摘要:

AbstractThis paper describes the selective isolation of dedifferentiated variants from a well‐differentiated rat hepatoma cell line (Fao). The well‐differentiated cells express the gluconeogenic enzymes, FDPase and PEPCK, and so can grow in a glucose‐free medium. By using glucose‐free medium in conjunction with the BudR‐visible light suicide technique it was possible to isolate two different classes of dedifferentiated variants from a mutagenized population of Fao cells. The variants of the first class resemble those previously described in that they display (1) an altered cellular morphology, (2) a pleiotrophic loss of all (or most) of the hepatic functions routinely analyzed in this laboratory, and (3) an extremely low reversion frequency (⩽ 10−8). The variants of the second class are characterized by an unstable phenotype and uncoordinated expression of the hepatic functions. Unstable variants can give rise to stable dedifferentiated variants, suggesting that the unstable variants may actually represent an intermediate in a two‐step dedifferen

ISSN:0021-9541    

DOI:10.1002/jcp.1041110102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractIt is shown that the competence of differentiation‐defective clones of mouse myeloid leukemic cells (MGI−D−clones) to be induced for some differentiation‐associated properties can be modulated. For certain inducers this was effected by changing the type of serum and for another inducer by removing serum from the culture medium. All the clones which could not be induced to differentiate by the differentiation‐inducing from of the normal macrophage and granulocyte inducing proteins MGI (MGI‐2), lipopolysaccharide (LPS), or dexamethasone (Dex) in serum‐free medium or in medium with horse, calf, rabbit, or sheep serum (nonpermissive serum) could be induced, by all three inducers, for the differentiation‐associated properties of lysozyme and Fc and/or C3 rosettes in syngeneic or allogeneic mouse serum or rat serum (permissive serum). The competence for induction of differentiation was altered merely by washing the cells and changing the type of serum, and addition of nonpermissive serum blocked the induction in permissive serum. This increased competence was associated with an increase in the ability for cap formation by concanavalin A. Removal of serum allowed some MGI−D−clones to be induced for lysozyme, Fc, and C3 rosettes by insulin. However, neither the changes in type of serum nor the removal of serum modulated in these clones the inducing effect of the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate. In contrast to the MGI−D−clones, changing the serum type or removing the serum did not modulate inducibility by MGI‐2, LPS, Dex, or insulin in other clones of myeloid leukemic cells that could be induced by MGI‐2 to differentiate either partly (MGI+D−clones) or completely (MGI+D+clones) in horse or calf serum. The results indicate that by the appropriate modulation all MGI−D−clones tested could be induced for some differentiation‐associated properties; that there are factor(s) present in permissive sera which allow MGI−D−clones to be induced by MGI‐2, LPS, and Dex; that there are other factor(s) in nonpermissive sera which block this induction; and that in serum‐free medium cells of the appropriate clone can become susc

ISSN:0021-9541    

DOI:10.1002/jcp.1041110103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractRetinoblastoma is a rare malignant eye tumor found almost exclusively in young children. In 30% of cases, the tumor is bilateral and is inherited as an autosomal dominant trait. In such patients, all of the cells in the body must carry the mutation predisposing to retinoblastoma. To search for the expression of the gene in cells outside the retina, we have studied several in vitro properties of skin fibroblasts from patients with bilateral retinoblastoma. Measurement in low concentrations of fetal calf serum of the initial growth rate and the plating efficiency show that fibroblasts from retinoblastoma donors grow significantly better than those from normal donors. However, we were unable to confirm the results of other investigators that fibroblasts from donors with bilateral retinoblastoma are unusually sensitive to ionizing radiation. In family studies, skin fibroblasts from normal siblings had the same radiation sensitivity as fibroblasts from sibling with retinoblastoma.

