Development of a competitive double antibody lateral flow assay for the detection of antibodies specific to glycoprotein B of Aujeszky's disease virus in swine sera.

Three lateral flow assays (LFAs) for the detection of antibodies against glycoprotein B (gB) of Aujeszky's disease virus (ADV) in swine sera: a competitive double antibody sandwich LFA without a preincubation step (CDAS-gB-LFA), a CDAS-gB-LFA with a preincubation step (pCDAS-gB-LFA), and a competitive direct gB-LFA have been developed and were compared with each other and with a gB-ELISA. The assays are based on monoclonal antibodies to immunodominant epitopes of ADV gB. The pCDAS-gB-LFA proved to be the most specific and sensitive assay to detect antibodies directed to ADV gB. The specificity and sensitivity of the pCDAS-gB-LFA with the use of an LFA reader for test line intensity measurements were 97.6 and 94.9%, respectively. The lower diagnostic sensitivity of the pCDAS-gB-LFA compared to a gB-ELISA reflects its reduced analytical sensitivity, which was shown in titration experiments with positive sera. The pCDAS-gB-LFA, using the reader-based and visual detection modes, showed good agreement in respect to specificity; however, the LFA reader detection provided a higher diagnostic and analytical sensitivity compared to visual detection. The developed pCDAS-gB-LFA is a rapid, sensitive, and specific method for the detection of antibodies to ADV gB and can be used for screening ADV-infected swine in unvaccinated herds.

Phospholipids of liver cell nuclei during hibernation of Yakutian ground squirrel.

In hibernating Yakutian ground squirrels S. undulatus, the content of total phospholipids in the nuclei of liver increased by 40% compared to that in animals in summer. In torpid state, the amount of sphingomyelin increased almost 8 times; phosphatidylserine, 7 times; and cardiolipin, 4 times. In active "winter" ground squirrels, the amount of sphingomyelin, phosphatidylserine, and cardiolipin decreased compared to the hibernating individuals but remained high compared to the "summer" ones. The torpor state did not affect the amount of lysophosphatidylcholine and phosphatidylinositol.

Ornithine decarboxylase activity in rat organs and tissues under artificial hypobiosis.

The influence of hypothermia-hypoxia-hypercapnia on ornithine decarboxylase (ODC, EC 4.1.1.17) activities in rat organs and tissues and also on the thymocyte distribution throughout the cell cycle stages was studied. The state of artificial hypobiosis in rats on decrease in the body temperature to 14.4-18.0°C during 3.0-3.5 h was accompanied by drops in the ODC activities in the neocortex and liver by 50-60% and in rapidly proliferating tissues (thymus, spleen, and small intestine mucosa) by 80% of the control value. In kidneys the ODC activity raised to 200% of the control level. Twenty-four hours after termination of the cooling and replacing the rats under the standard conditions, the ODC activities in the neocortex, liver, kidneys, spleen, and intestinal mucosa returned to the control values, but remained decreased in the thymus. Forty-eight hours later the ODC activities in the thymus and spleen exceeded the normal level. The distribution of thymocytes throughout the cell cycle stages did not change in rats in the state of hypothermia (hypobiosis); 24 and 48 h after termination of the cooling the fraction of thymocytes in the S stage was decreased and the fraction of the cells in the G(0)+G(1) stage was increased. The normal distribution of thymocytes throughout the cell cycle stages recovered in 72 h. Thus, in the thymus the diminution of the ODC activity preceded the suppression of the cell proliferation rate. The tissue-specific changes in the ODC activity are suggested to reflect adaptive changes in the functional and proliferative activities of organs and tissues during the development of hypobiosis under conditions of hypothermia-hypoxia-hypercapnia.

Cytotoxic activity of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide is underlain by DNA interchain cross-linking.

Currently, chemical bifunctional cross-linkers are regarded as promising therapeutic agents capable of affecting cell metabolism. Depending on the nature of the active groups and on the length of their mediating spacer, these cross-linkers have been shown to influence mitochondrial functions, the cell cycle and cell death. The current study was aimed to assay cellular effects of a cross-linker with 'zero'-length spacer, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). When added to cultures of transformed cells, EDC induced a G2/M blockade followed by cell death. Analysis of the molecular targets revealed that alteration of the cell cycle was caused by EDC-induced interchain cross-linking within double-stranded DNA. Administration of EDC to animals with experimental tumors increased their life span. The analysis of tumor cells from EDC-treated mice showed up-regulation of p21/WAF1, disturbance of tumor cell cytokinesis and, hence, cell death. Thus, both in vitro and in vivo, EDC exhibits cytotoxic activity, which may be of potential therapeutic use.

