Functional alterations induced by fusarium T-2 toxin in Madin-Darby bovine Kidney (MDBK) and primary fetal bovine Kidney (PFBK) cultures

We recently reported that primary fetal bovine Kidney (PFBK) cells were consistently more sensitive to the cytotoxic effects of fusarium T-2 toxin than Madin-Darby bovine kidney (MDBK) cells in culture. The present report examined the influence of T-2 on selected biochemical parameters of these two culture types. T-2 toxin inhibited incorporation of labeled thymidine, uridine, and leucine in both culture types; at lower concentrations of the toxin, PFBK cells were affected to a greater extent than MDBK cells. T-2 toxin inhibited both the transport of thymidine as well as thymidine incorporation into macromolecules in MDBK cells during initial periods, but did not affect uridine incorporation. The cellular enzymes, K⁺- dependent phosphatase and succinic dehydrogenase were inhibited in MDBK but not in PFBK cultures; acid phosphatase was not influenced in either culture types. In a cell-free system none of the above enzymes were affected by T-2 until the toxin concentration exceeded 10^?5M.     

Development of Hammondia heydorni in cultured bovine and ovine cells

Sporozoites were excysted from oocysts of Hammondia heydorni obtained from a naturally-infected dog and inoculated into monolayer cultures of bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney (MDBK) cells, bovine monocytes (M617), or ovine monocytes (WOMO). Sporozoites penetrated all four cell lines and underwent asexual reproduction by endodyogeny (as determined by electron microscopy) to form cyst-like structures at four to nine days after sporozoite inoculation (DAI). At 4-10 DAI, considerably more zoites were harvested from M617 cultures (80.1 x 10(6) zoites) than from CPA (17.4 x 10(6], MDBK (47.3 x 10(6], and WOMO (53.5 x 10(6]. Little or no parasite multiplication occurred at 10-16 DAI. Zoites harvested at 7 DAI and transferred to freshly prepared cultures did not penetrate cells nor develop further.     

Effects of Fusobacterium necrophorum subspecies necrophorum on extracellular matrix of tissue-cultured bovine kidney cells

The effects of Fusobacterium necrophorum subsp. necrophorum on the extracellular matrix were investigated. The toxic preparation from the culture induced reduction in the number of tissue-cultured bovine kidney cells. The exposed cells often manifested partial loss of cytoplasm and were morphologically irregular. Scanning electron microscopy demonstrated partial loss of the microvilli on the exposed cells and roughness of the cell surfaces. Finally, sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles revealed complete degradation of bovine collagen type 1 after treatment with the toxic preparation. This degradation was inhibited by the addition of homologous antiserum. These findings indicate that the degradation may contribute to the establishment of the infection caused by F.n. subsp. necrophorum.     

Immunotoxicity of repeated low level exposure to T-2 toxin,a trichothecene mycotoxin,in CD-1 mice

Male CD-1 mice were gavaged with T-2 toxin (0.0–5.0 mg/kg body weight) every third day. Body weight gain was depressed by exposure to 2.5 mg/kg, or greater, T-2 toxin; this was not associated with decreased food intake. The weights of the liver, kidney, spleen, and thymus were affected by two weeks exposure to T-2 toxin. However, a persistent effect after four weeks was observed only for the thymus. Peripheral leucocyte counts were elevated in the highest dose groups after two and four weeks. Thymidine uptake by cells not simultaneously exposed to mitogen was increased in splenic cell cultures of mice exposed to 2.5 mg/kg T-2 toxin for two or four weeks. Phytohemagglutinin stimulation of splenic lymphocytes following two weeks of exposure was depressed in the 2.5 mg/kg dose group; this phenomenon was not observed after four weeks exposure. Response to pokeweed mitogen increased after four weeks of exposure to 2.5 mg/kg T-2 toxin. A delayed-type hypersensitivity response decreased following two weeks exposure to levels greater than 0.02 mg/kg. Production of I g M class antibodies by splenic lymphocytes, evaluated by a hemolytic plaque response to sheep erythrocytes, was depressed in the 2.5 mg/kg dose group after two weeks exposure to T-2 toxin. The sensitivity and specificity of T-2 toxin immunotoxicity was indicated by the various parameters evaluated.     