ISSN:0021-9541    

DOI:10.1002/jcp.1041110104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractModulation of epithelial cell proliferation by the dissolved oxygen concentration (PO2) of the growth medium was assessed with primary human foreskin epithelium and a continuous monkey kidney epithelial cell line (LLC‐MK2). Direct measurement of the growth medium PO2provides the first quantitative evaluation of epithelial cell proliferation as a function of PO2provides the first quantitative evaluation of epithelial cell proliferation as a function of PO2. Sustained proliferation of LLC‐MK2cells occurs in serum‐free medium equilibrated with a gas phase containing 18% or 30% O2v/v. Mid‐logarithmic phase cultures rapidly consume dissolved oxygen; this results in a 60–70 mm Hg decline in PO2and leads to a stable growth medium PO2between 70 and 100 mm Hg, well above anoxic values. In contrast, if culture medium is equilibrated with a gas phase containing 0% or 1% O2v/v to yield a growth medium PO2∼ 20–40 mm Hg, proliferation of LLC‐MK2and primary foreskin epithelial cells is retarded, and LLC‐MK2cells use little dissolved oxygen. Gentle, continuous rocking to prevent diffusion gradient formation enhances proliferation slightly at the higher PO2, but neither periodic fluid renewals nor continued rocking stimulates cells retarded by a lowered oxygen concentration to resume proliferation. The data collectively demonstrate that epithelial cell proliferation requires a PO2>40 mm Hg, and threshold requirements are probably closer to 70 mm Hg. Glycolysis continues at a PO2insufficient for proliferation, but more lactic acid accumulates in actively proliferating cultures than in cultures equilibrated with 0% oxygen. We conclude that epithelial cells in vitro both consume more oxygen and require a higher PO2for continued proliferation, and that the oxygen requirement for epithelial cell proliferation exceeds that of a comparable population of fibroblasts for which low oxygen may enhance survival

ISSN:0021-9541    

DOI:10.1002/jcp.1041110105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractPyrimidine excretion by macrophages was studied in order to identify the potential immunoregulatory effector molecules. Deoxycytidine was found in the culture medium of thioglycollate‐elicited mouse peritoneal macrophages, along with thymidine, which was shown by others to be a possible immunoregulatory substance. The identification of deoxycytidine was based on: (1) cochromatography with the authentic compound in four different solvents, (2) UV absorption spectral analysis, and (3) the enzymatic peak shift method. Phagocytosis of nucleated chicken erythrocytes, but not enucleated sheep erythrocytes, increased deoxycytidine excretion. The macrophages lacked both deoxycytidine kinase and deoxycytidine deaminase, which is consistent with their excretory pattern. Since it has been known that deoxycytidine can protect cells against cytotoxic effects of thymidine, we propose that deoxycytidine has a role in preventing immunosuppression by thimidine. In patients with adenosine deaminase deficiency, however, immunosuppression caused by combined toxicity of thymidine and deoxyadenosine may not be adequately prevented by deoxycytidin

ISSN:0021-9541    

DOI:10.1002/jcp.1041110106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractIn the present study, the possible association of thymidine kinase (TK) with mitochondria inNaegleriawas investigated by treating growing and differentiating cells with chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis. In some systems, CAP causes an overproduction of mitochondrial proteins coded for in the nucleus. The present results show that in growingNaegleria, CAP stimulates a dramatic increase in TK activity while growth and division is gradually inhibited. CAP does not stabilize the enzyme in vivo or in vitro. The stimulation is cycloheximide (CHI)‐sensitive and specific since nucleoside phosphotransferase activity does not increase. In cells stimulated to differentiate, CAP does not prevent differentiation or the expected decrease in TK activity. Using polyacrylamide gel electrophoresis, a comparison of TK in mitochondrial and postmitochondrial fractions of CAP‐treated and untreated cells was made. Results suggest some processing of the enzyme, resulting in a slight change in electrophoretic mobility. No mitochondrial TK was found. The stimulation of a cytoplasmic enzyme by CAP suggests a form of mitochondrial control of nuclear transcription for other than mitochondrial proteins. DNA synthesis in CAP‐treated cells was not stimulated, suggesting (since TK and DNA synthesis are usually tightly coupled) an uncoupling of these two events, most likely, at the beginning of the S

