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PubMed2002

Occurrence of Cu-ATPase in Dictyostelium: possible role in resistance to copper.

Dictyostelium discoideum amoebae showed an uncommon resistance to Cu(2+), as pointed out through cell growth rate (EC(50) = 469 +/- 30 microM) and the neutral red cytotoxicity assay (EC(50) = 334 +/- 45 microM). Although no evidence of Cu-inducible metallothionein was found, Cu-dependent ATPase activity was cytochemically detected on pelletted, resin-embedded amoebae. This activity required Cu(2+) in the incubation medium, was sensitive to TPEN, vanadate and temperature, and showed dose-dependent increase after exposure of amoebae to 10-500 microM Cu(2+) for 7 days. Accordingly, immunofluorescence and Western blotting revealed the occurrence of a Cu-inducible, putative homologue of human Menkes (MNK) Cu-P-type ATPase. To verify if Cu-ATPase is involved in copper resistance, amoebae were exposed to low concentrations of Cu(2+) and vanadate followed by the neutral red assay. Exposure to either treatment showed no effect, while a combination caused a dramatic increase of Cu toxicity, possibly depending on Cu-ATPase inhibition.

Adenosine Triphosphatases↗

PubMed1984

The DNA repair host-mediated assay as a rapid and sensitive in vivo procedure for the determination of genotoxic factors present in various organs of mice. Some preliminary results with mitomycin C.

The DNA repair host-mediated assay, in which repairable DNA damage is determined in E. coli cells present in various organs of mice exposed to genotoxic agents, was further developed to broaden the range of organs under study and to simplify the procedure of assessing differential bacterial cell survival. A pair of derivatives of E. coli K-12 strain 343/113 was constructed which differed vastly in DNA repair capacity (uvr+/rec+ vs uvrB/recA), as a means of assessing DNA damaging effects; furthermore, the strains differed in their ability to ferment lactose (delta Lac vs Lac+), so that the individual survival of both strains could be determined on a single agar medium (containing neutral red as pH indicator), on which the strains had different colony colour morphology (red, Lac+ vs white, Lac- colonies). Finally, the strains were made streptomycin-dependent, to prevent uncontrolled growth of the bacterial cells within the various organs and also to inhibit contamination of the survival agar medium by representatives of the normal intestinal microflora. The experimental procedure consisted of injecting mixtures of stationary cells of the two strains (ca. 3-5 X 10(8) viable cells per mouse) both intravenously and orally into mice, either pretreated or subsequently treated with test chemicals. Ninety minutes after injection of the bacteria, the liver, spleen, lungs, kidneys, stomach, intestine, colon, and ca. 50 microliter blood, were removed, suspended in buffer, homogenized, and the survival of the two strains determined on neutral red agar supplemented with streptomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Influence of blood and synovial fluid immune complexes of patients with rheumatoid arthritis on production of nitric oxide and growth and viability of chondrocytes.

OBJECTIVE: To determine whether blood and synovial fluid (SF) immune complexes (IC) of patients with rheumatoid arthritis (RA) influence the production of nitric oxide (NO) and growth and viability of chondrocytes. METHODS: IC were precipitated and IgM and IgG were determined in the precipitates by ELISA and nephelometry, respectively. Primary cultures of bovine articular chondrocytes were incubated with the IC precipitates. After 48 h NO was determined as nitrite. After 7 days, growth was determined by incorporation of tritiated thymidine and viability was detected by neutral red uptake. RESULTS: Patient sera were positive in 8/12 for IgM IC and 9/12 for IgG IC, and 1/8 control sera was slightly positive for IgM IC. Seven of 12 SF samples were IgM IC and 10/12 IgG IC positive. With and without additional interleukin 1alpha (IL-1alpha) stimulation, NO production by chondrocytes was significantly higher with SF IC precipitates than with control serum precipitates (p = 0.03, p = 0.04, respectively). NO production by chondrocytes that were not stimulated with IL-1alpha was significantly increased with SF IC precipitates compared to RA serum IC precipitates (p = 0.03). SF IC significantly inhibited growth compared to control serum precipitates (p = 0.04) and RA serum IC (p = 0.012). Neutral red uptake by chondrocytes was significantly decreased when incubated with RA serum IC in comparison with control serum IC (p = 0.012) and SF IC (p = 0.006). With and without additional IL-1alpha stimulation, NO production by chondrocytes after incubation with SF derived IC was positively correlated to the Ritchie score (r = 0.8, r = 0.7, respectively) and the number of swollen joints (r = 0.8, r = 0.6, respectively). CONCLUSION: These results support the hypothesis that, especially in active RA, SF derived IC stimulate NO production and inhibit chondrocyte growth.

Antigen-Antibody Complex↗

PubMed1997

Apoptotic death in cerebral hemisphere cells is density dependent and modulated by transient oxygen and glucose deprivation.