Toxicity of the mycotoxins fumonisins B1 and B2 and Alternaria alternata f. sp. lycopersici toxin (AAL) in cultured mammalian cells

Fumonisins B₁ and B₂ and AAL toxin are a series of structurally related mycotoxins. Fumonisins B₁ and B₂, produced by Fusarium moniliforme Sheldon induce toxic hepatitis and hepatomas in rats and leukoencephalomalacia in horses. The cancer-promotion assay which has been used to guide their purification is slow and consumes large amounts of sample. We have examined a series of cultured mammalian cell lines in order to develop a more rapid and sensitive bioassay system, which may be useful for examining structure-activity relationships and the mechanism(s) of action of these toxins. Of 9 rat hepatoma cell lines tested, all except the two most de-differentiated lines were sensitive to the three toxins, with a toxic response visible by 48 h. Approximate IC₅₀ values for the most sensitive hepatoma line, H4TG, were 4, 2 and 10 g/ml for fumonisins B₁, B₂ and AAL toxin, respectively in 100 l cultures. Among 15 cell lines from other sources, only MDCK dog kidney epithelial cells were sensitive (IC₅₀ = 2.5, 2 and 5 g/ml, respectively). Studies in co-cultures of sensitive and insensitive cell lines and in cultures of a sensitive cell line over a range of cell densities indicated that cytotoxicity of fumonisins B₁ and B₂ does not involve metabolite activation to a derivative stable enough to diffuse to adjacent cells.Abbreviations AAL toxin Alternaria alternata f. sp. lycopersici toxin - IC₅₀ concentration giving 50% inhibition of cell proliferation     

Immunocytochemical localization of tryptophanyl-tRNA-synthetase in a line of bovine kidney cells and in sublines with elevated enzyme levels

Intracellular localization of tryptophanyl-tRNA synthetase (E. C. 6.1.1.2) has been studied immunocytochemically using monospecific antibodies in cultured bovine kidney cells (strain MDBK) and in substrains with elevated enzyme levels. Both light and electron microscopy were used and native or detergent-treated cells were examined. The product of cytochemical reaction was revealed on free polyribosomes, polyribosomes attached to membranes of granular endoplasmic reticulum, on cytofilaments and in the nucleus as well.     

Cytotoxicity of trichothecene mycotoxins isolated from Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum in baby hamster kidney (BHK-21) cells

Twenty-six trichothecene mycotoxins produced by Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum were screened for relative cytotoxicity in cultured baby hamster kidney (BHK-21) cells. The relative cytotoxicity was measured as LC₁₀₀. The most cytotoxic trichothecenes were T-2 toxin (5 ng/ml) and the recently isolated 4-propanoyl HT-2 (5 ng/ml) and 3-hydroxy T-2 toxin (5 ng/ml). T-2 tetraol (1 × 10⁴ ng/ml), 8--hydroxytrichothecene (1 × 10⁴ ng/ml), sporotrichiol (2 × 10⁴ ng/ml), 8-oxodiacetoxyscirpenol (6 × 10⁴ ng/ml) and 8-acetyl T-2 tetraol (1 × 10⁵ ng/ml) were the least toxic of the regular trichothecenes. None of the modified trichothecenes or the apotrichothecene were very cytotoxic: 8--hydroxysambucoin (2 × 10³ ng/ml), FS-1 (5 × 10³ ng/ml), 8--hydroxysambucoin (8 × 10⁴ ng/ml) and trichotriol (1 × 10⁵ ng/ml). The modified trichothecenes, FS-2 and FS-3, were not toxic even at 1 × 10⁵ ng/ml. The baby hamster kidney cell bioassay proved to be a very sensitive and reproducible means of screening new trichothecene mycotoxins for relative cytotoxicity.     

Anti-herpesvirus bovine type 5 activities of extracts obtained from Plocamium brasiliense

Bovine herpesvirus type 5 (BoHV-5) is an important etiologic agent of meningoencephalitis in cattle and has been frequently identified in outbreaks of neurological disease in bovine in the southern hemisphere including Brazil. This study aimed to evaluate the cytotoxic effect and the antiviral properties of extracts obtained from Plocamium brasiliense (Greville) Howe and Taylor in BoHV-5 RJ42/01 replication. The cytotoxic effects were measured in Madin-Darbin bovine kidney cells (MDBK) and cytotoxic concentration (CC₅₀) values have been determined for acyclovir (ACV) (223 μg?±?2.0), ethyl acetate extract from P. brasiliense (2,109 μg?±?10), hexane extract from P. brasiliense (7.181 μg?±?5), dichloromethane extract from P. brasiliense (2.356 μg?±?6.5), and hydroalcoholic extract from P. brasiliense (1.408 μg?±?5.8). As a first approach to characterize the action of these extracts on BoHV-5 RJ42/01, a virucidal assay activity was performed. A virus suspension containing 1?×?10⁵ plaque-forming units (PFU) of the BoHV-5 RJ42/01 was mixed with 600 μg of extract and acyclovir and kept at room temperature (24 °C) for 3 h. Meanwhile, a control of untreated infected virus was performed in the same conditions. Then, treated virus suspension and untreated control were diluted, and percentage of inhibition of infectivity was determined by plaque assay: ethyl acetate extract (99 %), hexane extract (90 %), dichloromethane extract (99 %), and hydroalcoholic extract (27 %). Acyclovir had a slight virucidal activity on viral particle. The inhibition of attachment was performed in MDBK cells inoculated with 100 PFU of BoHV-5 RJ42/01 in the presence or absence of various concentrations of extracts (0.3, 0.9, and 1.5 μg mL^?1). Acyclovir was also assayed at 2.8 and 11.25 μg mL^?1. The inhibition of adsorption was also tested in MDBK cells treated with the same concentrations of the extracts before virus inoculation. Results: hexane extracts inhibited virus attachment in pre-treated cell 0.9 μg (55 %) and 1.5 μg (71 %) and untreated MDBK cell only with 1.5 μg (63 %). Ethyl acetate extract on cell pre-treated with 0.3 μg (67 %), 0.9 μg (81 %), and 1.5 μg (91 %). Ethyl acetate extract on pre-treated cell 0.3 μg (67 %), 0.9 μg (81 %), and 1.5 (91 %) but discrete inhibition on cell untreated. Dichloromethane extract and acyclovir slightly inhibited virus binding on MDBK cell.     

