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A new model system is delineated that will enable study of CD4 cofactors and gp120 binding proteins other than CD4. We have previously described a nontransformed rat fibroblast cell line that can efficiently produce HIV-1 upon transfection with an HIV-1 infectious clone, in contrast to other nonhuman mammalian cell lines tested. In the present study we analyzed the susceptibility to HIV-1 infection of Rat2 cells expressing the human CD4 protein. We have used the mammalian expression vector pKS286, in which HIV-1 LTR drives the expression of the CD4 gene. We show that the Rat2 cell line, expressing the human CD4 (Rat2/CD4), is susceptible to fusion with and infection by HIV-1. The virus produced by the Rat2/CD4 cells was infectious. CD4 expression in the Rat2/CD4 was down-regulated over time, similarly to HIV-1 expression in HIV-1-transfected Rat2 cells. Transfection of the Rat2/CD4 cells with a tat expression vector reestablished the CD4 expression on the surface of those cells, as expected. We conclude that the expression of a gp120 binding protein on the Rat2 cells' surface suffices to render the Rat2 cells susceptible to HIV-1 infection.

Bcl-2 and Bax are members of a family of cytoplasmic proteins that regulate apoptosis. The two proteins have highly similar amino acid sequences but are functionally opposed: Bcl-2 acts to inhibit apoptosis, whereas Bax counteracts this effect. The antagonism appears to depend upon dimerization between Bcl-2 and Bax, but its mechanism is otherwise unknown. Here we report that overexpressing Bax induces apoptosis in a mammalian fibroblast cell line, and we identify a novel, short “suicide domain” in Bax that is required for this effect. Inserting this domain in place of the corresponding, divergent sequence in Bcl-2 converts Bcl-2 from an inhibitor into an activator of cell death. These findings imply that a specific region in Bax confers an active propensity for apoptosis in mammalian cells and support the view that Bcl-2 may block death primarily by suppressing Bax activity.

8-hydroxyguanine (8-OH-Gua) is a major oxidative DNA damage product. Its repair activity was determined in the human fibroblast cell line, TIG-3, during the aging process. The repair activity increased in the first part of the aging process and decreased in the latter part. The highest repair activity was observed at the age of 30 PDL. These results suggest that the defense capacity for replication errors due to oxidative DNA damage increases greatly when the cells are rapidly dividing.

International audience; pAntp, a 60 amino acid long peptide corresponding to the homeodomain of the Drosophila Antennapedia protein, translocates through neuronal membranes when added exogenously to neurons in culture, where it accumulates in the nucleus and promotes neurite outgrowth. We proposed that the peptide, once internalized, may compete for homeoprotein DNA binding sites. To investigate this point, we have produced a permanent fibroblast cell line which carries a luciferase reporter gene under the control of a 93 bp genomic region of the HOXD9 promoter with binding sites for homeoproteins. Externally added pAntp specifically down‐regulates the expression of the reporter gene, suggesting that the neurotrophic effects observed previously are mediated by direct binding of pAntp to homeoprotein target sites

The therapeutic effects of Taxus baccata for its anti-carcinogenic properties were explored. The study included the in vitro, cytotoxic, cytopathic effect and phytochemical analysis of aqueous, ethanolic, methanolic and ether extract of plant T. baccata on BHK-21. In vitro cytopathic and cytotoxic effects were determined for leaves and bark of T. baccata on BHK-21 fibroblast cell line. Highest cytotoxicity was found at 10 mg/ml extract and its combination with cow urine distillate (CUD) gives much better results. The viable cells in blank were 95.2 ± 5.66, which were reduced to 55.64±4.67 and 57.32±4.76 when treated with aqueous and ethanolic extract of leaves. These two are better than methanolic and ether extract of leaves alone and in combination with CUD. However, in case of bark, methanolic and ether extarcts gives better results, that is, 60.22±4.82 and 64±4.95, respectively than its aqueous and ethanolic extract. Methanolic and ether extract extract give much better result when used alone and in combination with CUD than aqueous and ethanolic extract of bark.

The mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites was investigated in the reverse mutation assay using S. typhimurium strains and the chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line, CHL. BHA, tert-butylhydroquinone (BHQ), tert-butylquinone (BQ) and BHA dimer (diBHA) did not show any mutagenic potential with and without S9 mix in the reverse mutation assay. In addition to the above 4 chemicals, 3-tert-butyl-4,5-dihydroxyanisole (BHA-OH), 3-tert-butylanisole-4,5-quinone (BHA-o-Q), and tert-butylquinone oxide (BQO) were tested in the chromosomal aberration test. BHA, BHQ and BQ induced chromosomal aberrations only in the presence of S9 mix, while BHA-OH, BHA-o-Q and BQO induced chromosomal aberrations only without S9 mix. DiBHA, however, showed no clastogenic potential with and without S9 mix. The present findings suggest that BHA-OH, BHA-o-Q or BQO may contribute to the clastogenicity of BHA in the presence of S9 mix.

We present here a historical documentation of a female with X-linked hypohidrotic ectodermal dysplasia (XHED) and a de novo X/9 chromosome translocation. The patient was verbally reported by Dr. P.L. J. Cook to the HGM conference in 1973, but was subsequently lost to follow up. We have since traced her and confirmed the diagnosis of XHED with moderately severe mental retardation. According to Dr. P. L. J. Cook's records, fibroblast cell line AnLy GMO 705, was derived from this patient. Another female with a de novo X/12 chromosome translocation and hypohidrotic ectodermal dysplasia was recently reported. In both cases, the X chromosome breakpoint appears to be at Xq13.1.

Apoptosis involves a highly organized and programmed series of events aimed at maintaining genomic stability by eliminating defective host cells. The purpose of this study was to determine the threshold doses and environmental UV-A and UV-B exposure times necessary to produce apoptosis and necrosis in the normal cells of a human fibroblast cell line. Environmental UV-A and UV-B doses were measured over a 6 year period with a four-channel UV radiometer. The fibroblasts were irradiated once using an Oriel UV Solar Simulator with six doses of environmentally-based UV. Doses corresponded to 0, 11, 19, 23 and 45 min of average environmental UV-A and UV-B radiation at solar noon in Puerto Rico. The Annexin-V binding method was used to differentiate between normal fibroblasts and apoptotic or necrotic fibroblasts. The threshold dose from apoptosis to necrosis was found between 24–28 kJ/m2, which corresponded to 19 and 23 min of environmental UV-A and UV-B exposure. This study provides the first data tha...

The effects of growth factors extracted from a newly established fetal lung fibroblast cell line (PMR-GF) on the melanocytes cultured from the perilesional and depigmented skins of vitiligo subjects and from normal healthy donors have been investigated. Melanocytes from normal subjects grown in the presence of 10 ng/ml of 12-0-tetradecanoyl phorbol 13-acetate and 10 -11 M cholera toxin grew exponentially immediately after seeding the epidermal cell suspensions. Exogenous addition of PMR-GF to these cells enhanced their growth rates. The perilesional skin melanocytes of vitiligo subjects in most cases did not manifest any growth when cultured in the presence of 12-0-tetradecanoyl phorbol 13-acetate and cholera toxin. PMR-GF induced a brief burst of growth in these cells after a lag of 15 days. Vitiligo lesions gave rise to a few unpigmented dendritic cells that did not manifest any growth in the presence or absence of PMR-GF. Morphologically the perilesional skin melanocytes of most vitiligo subjects, when cultured in 12-0-tetradecanoyl phorbol 13-acetate and cholera toxin, appeared to be larger and hyper-melanotic as compared to those of normal individuals. In the presence of PMR-GF these melanocytes appeared to be normal in size and less hyper- melanotic. Our results indicate that the melanocytes from vitiligo subjects are defective and thus the basic defect in vitiligo could be with the melanocytes themselves.

A method is described for the isolation and purification of trypomastigotes and amastigotes of Trypanosoma cruzi from cell cultures. L-A9, a transformed fibroblast cell line, and J774G8, a macrophage-like cell line of tumor origin, were used. Both cell lines were infected with bloodstream trypomastigotes of T. cruzi, which once within host cells transform into dividing amastigotes. After 6--8 days infection the host cells ruptured, spontaneously liberating parasites into the culture medium. L-A9 cells liberated mainly trypomastigotes while J774G8 cells liberated amastigotes. The parasites were collected and purified by centrifugation in a gradient of metrizamide. The purity of the preparation as well as the morphology of the parasites and the host cells were analysed by electron microscopy.

