome of infection results in apoptotic cell death or cell death by necrosis. Infected cells were observed for changes marking apoptosis. Observations of alterations in nuclear morphology, membrane changes, apoptotic body formation, membrane phosphatidylserine inversions, caspase activation and cell DNA laddering in infected cells were not indicative of apoptosis. On the other hand, at the end of the virus replication cycle, infected cells released viral haemagglutinin and infectious virus particles, as would be expected from cell membrane failure. Moreover, the infected cells released lactate dehydrogenase (LDH), release of which is a marker of necrosis. LDH release into the cell medium correlated directly with viral m.o.i. and time post-infection. Furthermore, assessment of mitochondrial dehydrogenase activity was consistent with cell death by necrosis. Taken together, these findings indicate that cell death in BPV-infected EBTr cells is due to necrosis, as defined by infected-cell membrane failure and release of the cell contents into the extracellular environment.

Tail profile: a more accurate system for analyzing DNA damage using the Comet assay.

The Comet assay (single cell gel electrophoresis assay) measures DNA strand breaks in individual cells. In the assay cells are embedded in agarose, lysed, and electrophoresed under low voltage, allowing migration of damaged DNA. The DNA is stained and subsequently viewed with an epifluorescent microscope. If DNA damage has occurred the electrophoresed DNA fragments appear as a diffuse tail behind the nucleus known as a "comet". Many computer-aided analysis systems are currently in use to quantify the amount of DNA damage that is represented by a comet image. Here, we present a novel method of analysis known as "tail profile". This method of analysis provides several advantages over currently employed methods, which rely primarily on the "tail moment" method of analysis. We compared the amount of DNA damage reported from both the tail profile and tail moment methods of analysis and observed a 26% (P<0.0001) increase in damage detected by tail profile across the 10-25 microm range of tail length, where the majority of the relevant comet data is concentrated. We further report that this increase in sensitivity is not only limited to assessing DNA damage, but also to gathering data from DNA repair assays. Furthermore, we demonstrate increased functionality and extended data analysis capabilities with the use of a compressed collection of images called a "comet chip" and through a visual representation of data called a "profile plot". Use of the custom macros enabled us to detect an unexpected characteristic of the electrophoretic profile, giving us novel insight into the nature of comet analysis. In addition to the increased analytical sensitivity proffered by this system, the tail profile macros are upgradeable and platform independent.

Decreased DNA repair rates and protection from heat induced apoptosis mediated by electromagnetic field exposure.

In this study, we demonstrate that electromagnetic field (EMF) exposure results in protection from heat induced apoptosis in human cancer cell lines in a time dependent manner. Apoptosis protection was determined by growing HL-60, HL-60R, and Raji cell lines in a 0.15 mT 60 Hz sinusoidal EMF for time periods between 4 and 24 h. After induction of apoptosis, cells were analyzed by the neutral comet assay to determine the percentage of apoptotic cells. To discover the duration of this protection, cells were grown in the EMF for 24 h and then removed for 24 to 48 h before heat shock and neutral comet assays were performed. Our results demonstrate that EMF exposure offers significant protection from apoptosis (P<.0001 for HL-60 and HL-60R, P<.005 for Raji) after 12 h of exposure and that protection can last up to 48 h after removal from the EMF. In this study we further demonstrate the effect of the EMF on DNA repair rates. DNA repair data were gathered by exposing the same cell lines to the EMF for 24 h before damaging the exposed cells and non-exposed cells with H2O2. Cells were allowed to repair for time periods between 0 and 15 min before analysis using the alkaline comet assay. Results showed that EMF exposure significantly decreased DNA repair rates in HL-60 and HL-60R cell lines (P<.001 and P<.01 respectively), but not in the Raji cell line. Importantly, our apoptosis results show that a minimal time exposure to an EMF is needed before observed effects. This may explain previous studies showing no change in apoptosis susceptibility and repair rates when treatments and EMF exposure were administered concurrently. More research is necessary, however, before data from this in vitro study can be applied to in vivo systems.