Effects of T-2 toxin and its congeners on membrane functions of cultured human fibroblasts

Cytotoxicity of T-2 toxin, HT-2 toxin, acetyl T-2, neosolaniol, and T-2 tetraol was compared between normal human fibroblasts and mutant I-cell human fibroblasts, which only produce 10 to 15% of lysosomal hydrolases present in normal fibroblasts. Both cleavage of 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and cell count by hemocytometer were used for evaluations. For all toxins, dose-related effects on both types of cultures were evident. Cytotoxicity of the above mycotoxins on both cell lines were similar, indicating that lysosomal enzymes were not involved in the toxicity of T-2 toxin and its congeners. An inhibitor of lysosomal cysteine proteases (E-64) did not alter the cytotoxicity of T-2 toxin. The decreasing order of toxicity was T-2 toxin, HT-2 toxin, neosolaniol, acetyl T-2 toxin, and T-2 tetraol in both cell lines. When normal human fibroblasts were loaded with the fluorescent dye Lucifer yellow CH (LY), a subsequent treatment of T-2 toxin did not disrupt lysosomal membranes. The uptake of LY was not affected by T-2 toxin, which indicated that T-2 toxin did not interfere with the endocytic pathway. Results indicate that T-2 toxin and its congeners do not exert their primary toxic effect through lysosomal enzymes, membranes, or via the endocytic pathway.     

Development of Hammondia heydorni in Cultured Bovine and Ovine Cells1

Sporozoites were excysted from oocysts of Hammondia heydorni obtained from a naturally-infected dog and inoculated into monolayer cultures of bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney (MDBK) cells, bovine monocytes (M617), or ovine monocytes (WOMO). Sporozoites penetrated all four cell lines and underwent asexual reproduction by endodyogeny (as determined by electron microscopy) to form cyst-like structures at four to nine days after sporozoite inoculation (DAI). At 4–10 DAI, considerably more zoites were harvested from M617 cultures (80.1 times 10⁶ zoites) than from CPA (17.4 times 10⁶), MDBK. (47.3 times 10⁶), and WOMO (53.5 times 10⁶). Little or no parasite multiplication occurred at 10–16 DAI. Zoites harvested at 7 DAI and transferred to freshly prepared cultures did not penetrate cells nor develop further.     

The systematic significance of achene micromorphology in Carex retrorsa (Cyperaceae)

Scanning electron microscopy (SEM) was used to examine achene epidermal features of nine Carex species within sections Vesicariae and Lupulinae. Two major epidermal features are recognized in this study: the epidermal layers with one or two primary silicon bodies in each cell and the absence of any primary silicon body. Comparative data of achene morphology using SEM, and evidence compiled from previous investigations, support the placement of Carex retrorsa within section Vesicariae.     

Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33–258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.This work was supported by a grant from the Institute for General Medicine [GM18924 (M. W. T.)]. V. G. C. was a predoctoral fellow on Genetic Training Grant No. GM12 from the National Institute for General Medicine.     

Atlas of foliar surface features in woody plants,VI. Carya (Juglandaceae) of north America

Scanning electron microscopy was used to examine foliar surface features such as trichome types and the micro-relief formed by cell contours, cuticular patterns, and epicuticular wax in the genusCarya. The fourteen taxa native to Mexico and eastern United States were sampled throughout their geographical ranges, and are represented here by specimens that include segregate taxa, interspecific hybrids, and mature and immature leaves of each. Six trichome types are recognized in this survey: acicular, fasciculate, multiradiate, solitary, capitate-glandular, and peltate scale. There are no consistent differences between representatives of either the two Sections of the genus or the two ploidy levels. Although there is significant inter- and intra-specific variation, the use of scanning electron microscopy provides few taxonomically useful characters or character-states beyond those already available through light microscopy. Foliar surfaces of the fourteen taxa and three hybrids are illustrated.     

