Differential feulgen-deoxyribonucleic acid hydrolysis patterns of Herpes simplex virus type 1 and type 2 infected cells.

Infection of human embryonic lung cells with herpes simplex virus type 1 (HSV-1) and herpes simplex type 1 (HSV-2) resulted in: (a) qualitative (nuclear cytopathologic) alterations and quantitative (nuclear area) differences in infected compared to control nuclei; (b) increased Feulgen-deoxyribonucleic acid (F-DNA) amounts in infected cells, probably due to viral DNA; (c) higher F-DNA levels in HSV-2 infected cells; and (d) increased rates of F-DNA hydrolysis in viral-infected as compared to uninfected nuclei.

Metabolism of T-2 mycotoxin by cultured cells.

T-2 mycotoxin is a small (i.e. mol. wt 466), non-protein toxin. We studied its metabolism in Chinese hamster ovary (CHO) cells, African green monkey kidney (VERO) cells, human fibroblasts and mouse connective tissue cells (L-929). Confluent cells were exposed to [3H]-T-2(0.01 micrograms/ml) for 1 hr at 37 degrees C. The toxin was removed, cells rinsed, and unlabeled culture media added for 4 hr (37 degrees C). Cell monolayers were extracted and media and cell extracts were spotted on thin-layer chromatography plates with known standards. Thin-layer plates were developed and scanned for radioactivity, and metabolites were identified based on co-migration with known standards. CHO and VERO cells metabolized T-2 to a greater per cent and to a wider variety of metabolites than the other two cell types. In CHO, fibroblast and L-929 cells, the major metabolite was HT-2 toxin, while in VERO cells an unknown metabolite, more polar than T-2, was the major metabolite. Cell and media extracts of CHO and VERO cells revealed smaller amounts of T-2 triol, T-2 tetraol and several unknowns. In both cell types, metabolites were detected in labeled media by 1 hr and in increasing amounts in unlabeled media by 4 hr. Under the above conditions, 37-58% of the radioactivity remained as T-2 toxin after 4 hr in both cell types. The data suggest that some cultured cell lines possess enzyme systems capable of limited metabolism of T-2 mycotoxin to a variety of known and some as yet unidentified metabolites.

Effects of sodium fluoride on uptake of T-2 mycotoxin in cultured cells.

We examined the effect of sodium fluoride on uptake of tritium-labeled T-2 toxin (molecules of toxin/cell) in Chinese hamster ovary (CHO) and African green monkey kidney (VERO) cells. Correlations were made to temperature (22 and 37 degrees C) and toxin concentration (0.001 and 0.01 microgram/ml) over time (0-180 min). As expected, toxin uptake increased in both cell types with increasing time and temperature. VERO cells exhibited significant (P less than 0.05) increases in the rate (i.e. slope) of toxin uptake under all parameters, while the rate of toxin uptake in both cell types was generally greater at 37 degrees C compared to 22 degrees C. The rate of equilibrium was affected by both temperature and sodium fluoride. At 37 degrees C toxin uptake plateaued by 30 min in the presence of sodium fluoride. At 22 degrees C the rate of toxin uptake was slower, with or without sodium fluoride present. Statistical analysis of individual time points along the curve demonstrated that sodium fluoride significantly increased cell-associated toxin at most time points. Analysis of the slopes of uptake curves from 0 to 20 min indicated significant (P less than 0.05) differences in the rates of T-2 uptake in both cell types and toxin doses in the presence of sodium fluoride. The increase in toxin uptake in the presence of sodium fluoride was not due to altered cell membrane permeability caused by sodium fluoride. This study demonstrates that sodium fluoride significantly increases cell-associated T-2 toxin and the rate of toxin uptake in two cultured cell lines.

Ultrastructural effects of T-2 mycotoxin on rat hepatocytes in vitro.