Flow cytometry, light and fluorescence microscopy, and designated biochemical techniques were used to examine the type of death which occurs in cerebral cortex cells when grown under crowded vs. sparse conditions or after brief anoxia/hypoglycemia. A 4 hr episode of anoxia combined with glucose deprivation enhanced apoptotic cell death as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and reduced neutral red eye uptake. An additional form of cell death involving exclusion of the nucleus was recorded by time lapse cinematography and DAPI stain. The presence of the endonuclease inhibitor aurintricarboxylic acid (0.1 mM) reduced cell death by 56.6%, while the protein and RNA synthesis inhibitors actinomycin D and cycloheximide (each at 5 micrograms/ml) effectively decreased cell death by 83.3% and 90.6%, respectively. In contrast, 5 mM glutamate had no effect on cell death in accord with the immature state of the cells. Growth of cells under crowded conditions improved cell survival; after 2 h or 4 days in culture, cells seeded at high density (34 microgram cellular DNA/cm2) showed a nearly 3-fold decline in the amount of cell death in comparison to cells seeded at low density (5 micrograms cellular DNA/cm2). At high cell density, anoxic episodes enhanced cell death most likely by preventing a cell density-mediated rescue. Neutral red dye uptake, an index for cell viability, was enhanced with increasing cell density and in vitro maturation, but was reduced in dense cultures exposed to anoxic/hypoglycemic conditions. The data suggest that cell density may play a critical role in brain organogenesis and that anoxic stress is more deleterious in dense than sparse cell assemblies.

Animals↗

PubMed1986

Interdigital tissue chondrogenesis induced by surgical removal of the ectoderm in the embryonic chick leg bud.

In the present work, we have analysed the possible involvement of ectodermal tissue in the control of interdigital mesenchymal cell death. Two types of experiments were performed in the stages previous to the onset of interdigital cell death: removal of the AER of the interdigit; removal of the dorsal ectoderm of the interdigit. After the operation embryos were sacrificed at 10-12 h intervals and the leg buds were studied by whole-mount cartilage staining, vital staining with neutral red and scanning electron microscopy. Between stages 27 and 30, ridge removal caused a local inhibition of the growth of the interdigit. In a high percentage of the cases, ridge removal at these stages was followed 30-40 h later by the formation of ectopic nodules of cartilage in the interdigit. The incidence of ectopic cartilage formation was maximum at stage 29 (60%). In all cases, cell death took place on schedule although the intensity and extent of necrosis appeared diminished in relation to the intensity of inhibition of interdigital growth and to the presence of interdigital cartilages. Ridge removal at stage 31 did not cause inhibition of the growth of the interdigit and ectopic chondrogenesis was only detected in 3 out of 35 operated embryos. Dorsal ectoderm removal from the proximal zone of the interdigit at stage 29 caused the chondrogenesis of the proximal interdigital mesenchyme in 6 out of 18 operated embryos. The pattern of neutral red vital staining was consistent with these results revealing a partial inhibition of interdigital cell death in the proximal zone of the interdigit. It is proposed that under the present experimental conditions the mesenchymal cells are diverted from the death programme by a primary transformation into cartilage.

Animals↗

PubMed2002

In vitro cytotoxicity of protocatechuic acid to cultured human cells from oral tissue: involvement in oxidative stress.

Data on the biologic activity of protocatechuic acid are contradictory; some studies have shown that it acts as an antioxidant and suppresses chemical-induced carcinogenesis and others that it induces oxidative stress and promotes tumour formation. The anticarcinogenicity of protocatechuic acid was postulated to be related, in part, to its specific suppression of neoplastic hyperproliferation. To determine whether protocatechuic acid was preferentially antiproliferative to malignant cells, non-malignant and carcinoma cells were exposed for 24 hr to protocatechuic acid (2.5 to 25 mM) and viability was assessed with the neutral red assay. The cell lines were derived from tissues of the human oral cavity, the initial site of exposure upon ingestion of dietary protocatechuic acid, and included normal GN61 gingival fibroblasts, immortalized, non-tumorigenic S-G gingival epithelial cells, and malignant HSG1 cells derived from the salivary gland, HSC-2 cells from the floor of the oral cavity, and CAL27 cells from the tongue. Selective toxicity of protocatechuic acid to malignant cells was not observed. Furthermore, using a total cellular protein determination to quantitate cell growth, no differences in comparative sensitivities of S-G epithelial cells and HSG1 carcinoma cells were noted in a 3 day continuous exposure to 2.5 to 12.5 mM protocatechuic acid and in recovery from a 24 hr exposure to 3 to 15 mM protocatechuic acid. The S-G and HSG1 cells were then used to study the effects of elevated concentrations of protocatechuic acid on oxidative stress. For both cell types, protocatechuic acid induced oxidative stress, presumably through its bioactivation by a tyrosinase pathway. A brief exposure to 25 mM protocatechuic acid lowered the levels of intracellular glutathione and potentiated Fe2+-induced lipid peroxidation of the cells. As determined with the neutral red assay, S-G and HSG1 cells exposed briefly to a non-toxic level (0.5 mM) of the glutathione depleter, 1,3-bis(2-chloroethyl)-N-nitrosourea, were hypersensitive to a subsequent challenge with 10 mM protocatechuic acid and preexposure of the S-G and HSG1 cells to a nontoxic level of protocatechuic acid (2.5 mM) enhanced their sensitivity to a subsequent exposure to tert-butyl hydroperoxide. These findings were consistent with protocatechuic acid, at high levels (> or = 10 mM), acting as an inducer of oxidative stress.

Anticarcinogenic Agents↗

PubMed1973