Ergovaline's role in the direct causation of fescue toxicosis first requires establishment of its dietary absorption. Therefore, ergovaline movement across human intestinal cells was assessed using Caco-2 cells derived from human colon carcinoma. A pre-equilibrated mixture of ergovaline/ergovalinine (60:40 ratio of isomers) was added to the apical compartment, and isomer movements were assessed by high-performance liquid chromatography (HPLC) of extracted media (initial pre-isomerized ergovaline concentrations of 6 and 22 μM, two doses). Mathematical models for ergot alkaloid movement were developed. Rates of movement were not different for the isomers. In the absence of cells, basal accumulation of isomers was essentially linear for 3 h regardless of loading concentration, after which basal accumulation of ergovaline/ergovalinine plateaued. Rates of ergovaline/ergovalinine movement in the presence of cells slowed to about 25% the rate of movement in the absence of cells (22 μM, kt = 0.0133 no cells, 0.0036 with cells, P < 0.05). Mass transfer rate was 7.5 ng·cm⁻²·min⁻¹ and was similar to that reported for ergovaline using a parabiotic chamber with sheep omasum. After 6 h in the presence of cells, ∼25 and 40% of the total ergovaline/ergovalinine administered had accumulated in the basal compartment for 6.6 and 22 μM treatments, respectively. Ergovaline and its naturally occurring isomer, ergovalinine, readily crossed intestinal cells intact and at similar rates. Either isomer, or a combination of both, could be involved in the pathogenesis of fescue toxicosis at sites distal to the intestine.

The wound healing response to injury can be affected by many factors such as cell migration and extracellular matrix elaboration. The objective of this study was to examine the serum- and age-dependent effects on cell migration, adhesion, and collagen expression by skin fibroblasts. Dermal fibroblasts were isolated and plated with and without serum for up to 7 d. Cell migration was determined by quantitative image analysis, adhesion was quantified using a centrifugation assay, and collagen expression was assessed by PCR and immunohistochemical staining. Both adult and fetal fibroblasts migrated significantly faster in serum-containing medium compared to serum-free medium. There was no significant difference in migration between the two cell types in either serum-containing or serum-free medium. There was no significant difference in adhesion in the presence of serum, although there was a greater fraction of adherent fetal skin fibroblasts than adult fibroblasts in serum-free medium. Moreover, the adherent fraction of fetal fibroblasts in serum-free medium was not significantly different from that in serum-containing medium, suggesting that fetal skin fibroblasts possess serum-independent adhesion properties. Collagen mRNA expression was significantly up-regulated in serum-free compared to serum-containing medium for both cell types. With respect to collagen immunohistochemistry, both dermal fibroblast populations exhibited greater type I collagen compared to type III collagen staining. Quantitative assessment of collagen staining indicated significantly enhanced type I collagen secretion in the presence of serum by fetal skin fibroblasts. These findings suggest that intrinsic cellular characteristics may govern the observed differences in adult and fetal wound healing.

Anurans are important animal models for studying the effects of anthropogenic chemical contamination of the environment. Two-compartment Teflon flow-through diffusion cells can be used to study percutaneous absorption of xenobiotics across harvested skin. However, such an approach currently necessitates that skin be harvested just before experimentation, a requirement that calls for the continuous growth and housing of living animals. The ability to preserve and store skin would allow more efficient use of animals and more flexibility in experimental design. To this end, we examined the viability of harvested anuran skin stored under various protocols consistent with current practices of mammalian skin preservation. Skin from the American bullfrog maintained 80–85% viability after 28 d, whereas viability of skin from the marine toad was only maintained for 7–10 d.

