Fibroblasts in vitro can acquire myofibroblast phenotype by the development of several biochemical and morphological properties of smooth muscle cells, particularly the expression of alpha-smooth muscle actin. These cells play a major role in inflammatory responses and in wound repair through their production of growth factors, cytokines, and other soluble mediators. Lipid bodies (LB) are lipid-rich cytoplasmic inclusions and have been recognized as specialized intracellular domains involved in the formation of paracrine mediators of inflammation. The aim of the present study was to investigate the occurrence and distribution of LB during differentiation of rat fetus skeletal fibroblasts into myofibroblasts in vitro. Primary cultures of fibroblasts were obtained from skeletal muscles of 18-d-old Wistar strain rat fetus by enzymatic dissociation. At 1–7 d, the cells were stained with Nile red vital dye to identify LB and then observed under a Zeiss CLSM-310. Our results showed that there was an accentuated increase in the number of LB during the differentiation of skeletal fibroblasts into myofibroblasts and that these inclusions were scattered at the cytoplasm.

Animal models have long been used to elucidate the mechanisms responsible for osteoporosis in humans. The American black bear, an animal that does not experience extensive bone loss normally associated with long-term immobilization (when hibernating), may provide an insight into the nature of the pathogenesis of the disease. Circulating growth and differentiation factors present in the serum may facilitate continued proliferation of bone-forming cells. The aim of our study was to determine the effects of bear serum on human osteoblasts when cultured for extended periods of time. Unexpectedly, exposure to the bear serum in vitro led to the detachment of osteoblasts from the surface of the culture plate after 3 d of incubation. The osteoblasts pulled off the polystyrene surface in sheets and aggregated into floating conglomerations of viable cells. In contrast, osteoblasts cultured in fetal calf serum maintained adherence to the surface of the culture plate. Detachment of osteoblasts propagated in bear serum was time dependent and was associated with an increased expression of integrins compared with osteoblasts propagated in fetal calf serum, as indicated by reverse transcriptase–polymerase chain reaction and immunostaining.

This article describes the culture of epithelial cells from anterior and posterior midgut regions of adult Dendroctonus valens. Culture conditions were established, and cell adherence was improved by means of a new technique that allowed the cells to grow between two glass coverslips. Cytoplasmic projections occur as anterior midgut cells grow to confluence; these projections were not observed in cells of the posterior midgut. The optimal culture medium for the maintenance of these epithelial cells was Roswell Park Memorial Institute 1640 medium at 25° C. Cells in Grace's medium died in 24 h. Cultures did not require CO₂ atmosphere, but culture development was favored by the microaerophilic environment and the dark conditions in which the cells were grown, between the coverslips.

We have established an explant-cell culture system for mammary gland tumors from c-myc oncogene–expressing transgenic mice and potentially other transgenic strains. By coating culture dish surfaces with fetal bovine serum and using culture media supplemented with low serum and growth factors, the mammary tumor specimens could be maintained in culture for over 3 mo. Throughout the culture period, the explants produced abundant outgrowths of epithelial cells. As the outgrowths of epithelial cells filled the dishes, the explants were serially transferred from one dish to another—a process that could be repeated at least six times, thus providing a continuous supply of primary tumor cells. This culture system provides a useful tool for studying the biology of mouse mammary gland tumors and possibly tumors from other organ sites.

Human proximal tubule epithelial cell lines are potentially useful models to elucidate the complex cellular and molecular details of water and electrolyte homeostasis in the kidney. Samples of normal adult human kidney tissue were obtained from surgical specimens, and S1 segments of proximal convoluted tubules were microdissected, placed on collagen-coated culture plate inserts, and cocultured with lethally irradiated 3T3 fibroblasts. Primary cultures of proximal tubule epithelial cells were infected with a replication-defective retroviral construct encoding either wild-type or temperature-sensitive simian virus 40 large T-antigen. Cells forming electrically resistive monolayers were selected and expanded in culture. Three cell lines (HPCT-03-ts, HPCT-05-wt, and HPCT-06-wt) were characterized for proximal tubule phenotype by electron microscopy, electrophysiology, immunofluorescence, Southern hybridization, and reverse transcriptase–polymerase chain reaction. Each of the three formed polarized, resistive epithelial monolayers with apical microvilli, tight junctional complexes, numerous mitochondria, well-developed Golgi complexes, extensive endoplasmic reticulum, convolutions of the basolateral plasma membrane, and a primary cilium. Each exhibited succinate, phosphate, and Na,K– adenosine triphosphatase (ATPase) transport activity, as well as acidic dipeptide– and adenosine triphosphate–regulated mechanisms of ion transport. Transcripts for Na –bicarbonate cotransporter, Na –H exchanger isoform 3, Na,K-ATPase, parathyroid hormone receptor, epidermal growth factor receptor, and vasopressin V2 receptor were identified. Furthermore, immunoreactive sodium phosphate cotransporter type II, vasopressin receptor V1a, and CLIC-1 (NCC27) were also identified. These well-differentiated, transport-competent cell lines demonstrated the growth, immortalization, and differentiation potential of normal, adult, human proximal tubule cells and consequently have wide applicability in cell biology and renal physiology.

