A cell line derived from the larval-fat body tissues of the wax moth, Galleria mellonella Linne, was established in MGM-450 medium. The cells grew in suspension and were mainly spherical in shape. Population doubling time was between 1.4 and 1.7 d over a range of 15 to 35° C, and the maximum growth rate was at 25° C. The chromosome number ranged from 70–239, with a mode of 170. The cells were sensitive to 20-hydroxyecdysone, which stimulated their growth and induced morphological changes. The cell line was designated GaMe-LF1.

Skeletal fibroblasts in vitro can acquire myofibroblast phenotypes by the development of biochemical and morphological features, mainly the expression of alpha–smooth-muscle actin (α-SMA). Myogenic differentiation is a central event in skeletal muscle development, and has commonly been studied in vitro in the context of skeletal muscle development and regeneration. Controlling this process is a complex set of interactions between myoblasts and the extracellular matrix. Osteopontin (OPN) is an acidic, phosphorylated matrix protein that contains an Arg-Gly-Asp (RGD) cell attachment sequence and has been identified as an adhesive and migratory substrate for several cell types. The aim of this study was to investigate osteopontin expression during the differentiation of skeletal fibroblasts into myofibroblasts and during myogenesis in a coculture model. Fibroblasts and myoblasts were obtained from skeletal muscle of 18-d-old Wistar strain rat fetuses by enzymatic dissociation. At 1 and 9 d, cocultures were immunolabeled, and the cells were also separately subjected to Western blotting to analyze OPN expression. Our data using confocal microscopy showed that myoblasts displayed a strong staining for OPN and that this labeling was maintained after myotube differentiation. Conversely, during fibroblast differentiation into myofibroblasts, we observed a significant increase in OPN expression. The results obtained by immunolabeling were confirmed by Western blotting. We suggest that OPN is important mainly during early stages of myogenesis, facilitating myoblast fusion and differentiation, and that the increased expression of OPN in myofibroblasts might be related to its effects as a key cytokine regulating tissue repair and inflammation.

UDP glycosyltransferases (UGTs) and sulfotransferases (SULTs) are phase II enzymes that interact with a number of xenobiotics in humans and animals. Species differences in enzymatic characteristics have seldom been investigated. Liver S9 fractions are commonly used for studying phase II metabolism in vitro. The objective of this study was to characterize the UGT and SULT activities in liver S9 fractions from various species including humans, monkeys, dogs, and rats. A single substrate, 7-hydroxycoumarin (7-HC), at several concentrations was incubated at 37° C with the S9 reaction matrices along with necessary cofactors. The rate of formation of two metabolites, 7-HC-glucuronide (7-HC-G) and 7-HC-sulfate (7-HC-S), was determined with Liquid Chromatography/Tanderm Mass Spectrometry (LC/MS/MS). Apparent K_m and V_max values were calculated for each species. For the UGTs, the apparent K_m and V_max for 7-HC-G formation varied greatly among different species, with dog UGTs having both the highest K_m and V_max values. In contrast to UGTs, the K_m for 7-HC-S formation showed no significant difference among humans, monkeys, and rats (approximately 3 μM). However, the K_m in dog was 8.7 μM. Species differences with respect to phase II metabolism must be carefully considered when selecting an in vitro model system to study various aspects of drug metabolism.

Tiny particles were found in the medium in the presence of the human leukemia cell line Kasumi-6. The particles were separated from human cells by filtration and incubated in RPMI1640 supplemented with 10% fetal calf serum at 37 C. The particles increased in number very slowly in the liquid medium but did not reveal any biological activity. Transmission electron microscopy of the particles showed a spheroid or ovoid shape in ultrathin section. No specific polypeptides from the purified particles were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), except for bovine fetuin that adsorbed to the surface of the particles. X-ray diffractometry as well as Fourier transform infrared spectrometry suggested the particles consisted of hydroxyapatite. The mechanism of self-propagation of the hydroxyapatite particles in liquid medium is currently unknown. This type of particle has been overlooked for a long period because it is noncultivable. It will be necessary to examine its biological effects to the cultured cells.

This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation. This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.

Ethanolamine (Etn) stimulates hepatocyte proliferation in vivo and in vitro; however, the physiological function of Etn in hepatocytes has yet to be elucidated. In the present study, we examined the effect of Etn using a primary culture of rat hepatocytes. The level of membrane phosphatidylethanolamine (PE) significantly decreased when the hepatocytes were cultured without Etn but increased to the level found in the liver when the culture medium was supplemented with 20– 50 μM Etn. Moreover, Etn stimulated DNA synthesis in a dose-dependent manner and had a synergistic effect with epidermal growth factor (EGF). A binding assay and Western blotting showed that the number of EGF receptors was 22– 30% lower in cells grown in the absence of Etn compared to those grown in its presence, but the respective Kd values were almost the same. Furthermore, tyrosine phosphorylation of the EGF receptor was significantly lower in cells grown without Etn. Phosphatidylcholine (PC) synthesis in the liver is unique in that it occurs via stepwise methylation of PE. We found that without Etn supplementation, bezafibrate-induced inhibition of PE methylation increased the level of PE by decreasing its conversion to PC and stimulated DNA synthesis. Moreover, the function of EGF in stimulating DNA synthesis was significantly enhanced under Etn-sufficient conditions. These data suggest that Etn is a nutritional factor required for synthesis of adequate PE, levels of which are important for hepatocyte proliferation.