Cell death induced by chemical homobifunctional cross-linkers. Cross-linker induced apoptosis.

Physical association of proteins that underlies cytotoxic signal induction and transduction suggests a possibility of regulating cell response by modifying protein-protein interactions. For protein complexing, chemical cross-linking agents have been traditionally used. However, the ability of various cross-linkers to induce and modify cell responses, cell death in particular, is still obscure. We have undertaken the investigation to test the apoptosis-inducing and modifying properties of the homobifunctional cross-linkers-dimethyl suberimidate (DMS) and 1,5-bis(succinimido-oxycarbonyloxy)pentane (BSOCOP). The functional groups of these cross-linkers are different but both are able to interact with available amino groups. It was shown that bifunctional cross-linkers, unlike their monofunctional analogues, are capable of inducing cell death in transformed cells, thus indicating the crucial role of cross-linking in cell killing. DMS- and BSOCOP-treated cells were shown to undergo cell death by apoptosis, though the signaling pathways were distinct. DMS inhibited bcl-X(L) and bak but not bax gene expression, while BSOCOP potentiated bax mRNA synthesis immediately after application. Cell pre-incubation with DMS, but not with BSOCOP, resulted in an increasing sensitivity to TNF, although activities of anti-Fas cytotoxic antibodies were then inhibited. Thus, this study has demonstrated for the first time that chemical cross-linkers are capable of inducing apoptosis by themselves and modifying the TNF-dependent and Fas-mediated cell death that may have potential therapeutic significance.

The use of flow cytometry for the investigation of cell death.

Flow cytometry is more and more widely used for investigations of cell death, predominantly in the study of DNA degradation in cells dying by apoptosis. There are different interpretations of changes observed in DNA histograms of these cells. We describe an approach based on extraction of chromatin degradation products from fixed cells and subsequent staining with DNA specific dyes. Apoptotic cells containing fragmented DNA are observed in < 2C DNA region of DNA histograms. DNA histograms of irradiated thymocytes dying in vitro and stained without extraction of fragmented DNA do not differ from control. Under the same staining conditions DNA histograms of lymphocytes dying in thymus of irradiated animals reveal fluorescent material in < 2C DNA region, most likely due to formation of apoptotic bodies (cell fragments, some of them contain fragments of nuclei). Similar changes are observed in thymocytes dying upon glucocorticoid treatment. Our present results and other data indicate that reduced amount of DNA in dying cells is the main reason for changes of DNA histograms. Examples of application of the method described for the investigations of cell death modifiers are presented.

Apoptosis of murine BW 5147 thymoma cells induced by dexamethasone and gamma-irradiation.

The mode and the kinetics of the death of T-thymoma cells upon dexamethasone treatment and gamma-irradiation (10Gy) have been studied using flow cytometry and biochemical analysis. It has been shown that the hormone and gamma-irradiation induce cell death by apoptosis. In both cases the cells are initially blocked in G2/M and die only after overcoming the blockage and cytokinesis. A short exposure to dexamethasone results in a cytostatic effect, whereas a cytotoxic effect is absent. Reducing serum concentration to 2% causes more rapid death both following gamma-irradiation and dexamethasone. These results are discussed in relation to cell death and proliferation.

Flow cytometry analysis of DNA degradation in thymocytes of gamma-irradiated or hydrocortisone treated rats.

The pattern of DNA degradation in thymocytes of irradiated or hydrocortisone-treated rats has been studied by means of flow cytometry of the cells, treated with probes specifically bound to the AT or GC-pairs of DNA. It has been shown that the death of thymocytes is accompanied by a decrease in their DNA content. The main features of the occurrence and accumulation of cells with a DNA content less than the normal diploid level correspond with those of internucleosomal DNA fragmentation: such cells appear after a 1 hour lag-period, their accumulation is prevented by cycloheximide injection and is lower at 300 Gy than at doses of 10 to 30 Gy. At the same time, no increase in permeability of the cell membrane to ethidium bromide was observed up to the sixth hour after irradiation. Most of the thymocytes dying under the action of irradiation or hydrocortisone are in the G0 or G1 phases of the cell cycle. The method used allows detection of the cells with cleaved but not removed DNA.