Our study focused on investigating the mechanism of action of estrogen in regulating p53 levels within osteoblasts. In the studies reported here, we attempted to understand the role of estrogen receptors, ER-alpha and ER-beta, in the regulation of p53 and osteoblast differentiation. We stably expressed ER-alpha and ER-beta in ROS 17/2.8 cells and isolated several single cell clones. These clones were initially characterized for expression of the exogenous receptors, and representative clones from each type were chosen for further analyses. Cell proliferation, alkaline phosphatase activity, and the viability of these clones in culture were tested. The cells expressing exogenous ER-alpha exhibited more differentiated characteristics than cells expressing ER-beta. Morphologically, ER-beta–overexpressing cells were more rounded than the ER-alpha–overexpressing cells, which were more elongated and fibroblastic in appearance. The ER-beta–expressing cells had a higher survival and growth rate when compared with ER-alpha cells. The ER-alpha clones were not as viable as ER-beta clones, and some of the ER-alpha cell lines showed signs of senescence, with an increase in senescence-associated (SA) galactosidase activity. The basal levels of p53 functional activity were higher in cells expressing ER-alpha as was protein expression of the p53-regulated gene p21. The significance of these receptors to osteoblast differentiation and p53 regulation is discussed.

The objective of this study was to evaluate synchronous and asynchronous pronucleus (PN) formation and the related patterns of juxtapositional nucleolus (n) formation in immature (prophase I [PI] and metaphase I [MI]) and mature (metaphase II [MII]) oocytes after fertilization, both ultrastructurally and at the level of light microscope. A single dose of 15 IU gonadotrophin was injected subcutaneously to twenty four 26-wk-old, female Wistar rats to induce ovulation. Human chorionic gonadotrophin (4 IU) was administered 40 h later, and after 4–6 h the ovaries were dissected, and the oocytes were aspirated. A total of 214 rat oocytes were classified according to a maturation index as follows: group I, 80 PI oocytes; group II, 50 MI oocytes; and group III, 84 MII oocytes. Immature oocytes were in vitro matured for 18–36 h. Spermatozoa were acquired by microepididymal sperm aspiration and processed using swim-up technique. Intracytoplasmic sperm injection was performed on mature oocytes after 2 h of incubation and on in vitro matured (IVM) oocytes 4 h after maturation. Pronuclear synchronization [both pronucleases (PNs) centrally located, equal sized, with equal numbers and sizes of juxtapositional nucleoli (Nn)] was observed in fertilized oocytes. Asynchronous PN formation (diversity between male and female PNs, related to dimensions, localization, and the number of Nn) in groups I, II, and III was found in 75, 86, and 47% of preembryos, respectively. There was a significant difference of synchronous pronuclear formation between mature and IVM oocytes (P < 0.05). In IVM oocytes, asynchronous PN formation is high, and juxtapositional pronucleolar patterns are observed to be low by transmission electron microscope (TEM).

The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointments due to inadequate ES cell sources.

We investigated the effect of exogenous wild-type p53 on the radiation-induced cells apoptosis and necrosis at different levels of linear energy transfer (LET) to evaluate its mechanisms. The human melanoma cell line A375, which bears wild-type p53 gene status, was used, as well as the transfectant A375 cells (A375/p53) with adenoviral vector containing the wild-type p53 gene. We exposed these cells to X-rays and to accelerated carbon-ion (C-) beams. Cellular sensitivities were determined by using clonogenic assay. Apoptotic and necrotic cell deaths were determined morphologically by dual staining (acridine orange and ethidium bromide) using fluorescence microscopy. We discovered that (1) there was no significant difference in survival fraction between A375 cells and A375/p53 cells irradiated by C-beams with greater than 32 KeV/μm LET, (2) although apoptosis in the two kinds of cells increased in an LET-dependent manner, exogenous wild-type P53 induced cell apoptosis efficiently in A375/p53 relative to A375 cells with X-rays or high-LET irradiation, and (3) by high-LET irradiation, the number of necrosis in A375 cells increased significantly (P < 0.05) in comparison with A375/p53 cells. These results indicate that in high-LET irradiation apoptosis induction is p53 dependent partly and exogenous wild-type P53 plays an important role in modulating cell death type, although there was no significant difference in cellular radiosensitivities. Our observation in the study offers the potential application of high-LET radiation combined with p53 in the management of human patients with melanoma.