Cultures of the rainbow trout fibroblast cell line RTG-2 withstood temperatures from 0 degrees C to 28 degrees C. At 0 degrees C and 28 degrees C, no proliferation occurred, but cells persisted for at least 7 days. If the cultures were placed back at 22 degrees C, proliferation returned to normal in those that had been kept at 0 degrees C but was reduced in cultures that had been kept at 28 degrees C. Above 28 degrees C, cultures survived for only short periods. If RTG-2 cells that were grown routinely at 22 degrees C were shifted to 26, 28, and 30 degrees C, heat shock proteins (hsps) of 100, 87, 70, 68, 60, 39, 27, and 19 kilodaltons were synthesized. Synthesis was most pronounced at 28 degrees C, and at this temperature hsp synthesis was maximal by 2 hr and had returned to control levels by 36 hr. Individual hsps were synthesized maximally at slightly different times and temperatures, but under all conditions hsps 87 and 70 were most abundant. If cultures were shifted to 24 degrees C or 32 degrees C, hsp synthesis was not observed. Neither the placement of cultures at 5 degrees C nor the shift of cultures that had been maintained at 0 degrees C or 5 degrees C back to 22 degrees C induced the synthesis of hsps. However, cultures incubated at 5 degrees C for 24 hr did synthesize hsps at 26 degrees C, 28 degrees C, and 30 degrees C.

The effects of 1,6-dinitropyrene (1,6-DNP) on the fidelity of cell division were studied in the transformed human fibroblast cell line MRC5VA. Over a dose range of 0.1–10 μg/ml of 1,6-DNP, we observed significant increases in the levels of abnormal division stages, associated with damage to the spindle apparatus of the cell. Qualitative changes in spindle morphology and a quantitative decrease in pole-to-pole spindle length were also observed with increasing doses of 1,6-DNP. Such changes in the size and morphology of the spindle corresponded with an accumulation of cells blocked at metaphase. The presence of catalse did not modify the response, suggesting that the effects on the spindle apparatus and cell division were not caused by the generation of radicals but by the direct action of 1,6-DNP.

A number of methods for determining the general toxic effects of test chemicals on cells in culture are now at the validation stage. Three such methods based on measurement of total cell protein or neutral red uptake and on the detection of morphological effects have been compared. The cell line used was 3T3-L1 (a continuous fibroblast cell line derived from mouse embryos). The results obtained in a blind trial with 30 coded chemicals indicated a close correlation between the relative cytotoxicities of chemicals tested by all three methods.

— This study was designed to determine whether UV-induced tumors have a selective growth advantage in the autochthonous host by virtue of possessing a heritable resistance to UV-induced lethality. Several fibrosarcomas were induced either by repeated exposure of C3H mice to UV radiation from FS40 sunlamps or by subcutaneous injection of C3H mice with a chemical carcinogen (methylcholanthrene). Tissue culture lines of these tumors were tested in vitro for susceptibility to the lethal effects of UV radiation from an FS40 sunlamp. Lethality was assessed by measuring colony formation as a function of increasing dose of radiation. Cells from the UV-induced fibrosarcomas were not more resistant to the lethal effects of UV radiation than cells from methylcholanthrene-induced fibrosarcomas or cells from a nontumorigenic C3H fibroblast cell line. This suggests that UV-induced tumors do not arise from a subpopulation of UV-resistant cells.

The characteristics of the cytoskeleton of a Fischer rat embryo fibroblast cell line (3Y1) transformed by ultraviolet (UV)-irradiated HSV were studied by indirect immunofluorescence using anti-actin IgG. Parental 3Y1 cells possessed well-developed actin filaments, while 3Y1 cells transformed by UV-irradiated HSV also retained well-developed actin filaments. Transformed cells were divided into 2 groups according to tumorigenicity in newborn Fischer rats; one had a strongly tumorigenic potential and the other a weakly tumorigenic potential. Tumor-derived cell lines exhibited a highly tumorigenic potential, and were also divided into 2 groups, one with well-developed actin filaments and the other without well-developed actin filaments. Our results suggested that transformation or tumor formation by HSV is a multi-step process and that morphological loss of actin filaments in the cells is not essential to the tumorigenic potential of the cells transformed by HSV.