Binding and signaling of angiotensin-(1-7) in bovine kidney epithelial cells involves the AT(4) receptor

Angiotensin-(1-7) decreased mitogen-activated protein (MAP) kinase (Erks) activation in cultured Mardin-Darby bovine kidney (MDBK) epithelial cells. Also, saturable, high-affinity (125)I-angiotensin-(1-7) binding was detected in MDBK cell membranes. Together, the data suggested the possible presence of an angiotensin-(1-7) receptor. However, ligand structure-binding studies revealed that angiotensin-(3-7) and AT(4) receptor ligands competed with high-affinity for (125)I-angiotensin-(1-7) binding. Furthermore, angiotensin-(3-7) and AT(4) receptor ligands decreased MAP kinase activation in MDBK cells. These results demonstrate that NH(2)-terminal-deleted metabolites of angiotensin-(1-7) can bind with high affinity to the AT(4) receptor and regulate the MAP kinase/Erk signaling pathway in renal epithelial cells.     

Investigation of a toxic water-bloom of Microcystis aeruginosa (Cyanophyceae) in Lake Akersvatn,Norway

During the summer and fall of 1984 and 1985, the eutrophic Lake Akersvatn in south-eastern Norway, used as reserve drinking water reservoir, was found to produce heavy water-blooms of the colonial blue-green alga Microcystis aeruginosa. Samples of the water-bloom were found to be toxic using the mouse bioassay. No toxin was found free in the water as detected by HPLC and mouse bioassay. The toxic cells (minimum lethal dose 8–15 mg/kg body weight in mice) and purified toxin (minimum lethal dose 50 μg/kg body weight in mice) showed signs of poisoning in laboratory rats and mice identical to that of other hepatotoxin-producing M. aeruginosa blooms and strains reported from other parts of the world. The toxin has chemical properties similar to the cyclic heptapeptide reported for a South African M. aeruginosa toxin. The toxin from Lake Akersvatn bloom material has a molecular weight of 994. The toxic bloom of M. aeruginosa persisted from August to November in 1984 and reappeared in July of 1985. While water from Lake Akersvatn was not used for municipal water supply during this period, the presence of toxic blue-green algae in a drinking water reservoir indicates the need to develop monitoring and detection methods for toxic cells and toxin(s).     

Invasion of bovine epithelial cells by verocytotoxin-producing Escherichia coli O157:H7

The ability of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 to enter selected human (RPMI-4788 and HeLa) and bovine (MAC-T, mammary secretory; MDBK, kidney) epithelial cell lines was evaluated. All VTEC evaluated efficiently entered RPMI-4788 and MAC-T cell lines. VTEC entered MDBK cells at approximately 4% of MAC-T cells. VTEC were not able to invade HeLa cells. Presence of plasmid had no influence on efficiency of entry, nor did production of shiga-like toxin (SLT I or SLT II). Internalization required microfilaments, but not microtubules. Two types of adherence, localized and diffuse, were exhibited depending on isolate and cell line evaluated. Ability of VTEC to invade bovine mammary epithelial cells may be important in pathogenesis in the bovine, may indicate a route by which raw milk may potentially become contaminated, and may provide a reservoir of bacteria for the contamination of workers, equipment and carcass at time of slaughter.     

Notices

A virus isolated from the lung of an aborted Hereford fetus was shown to possess the physical and chemical properties of the herpesvirus group. The virus designated bovine herpesvirus (BH-1247) was isolated in cultures of Madin-Darby bovine kidney (MDBK) and primary bovine embryonic kidney (BEK) cells. Electron microscopic studies revealed typical forms of virus particles of the herpesvirus group. The virus was sensitive to chloroform and virus replication was inhibited by the addition of 5-iododeoxyuridine into the cell culture medium. The characteristic features of the cytopathic changes were syncytial formations with intranuclear inclusion bodies. Discrete plaques were formed in MDBK cell cultures overlaid with agar. Virus growth studies in BEK cells revealed infectious virus to be cell associated and replicated at low titer. By serum neutralization tests the virus was shown to be distinct from bovine herpesviruses; infectious bovine rhinotracheitis, DN-599, Movar 33/63, bovine mammallitis, malignant catarrhal fever and feline viral rhinotracheitis, equine herpesvirus I and pseudorabies. The isolate was nonpathogenic to mice inoculated subcutaneously, intracerebrally and intraperitoneally. Virus replication was not demonstrated when inoculated on the chorioallantoic membrane of embryonated eggs.