Serial passaging of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) in H. zea (Hz-AM1) insect cell cultures results in rapid selection for the few polyhedra (FP) phenotype. A unique HaSNPV mutant (ppC19) was isolated through plaque purification that exhibited a partial many polyhedra (MP) and FP phenotype. On serial passaging in suspension cell cultures, ppC19 produced fivefold more polyhedra than a typical FP mutant (FP8AS) but threefold less polyhedra than the wild-type virus. Most importantly, the polyhedra of ppC19 exhibited MP-like virion occlusion. Furthermore, ppC19 produced the same amount of budded virus (BV) as the FP mutant, which was fivefold higher than that of the wild-type virus. This selective advantage was likely to explain its relative stability in polyhedra production for six passages when compared with the wild-type virus. However, subsequent passaging of ppC19 resulted in a steep decline in both BV and polyhedra yields, which was also experienced by FP8AS and the wild-type virus at high passage numbers. Genomic deoxyribonucleic acid profiling of the latter suggested that defective interfering particles (DIPs) were implicated in this phenomenon and represented another undesirable mutation during serial passaging of HaSNPV. Hence, a strategy to isolate HaSNPV clones that exhibited MP-like polyhedra production but FP-like BV production, coupled with low multiplicities of infection during scale-up to avoid accumulation of DIPs, could prove commercially invaluable.

Insect cell lines from Arthropoda represented by Lepidoptera, Coleoptera, Diptera, and Homoptera were evaluated for their ability to support replication of AcMNPV. In addition, some of the cell lines that were refractive to AcMNPV were tested with AcMNPV hsp70 Red, a recombinant carrying the red fluorescent protein (RFP) gene, for their ability to express this protein after inoculation. Of the 10 lepidopteran cell lines tested, only three cell lines from Helicoverpa zea (BCIRL-HZ-AM1), Lymantria dispar (IPLB-LD 65), and Cydia pomonella (CP-169) failed to support detectable viral replication as measured by tissue culture infectious dose 50 (TCID₅₀) assay. Heliothis virescens (BCIRL-HV-AM1) produced the highest viral titer of 2.3 ± 0.1 × 10⁷ TCID₅₀/ml followed by Heliothis subflexa (BCIRL-HS-AM1) at 4.7 ± 0.1 × 10⁶ TCID₅₀/ml and Spodoptera frugiperda (IPLB-SF21) at 4.1 ± 0.1 × 10⁶ TCID₅₀/ml. None of the coleopteran, dipteran, or homopteran cell lines supported AcMNPV replication. However, when studies were performed using AcMNPV hsp70 Red, the dipteran cell lines Aedes aegypti (ATC-10) and Drosophila melanogaster (line 2), both expressed the RFP as well as the refractive lepidopteran cell lines from H. zea and L. dispar. No RFP expression was observed in any of the coleopteran or homopteran cell lines. Cell lines refractive to AcMNPV did not appear to be adversely affected by the virus, as judged by their ability to multiply, nor was there any indication of induced apoptosis, as assessed by deoxyribonucleic acid fragmentation profiles or cell blebbing, or both.

Injuries to the avascular region of the meniscus occur frequently and may be difficult to repair. This study was designed to determine whether growth factors could diffuse from a collagen sponge or a collagen gel into meniscal tissue and stimulate healing of defects using an in vitro model. The diffusion of platelet-derived growth factor (PDGF) from the collagen carriers into the medium was rapid with approximately 50% being released from the collagen sponge within the first hour. After 5 d of incubation, 8% of the PDGF was present in the meniscus, 11% in the collagen sponge, and 62% had been released into the medium. Similar results were obtained when a collagen gel was used as a carrier. Histological evaluation of the meniscal explants after 2 wk in culture revealed extensive proteoglycan staining in the areas surrounding defects treated with either hepatocyte growth factor (HGF) or PDGF compared with controls without growth factor. The HGF–PDGF treatment resulted in alignment and migration of meniscal cells toward the defect, which was not observed in untreated controls. At 3–7 d, increased number of cells were observed in defects treated with collagen gels (but not the sponge) with PDGF–HGF. At 4 wk, combined HGF–PDGF treatment resulted in the formation of tissue with birefringence by polarized microscopy, suggestive of organized collagen. The data suggest that use of specific PDGF–HGF may enhance the repair of meniscal injuries.