Effects of two tumor promoters, 12- O -tetradecanoyl phorbol-13-acetate (TPA) and p,p′ -dichlorodiphenyltrichloroethane (DDT), on cellular calcium regulatory mechanisms were studied by using the Chinese hamster V79 fibroblast cell line. Both compounds at concentrations known to inhibit cell-cell communication, increased the cellular 45 Ca 2+ uptake and inhibited its efflux. DDT inhibited Ca,Mg-ATPase of the plasma membrane, while TPA stimulated cellular uptake of Ca 2+ . A working hypothesis has been proposed that the increase in intracellular concentration of Ca 2+ by the action of these chemicals directly results in the closure of the central opening of the gap junction, and thereby causing a cessation of gap junction-mediated intercellular communication.

Summary. Aluminium release was investigated from a range of dental materials including several composed of aluminosilicate glass; with the exception of ASPA and Silicap, the release was negligible. The concentrations of aluminium released from these two materials were neither toxic to a fibroblast cell line nor to mouse peritoneal macrophages. Levels in the order of 40ppm were required to exert a toxic effect.

Although instrumentation for work with microbial cells is highly developed, the use of instrumentation for animal cell culture leaves much to be desired. This lack of progress was mainly due to the surface requirement of normal diploid cells. However, with the development of microcarriers, cells can be grown in suspension while obtaining high cell densities. The experimental system to be discussed utilizes a microcomputer for data acquisition and analysis. This computer is interfaced with sensors for the measurement of dissolved oxygen tension, pH, temperature, rpm, and conductance. These data are collected and analyzed for assessment of various metabolic activities as well as rate of cell growth. The system is presently used to monitor the growth and metabolism of a human fibroblast cell line (FS-4), which is also an interferon producer.

Thermotolerance was studied in the rainbow trout fibroblast cell line RTG-2. RTG-2 cultures that had been incubated at 28 degrees C for 24 h were better able to withstand ultimately lethal temperatures above 28 degrees C than RTG-2 cultures that had been maintained at the routine growth temperature of 22 degrees C. This thermotolerance developed rapidly between 3 and 6 h and was fully developed by 24 h at 28 degrees C. After development for 24 hr at 28 degrees C, thermotolerance showed little change over 72 h at 0 and 5 degrees C but approximately a 40 and 60% reduction at 10 and 22 degrees C, respectively. This is the first demonstration of heat-induced thermal resistance in the cells of a poikilothermic vertebrate.

SummaryThe sensitivity of adenovirus Types 2, 7, and 12 to moderate amounts of human tissue culture interferon was demonstrated in plaque-reduction assays using primary and secondary HEK cell cultures. The findings for Ad. Type-2 were extended to another species using rabbit serum interferon in yield-inhibition assays done in an established rabbit heart fibroblast cell line. These results indicate that at least some members of the adenovirus group are indeed susceptible to interferon, although to a clearly lesser extent than are many other viruses. The implications of these findings for the possible use of interferon in examining in vitro the mechanisms of viral oncogenesis are considered.

Background To evaluate and compare the effects of ethanolic extracts of Malaysian propolis and Brazilian red propolis at different concentrations on the migration and proliferation of fibroblast cells. Methods Malaysian and Brazilian red propolis crude samples were extracted using ethanol. Their wound healing effects were tested in vitro on the normal human fibroblast cell line CRL-7522. Cell migration and proliferation assays were carried out using propolis concentrations of 1, 10, 100, 250, 500 and 1000 μg/mL. The data were analyzed using one-way ANOVA and post hoc Bonferroni tests (α = 0.05). Results Malaysian and Brazilian red propolis followed a concentration-dependent increasing and decreasing trend. Malaysian propolis showed the fastest migration rate at 250 μg/mL which was statistically significant (p 0.05) compared to control. Brazilian red propolis showed a slight increase in migration and proliferation at 10 and 100 μg/mL, respectively with no significant difference (p > 0.05) compared to control, while concentrations above these conferred inhibitory effects. Conclusion Malaysian and Brazilian red propolis show potential to assist in wound healing, depending on their concentration.

SUMMARY Reticuloendotheliosis virus (REV) strains T and CS consistently induced focus formation in a quail fibroblast cell line designated QT35 that was maintained under agarose overlay following inoculation of REV. The foci were more distinct and better circumscribed than those that developed in QT35 cultures under fluid medium. The focus formation in QT35 cultures under agarose overlay was inhibited by REV antiserum, and there were a highly significant correlation (r = 0.966, P < 0.005) and a linear relationship between the number of foci that developed and relative virus concentration inoculated. This provides a valid focus assay for REVs. RESUMEN