The migratory responses of four human melanoma cell lines (A-2058, DEMEL, HTB-63, and HTB-72), using chemotaxis (CTX) and haptotaxis (HPTX) assays, were studied. The attractants were three extracellular matrix components (EMCs), fibronectin, laminin, and collagen type IV. The conditioned media (CM) of each cell line were used to study autocrine and paracrine responses. A screening and sensitive CTX assay was performed, using pertussis toxin (PTX)– treated A-2058 as responder cells; the other melanoma cells and normal cells were used as secretory cells. Autotaxin (ATX), a purified autocrine motility factor, was also used as a chemoattractant. Reverse transcriptase–polymerase chain reaction was used to detect the expression of ATX by all cell lines. The secretion of ATX was determined by Western blot. The invasive capacity of the cell lines was evaluated using Matrigel and ATX as attractant. Chemotaxis responses to EMCs varied. Except for the A-2058 cells, HPTX migration was low. Autocrine and paracrine responses also varied. The migration of PTX-treated A-2058 cells to ATX and to their own CM was abolished. All the melanoma cells expressed ATX, and except for the HTB-72 and normal cells, all secreted ATX. Matrigel was invaded by all the melanoma cell lines except the HTB-72 and normal cells. The migratory properties of human melanoma cells in vitro suggest that they could correlate to their metastatic potential in vivo.

In a first series of experiments, the biological response of a continuous cell line of the beet armyworm, Spodoptera exigua, was tested with different groups of insecticides with different modes of actions: acetylcholinesterase inhibitors, acetycholine receptor agonists, inhibitors and uncouplers of oxidative phosphorylation, site I electron transport inhibitors, gamma-aminobutyric acid receptor inhibitors, chitin synthesis inhibitors, and juvenile hormone analogues. From the concentration response curves, 50% inhibition concentration (IC₅₀) values were calculated. The most active compound in vitro was pyridaben with an IC₅₀ value of 0.0083 ppm. In a second series of experiments, the toxicity of these insecticide groups was determined on third-instar larvae of S. exigua, and lethal concentration with 50% kill (LC₅₀) values were used in the evaluation of their in vivo biological activity. Toxicity bioassays showed that lufenuron was the most toxic (LC₅₀ = 0.098 ppm). To explain the discrepancies in biological responses in vitro with insect cells compared with in vivo conditions with whole third-instar larvae, the significance of different detoxifying enzyme systems was tested. P₄₅₀ monooxygenases, esterases, and glutathione S-transferases were measured in third-instar larvae and cells of S. exigua. Data are discussed in terms of the usefulness of insect cell cultures as tools in the screening for novel insecticide actions.

The neurotransmitter serotonin (5HT) possesses developmental functions in vertebrates and invertebrates. Rodent embryos express 5HT receptors even before neural development, but the role of this neurochemical seems to be particularly important during axonal morphogenesis and differentiation and in neural crest cell migration. Moreover, 5HT inhibitors are teratogenic in mammals, inducing brain and heart abnormalities. The aim of this study was to investigate the effects of nonphysiological concentrations of 5HT (5HT excess as well as deprivation) on developing rat neural cells using the micromass method. This simple and rapid micromass method allows the culture of mesencephalic cells capable of achieving and maintaining a significant degree of differentiation. Mesencephalic cells from 13 d post coitum (pc) rat were cultured and exposed to exogenous 5HT (1, 10, 50, or 100 μM) or to the specific 5HT2 receptor inhibitor mianserin (0.5, 5, 25, or 50 μM) during the whole culture period (5 d). The micromass morphology, the cytoskeletal organization, the pathological apoptosis, and the differentiative capability of cultured mesencephalic cells have been analyzed. The results show that 10–100 μM 5HT and 0.5–50 μM mianserin are able to disrupt the normal micromass morphology; 5HT and mianserin are unable to interfere with the cytoskeletal structures; mianserin (but not 5HT) induces pathological apoptosis on micromass cells at concentration levels of 0.5–50 μM; 5HT (but not mianserin) alters the neural differentiation at concentration levels of 10–100 μM. In conclusion, our results demonstrate that an excess of 5HT inhibits the capability of mesencephalic neurons to differentiate as shown by the alterations of the expression of the neuronal differentiative proteins glial-derived neurotrophic factor and Neu-N; on the other hand, the blocking of 5HT2 receptors induces apoptosis in differentiating neurons.

New World primate-derived cell lines were instrumental in identifying the primary factors causing glucocorticoid resistance in these primate species. Their use is expanding because it has been recognized that some of these cell lines exhibit differential sensitivity to retroviral infection. To enhance their utility as cell models, we have further characterized one of these cell lines, squirrel monkey–derived B-lymphoblast (SML) cells, using PowerBlot™. PowerBlot™ is a high-throughput, proteomic screen designed to identify differentially expressed proteins. We compared proteins expressed in SML cells and in a human B-lymphoblast (HL) cell line. We found that, relative to HL cells, SML cells overexpress the calcineurin-activated transcription factor nuclear factor of activated T cells 1 (NFAT-1), which exists in a cyclosporine A (CsA)–sensitive dephosphorylated, constitutively active state. We show that there is increased binding of NFAT-1 to deoxyribonucleic acid and greater activity of an NFAT-sensitive human interleukin-2 (IL-2) promoter–luciferase reporter gene in SML compared with activity in HL cells. The increased NFAT activity does not likely result from calcium-dependent activation of calcineurin because cytosolic calcium levels were not different in SML and HL cells. Rather, SML cells express a truncated form of the catalytic subunit of calcineurin that we propose is responsible for the increased activity of the NFAT pathway. Thus, these novel findings first uncovered by a proteomic screen will enhance the value of these New World primate cell lines as “experiments of nature” to gain insight into mechanisms of NFAT activation.