Cultured rat hepatocytes were treated with several doses of T-2 mycotoxin for either 1 or 12 hr and with or without a 12 hr recovery period. Inhibition of protein synthesis and release of lactate dehydrogenase were measured and correlated with ultrastructural changes, as assessed by transmission electron microscopy. Results indicated that at a dose of 0.01 microgram/ml protein synthesis was inhibited 75% within 1 hr, but recovered to near control levels with or without the continual presence of toxin. At the higher toxin dose of 1.0 microgram/ml hepatocytes were able to recover from a 1 hr, but not a 12 hr, exposure. Cell damage, as assessed by release of lactate dehydrogenase, lagged behind inhibition of protein synthesis. Only at a T-2 concentration of 1.0 microgram/ml for 12 hr followed by a 12 hr recovery period was release of lactate dehydrogenase significantly elevated over control values. Under the same parameters, protein synthesis was inhibited 94%. The ultrastructural appearance of the cell membrane, nucleus, lysosomes, peroxisomes and smooth endoplasmic reticulum remained unchanged. The two organelles which appeared altered by T-2 exposure were the rough endoplasmic reticulum and the mitochondria. Endoplasmic reticulum changes were limited to degranulation of attached ribosomes without dilation of the cisternae. Alterations were seen as early as 1 hr at a T-2 dose of 0.01 microgram/ml. After a dose of 1.0 microgram/ml T-2 for 12 hr some mitochondria displayed one or more non-membrane-bound translucent foci, some of which contained electron-dense cores.

Morphological changes in CHO and VERO cells treated with T-2 mycotoxin. Correlation with inhibition of protein synthesis.

Exposure of Chinese hamster ovary and African green monkey kidney cells to T-2 mycotoxin resulted in several morphological changes which were related to inhibition of protein synthesis, the basic in vitro mechanism of action of the toxin. These changes, which occurred in both cell types, included disassociation of polysomes and mitochondrial cristae alterations. In addition, CHO cells displayed membrane bleb formations similar to those found in CHO cells after exposure to established inhibitors of protein synthesis, puromycin and anisomycin. Blebs could be either a result of protein synthesis inhibition or a non-specific early pathological response. Bleb formations were not observed in VERO cells under any experimental condition.

Stability of T-2 mycotoxin in aqueous media.

3H-labeled T-2 mycotoxin was dissolved in various aqueous media and stored for up to 3 weeks at 4, 22, and 37 degrees C. At periods ranging from 1 to 21 days, aliquots were assayed by thin-layer chromatography. Thin-layer chromatography plates were scanned for breakdown products by use of a radioisotope scanner, and breakdown products were identified based on their comigration with known standards. Results indicated that T-2 toxin was more stable in tissue culture medium with or without serum, than in Hanks balanced salt solution (HBSS), at all temperatures. The metabolites HT-2, T-2 triol, and T-2 tetraol were detected as early as 1 day (HBSS; 37 degrees C) and as late as 3 weeks (HBSS; 4 degrees C) after storage. Stability of the toxin in aqueous media decreased with increasing temperature.

The effect of catheptic enzymes on chilled bovine muscle.

the toughening of meat which has been caused by cold shortening prior to the onset of rigor is of significant commercial importance. Various studies have shown that catheptic enzymes produce degradative changes to meat which are very similar to those which occur during the natural aging process and which lead to a more tender meat product. Because of the tenderizing action of cathepsins, this study was undertaken to determine what effect these enzymes had on the cold shortening process of bovine sternomandibulatis muscle. Samples which were cold shortened for 24 or 72 hrs were soaked in either a control solution or one containing catheptic enzymes. Microstructural observations and measurement of sarcomere length by laser diffraction, transmission and scanning electron microscopy, indicated that enzyme treatment hastened the change from rigor to aged muscle.

In vitro model for the study of platelet-vessel wall interactions following a freeze- thaw injury.

Platelet-endothelial cell interactions are important for maintaining normal hemodynamics. The intact endothelial cell lining is considered nonthrombogenic, but following disruption of the lining, platelets bind to the subendothelium. There is also much conjecture concerning the affinity of platelets for damaged endothelial cells. A model is described for the study of platelet-aorta vessel wall interactions following freeze-thaw insult. Using this model, we found that control aortas (37 degrees C) perfused with platelet-rich plasma or gel-filtered platelets showed no generalized platelet-endothelial cell interactions, although some platelets did adhere to areas of exposed subendothelium. Following freeze-thaw insult (-15 degrees or -20 degrees C), the endothelial lining was grossly disrupted. The remaining endothelium was severely damaged, demonstrating holes and pits in the plasma membranes and separation of adjacent cell borders. Platelets readily adhered to the basal lamina but were rarely noted in sole contact with the damaged endothelium. Platelet binding did not result in morphologic changes, degranulation, or aggregation. Using transmission electron microscopy, we noted platelets in contact with amorphous material and microfibrils but not collagen fibers of the subendothelium. It is concluded that this model is suitable for the in vitro study of certain hemodynamic phenomena associated with blood vessel freeze-thaw injury. In addition, freeze-thaw damage in this in vitro model indicated that platelet-vessel wall interactions were limited to areas of exposed subendothelium.

