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81.
Mercuric compound toxicity is well documented in animals and man for practically all organs. The recent development of cell culture techniques appeared as a novel fruitful tool in toxicology, especially in renal toxicology. Heavy metal induced renal cell alterations can be evaluated by membrane permeability damages.The present study evaluates mercuric chloride nephrotoxic effect in human kidney epithelial cells by measuring the release of two specific nephrotoxicity marker enzymes, Gamma Glutamyl Transferase (GGT) and Alkaline Phosphatase (ALP) in the culture medium. Cultured kidney epithelial cells were exposed to different HgCl2 concentrations (5, 10, 20, and 50 g). Cultures were examined after 6 and 24 hours exposure. A good correlation between mercury dose and toxic effect, and exposure time and toxic effect was found. Enzymes were significantly released into the culture medium for 5 g and 10 g HgCl2/ml after 6 hours exposure; and after 24 hours exposure, enzymes were released for 5 g/ml only.It appears that the specific tubular enzyme release in the culture medium is a good in vitro test for quantification of specific tubular damage. 相似文献
82.
Terry L. Riss Kenneth P. Karey B. Daniel Burleigh David Farker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1099-1106
Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant
insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on
poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's
modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin,
100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free
growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold
more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The
concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like
growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin,
ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth
factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
83.
Cyprian Weaver Robert L. Sorenson Brian Kobienia 《In vitro cellular & developmental biology. Plant》1988,24(2):108-116
Summary The purpose of this study was to develop a nonenzymatic method of isolating adult islets using atrophied pancreata from copper-deficient
rats and to analyze their morphologic characteristics and behavior in culture. This unusual model of isolation was studied
because islets remain intact in the course of dietary copper deficiency while the acinar glandular component of the pancreas
undergoes selective atrophy and lipomatosis. Small fragments containing islets were readily microdissected from atrophied
glands and placed in culture. Within 24 h the fragments congealed into small irregular- to spherical-shaped masses within
which the darker profile of islets could be distinguished. Within a period of 3 to 5 d, islet tissue began to bud from the
lipocytic mass until by Day 7 spherical aggregates of intact islet tissue separated from the residual fragments. Subsequent
to further in vitro treatment, these islets could be maintained as free viable spherical masses if periodically agitated,
as attached stationary islets which developed monolayer growth if left undisturbed and as aggregated masses of islet tissue
forming megaislets if combined in small groups. Grouped islets treated with actinomycin D and cycloheximide did not exhibit
aggregation when incubated with these inhibitors. This suggests that megaislet formation was an active process requiring protein-RNA
synthesis rather than passive clumping or aggregation that can accompany metabolically altered or dying islets undergoing
cellular shedding and adhesion. Immunohistochemical localization demonstrated that insulin, glucagon, somatostatin, and pancreatic
polypeptide-immunoreactive cell types were present within the islets derived from this technique. The cellular topography
of these islets was not unlike that described by others for islets cultured from enzymatic isolation. This culture model may
serve as a resource for mature, viable islets isolated without mechanical or enzymatic disaggregation which can have attenuating
effects on islet function.
This work was supported by a research grant from the Diabetes Research and Education Foundation. 相似文献
84.
John F. Foley Byron Th. Aftonomos 《In vitro cellular & developmental biology. Plant》1988,24(9):900-904
Summary Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described
as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to
the growth medium. We have observed that for a particular medium or serum system, the percentage of compact colonies remains
fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider
range. The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies
at the expense of other types. Furthermore, we have discovered that colonial morphology is influenced by cocultivation of
the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced
into the mixed cultures is increased. Subsequent investigation revealed that conditioned medium from confluent fibroblast
and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies. The factor
is nondialyzable, heat-stable and can be neutralized by serum. Recorded in this presentation are preliminary observations
on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate,
and the compact. The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s)
is removed and fresh medium added. 相似文献
85.
Enterochromaffin-like cells in the rat stomach: effect of α-fluoromethylhistidine-evoked histamine depletion 总被引:2,自引:0,他引:2
Kjell Andersson Duan Chen Rolf Håkanson Hillevi Mattsson Frank Sundler 《Cell and tissue research》1992,270(1):7-13
Summary In the rat, gastric histamine is stored predominantly in the enterochromaffin-like (ECL) cells, which are located basally in the oxyntic mucosa. The functional significance of histamine in the ECL cells is a matter of speculation. In this study the effect of depletion of histamine on the properties and ultrastructure of the ECL cells was examined. Histamine synthesis was inhibited with -fluoromethylhistidine (3 mg·kg-1·h-1) given via osmotic minipumps over a period of 24 h. The treatment reduced the histidine decarboxylase activity (approximately 20% remaining) and histamine concentration (less than 20% remaining) in the oxyntic mucosa, as well as the intensity of histamine- and chromogranin A-immunostaining in the ECL cells, compared to control rats. The cytoplasmic (secretory) granules/vesicles were greatly reduced in number and size following -fluoromethylhistidine administration. The histamine immunostaining of the mast cells, which occurs at the mucosal surface and in the submucosa, appeared unaffected. We conclude that ECL cell histamine accounts for at least 80% of the total oxyntic mucosal histamine in the rat and that it represents a more mobile pool than mast cell histamine. The reduction in the number and size of the ECL cell granules/vesicles following histamine depletion is in accord with the idea that they represent the storage site for histamine. 相似文献
86.
Summary Neuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.On leave from Department of Zoology, Attlia József University, Szeged, Hungary 相似文献
87.
M. Apiranthitou-Drogari C. Paganin S. Bernasconi G. Losa A. Maneo N. Colombo A. Mantovani P. Allavena 《Cancer immunology, immunotherapy : CII》1992,35(4):289-295
Summary Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized. 相似文献
88.
Pancreatic adenocarcinomas induced in Syrian hamsters by treatment with N-nitrosobis(2-oxopropyl) amine express blood group A antigen, which is absent in normal pancreatic cells. On membrane glycoproteins purified from tumors, blood group A antigen has been found to be expressed on multiantennary Asn-linked complex glycans. In this study, we investigated the effect of inhibitors of Asn-glycan processing on blood group A antigen bearing glycan structures in a cell line (PC-1) established from a primary induced pancreatic cancer. Expression of blood group A antigen on cells and in membrane preparations was blocked by treatment with 1-deoxymannojirimycin, an inhibitor of mannosidase I, but was retained after treatment with swainsonine, an inhibitor of mannosidase II. However, swainsonine treatment altered the glycan structure associated with blood group A antigen from an endoglycosidase H resistant type to a sensitive type, indicating that the blood group A structure might shift from a complex type to a hybrid type glycan by this treatment. These results demonstrate that Asn-linked glycans carry the major blood group A antigens in PC-1 cells. 相似文献
89.
G Milazzo G L La Rosa R Catalfamo R Vigneri A Belfiore 《Journal of cellular biochemistry》1992,49(3):231-238
The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
90.
It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA 相似文献