The pleiotropic drug resistance 5 gene (pdr5) encodes a multidrug membrane transporter and plays a very important role in the efflux of a broad range of chemicals in yeast cells. To study the possible function of pdr5 in insect cells, two stably pdr5-transformed lepidopteran insect cell lines, Sf21 and CF-203, were developed. Transcripts of pdr5 were detected in these two lines using Northern blotting and RT-PCR analysis. When cells were treated with the protein synthesis inhibitor diacetoxyscirpenol, the transformed Sf21 and CF-203 cell lines showed increased tolerance to this chemical. However, unlike in yeast cells, ecdysone agonist RH5992 could not be excluded by PDR5, probably because of low expression levels or imperfect incorporation of the recombinant protein in these transformed cell lines.

Adenocarcinoma of the prostate comprises 95% of all prostate cancer. Commercially available primary cultures of “normal” prostate epithelial cells, PrECs, have been used as a convenient model to investigate neoplastic transformation. Here PrECs were characterized for the expression of lineage- and developmental-specific markers cytokeratin (CK) 8 and 18, p63, chromogranin A, TMEPAI, S100P, NKX 3.1, ANKH, and FN 1 as well as androgen receptor and prostate-specific antigen by Western blot and Northern blot analyses, immunohistochemistry, reverse transcriptase–polymerase chain reaction (RT-PCR), and quantitative real-time PCR. Immunohistochemical staining detected PrECs positive in varying degrees for p63, CK 8, and CK 18, with only the rare cell being positive for chromogranin A. The PrECs also tested positive for p63 protein by Western blot analysis. RT-PCR with PrEC cDNA showed products for FN 1 and S100P but not for ANKH and androgen receptor or prostate-specific antigen. This profile of markers in PrEC cells is consistent with that expected for pubertal prostate epithelial cells.

One limitation to the widespread use of in vitro–produced embryos in cattle is their poor survival following cryopreservation. Two approaches for enhancing survival of in vitro–produced bovine embryos following cryopreservation were evaluated: culture in the presence of hyaluronic acid and alterations in the cytoskeleton through cytochalasin B treatment. The experiment was a 2 × 2 factorial design to test main effects of hyaluronic acid added to culture at day 5 after insemination ( or −) and cryopreservation treatment (control or cytochalasin B). Embryos used for cryopreservation were blastocysts and expanded blastocysts harvested on day 7 after insemination. Cytochalasin B increased the percent of embryos that re-expanded (P < 0.0001) and that hatched following thawing (P < 0.05). The hatching percent was 29.6% for embryos treated with cytochalasin B versus 9.1% for control embryos. There was no significant effect of hyaluronic acid on survival although there was a tendency for embryos cultured with hyaluronic acid to have higher percent hatching if not treated with cytochalasin B (12.7% for hyaluronic acid versus 4.5% for control; hyaluronic acid × cytochalasin B interaction; P = 0.09). In conclusion, cytochalasin B treatment before freezing improved cryosurvival of bovine embryos produced in vitro. Such a treatment could be incorporated into methods for cryopreservation of bovine embryos provided post-transfer survival is adequate. In contrast, culture with hyaluronic acid was of minimal benefit— the increased cryosurvival in the absence of cytochalasin B was not sufficient to allow an adequate number of embryos to survive.

The mammalian intestinal epithelium has been found, based on in vivo experiments, to be resistant to insecticidal Cry toxins, which are derived from Bacillus thuringiensis and fatally damage insect midgut cells. Thus, the toxins are commonly used as a genetic resource in insect-resistant transgenic plants for feed. However, Cry toxins bind to the cellular brush border membrane vesicle (BBMV) of mammalian intestinal cells. In this study, we investigated the affinity of Cry1Ab toxin, a lepidopteran-specific Cry1-type toxin, to the cellular BBMV of two mammalian intestinal cells as well as the effect of the toxin on the membrane potential of three mammalian intestinal cells compared to its effects on the silkworm midgut cell. We found that Cry1Ab toxin did bind to the bovine and porcine BBMV, but far more weakly than it did to the silkworm midgut BBMV. Furthermore, although the silkworm midgut cells developed severe membrane potential changes within 1 h following the toxin treatment at a final concentration of 2 μg/ml, no such membraneous changes were observed on the bovine, porcine, and human intestinal cells. The present in vitro results suggest that, although Cry1Ab toxin may bind weakly or nonspecifically to certain BBMV components in the mammalian intestinal cell, it does not damage the cell's membrane integrity, thus exerting no subsequent adverse effects on the cell.