Transmission and scanning electron microscopy of cell monolayers grown on polymethylpentene coverslips.

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized, separated from the coverslip by liquid nitrogen immersion, and sectioned either "en face" or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.

Characterization of freeze-thaw induced ultrastructural damage to endothelial cells in vitro.

The pathophysiology of endothelial cells is important to a variety of vascular conditions including coagulation and hemostasis resulting from clinical frostbite. Use of an in vitro model system demonstrated that when bovine endothelial cells were frozen at 1 degrees C or 20 degrees C/min and thawed immediately (20 degrees C/min), a variety of ultrastructural alterations occurred. Membranous structures were most extensively damaged, with mitochondria the most sensitive organelle. Low amplitude mitochondrial swelling, first evident at 0 degrees C, progressed to high amplitude swelling by -10 degrees C (frozen). In addition, the rough endoplasmic reticulum was dilated and formed large vesicles with a homogeneous matrix. Nuclear changes first occurred at -15 degrees C. These included separation and distortion of the nuclear membrane, changes in chromatin distribution, and disruption of the nucleolus. Scanning electron microscopy revealed perforated plasma membranes in some cells at -10 degrees C (frozen) and in most cells by -20 degrees C. Cultures frozen at 20 degrees C/min revealed mostly the same ultrastructural damage noted at 1 degrees C/min except a higher percentage of cells exhibited alterations. Data from the recovery index and lactic dehydrogenase (LDH) release correlated well with observed ultrastructural changes. Early swelling of mitochondria and dilation of rough endoplasmic reticulum was not lethal in the absence of freezing. Increased swelling in cytoplasmic organelles coupled with nuclear alterations at -15 degrees C resulted in a decreased survival rate and release of significant quantities of LDH by -20 degrees C. No unique morphological changes were temperature specific, but the total number of cells that displayed alterations increased as temperature decreased.

Platelet-endothelial cell interactions in vitro following freeze-thaw injury or detergent treatment.

Platelet interactions with cultured bovine endothelial cells were studied following freeze-thaw damage or detergent treatment. Platelets from whole blood, platelet-rich plasma, or gel-filtered plasma did not interact directly with freeze-thaw-damaged endothelial cells. Freezing and thawing did result in the exposure of an extracellular matrix located beneath the cells, which proved very thrombogenic. Platelets from all sources attached to both microfilament and amorphous components of the extracellular matrix, although only platelets from whole blood demonstrated aggregation and extensive pseudopodia formation. Treatment of cells with Triton-X detergent resulted in exposure of an intracellular cytoskeleton. Most platelets attached to the cytoskeleton were located near the cell border and had one or more pseudopodia either in contact with extracellular or intracellular material. Adhesion of platelets to the extracellular matrix may represent platelet-collagen or platelet-fibronectin interactions since both are produced by an incorporated into the extracellular matrix. Platelet interaction with endothelial cytoskeletons may represent contact of pseudopodia with the now exposed matrix located beneath the cells. The possibility that platelets also adhered to intracellular components could not be eliminated. These findings are in agreement with data from a freeze-thaw injury model of perfused aorta. In addition, they tend to indicate that physical insult is not sufficient to induce platelet interaction with the endothelial surface, but that chemical modification enhances platelet deposition.

Quantitative cytophotometry evaluated as a method for analyses of herpes simplex viral deoxyribonucleic acid synthesis.

Feulgen deoxyribonucleic acid (F-DNA) microspectrophotometry was evaluated as a potential tool for quantification of herpes simplex virus type 1 (HSV-1) and 2(HSV-2) DNA synthesis in single cells. Since HSV DNA synthesis has been extensively studied using incorporation of radioactive precursors into viral and cellular DNA, microspectrophotometric measures were correlated with biochemical data obtained using tritium-labeled thymidine ([3H]TdR). It was established that: (i) viral-induced increaae in F-DNA can be cytophotometrically detected between 1 and 6 h postinjection (p.i.), which corresponds to the initial incorporation of [3H) TdR into viral DNA; (ii) peak F-DNA levels occurred 8 h p.i., which supported cytological observations of a more rapid development of an inclusion body in HSV-2-infected nuclei. Despite the fact that F-DNA cytophotometry is unable to distinguish between cell and viral DNA, the overall study supports the existence of a good correlation between data obtained using microspectrophotometric and conventional isotope methods. Furthermore, cytophotometry complements biochemical evaluations in that it permits analyses of DNA changes on a single-cell basis or the detection of infection in very small numbers of cells.