ISSN:0021-9541    

DOI:10.1002/jcp.1041110107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractA nontransformed line of Chinese hamster ovary (CHO) cells (Pollard and Stanners, 1979) has been transformed by the Schmidt‐Ruppin subgroup D strain of Rous sarcoma virus (SR‐RSV). SR‐RSV transformed CHO cells are shown to differ from spontaneously transformed cells in that the virally transformed cells are more resistant to growth inhibition or changes in cell shape by 8‐Br‐cyclic AMP or cholera toxin. SR‐RSV transformed rat (NRK) cells also have a reduced sensitivity to growth inhibition by 8‐Br‐cyclic AMP. Cyclic AMP‐dependent protein kinase was examined in SR‐RSV transformed CHO cells, but no differences in enzyme level, activation by cyclic AMP, chromatographic behavior, or its ability to phosphorylate endogenous proteins in whole cells could be detected. It is concluded that transformation of CHO and NRK cells by SR‐RSV alters the cells in a manner different from spontaneous transformation, and that this alteration does not affect cAMP‐dependent

ISSN:0021-9541    

DOI:10.1002/jcp.1041110108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe myeloperoxidase‐mediated oxidation of methionine was studied using a purified canine myeloperoxidase preparation. The system required the simultaneous presence of myeloperoxidase, H2O2, and a halide anion. When 0.1 mM H2O2was used, the system has a pH optimum of approximately pH 5–5.5. Bromide and iodide were much more effective than chloride in the myeloperoxidase‐mediated oxidation of methionine. Horseradish peroxidase was unable to oxidize methionine whether chloride or iodide was used. In contrast, lactoperoxidase oxidized methionine in the presence of iodide but not chloride.Based on studies of (1) the effect of various inhibitors and singlet oxygen quenchers, as well as (2) the effect of D2O on the oxidation of methionine, by the myeloperoxidase system, OCl−, or methylene blue, it was shown that the oxidation of methionine by the myeloperoxidase system was not mediated by OCl−or1O2. The mechanism of the myeloperoxidase‐mediated oxidation of methionine remains unclear. However, it may be one mechanism by which the myeloperoxidase system damage mic

ISSN:0021-9541    

DOI:10.1002/jcp.1041110109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractVibration of hamster small intestinal segments in hypotonic media containing PVP is a rapid method for obtaining quantitative yields of viable intestinal epithelial cells. This preparation of epithelial cells offers a unique system for the study of epithelial cell function in vitro. The method for cell separation combines hypoosmotic swelling of cells, which separates them at the desmosomes, with mechanical agitation which releases the cells from the lamina propria. No chemical agents known to affect cell proteins and cell surfaces are employed in this procedure. Only a short time is elapsed between in vivo and in vitro conditions, i.e., a preparation time of approximately 75 minutes.Although the technique yields a pure population of epithelial cells, the cells are of different morphologies, are removed from different areas of the crypts and villi, and therefore presumably have different functions. Examination of the intestinal tissue remaining after several vibration intervals by light and scanning electron microscopy indicates that the sequences of release of cells is removal of: (1) cells from the villus based, (2) cells from the lower one‐half to two‐thirds of the villi, (3) cells from the villus tips (and some crypts), and (4) cells from the crypts. When pools of a + b cells are compared to pools of c + d cells, it is found that villus cells can be characterized by: (1) processes, such as monosaccharide absorption, associated with the brush border, and (2) synthesis of components (e. g., glycoproteins) of the brush border. Surprisingly, disaccharide hydrolytic activity is found in cells which transport monosaccharides poorly. The subpopulations of cells synthesize proteins equa

ISSN:0021-9541    

DOI:10.1002/jcp.1041110110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractIsolated hamster intestinal epithelial cells can be separated by velocity sedimentationion on 2–10% Ficoll gradients into three subpopulations of cells which differ in morphology, biochemistry, physiology, and membrane components. These subpopulations are not pure but are enriched in a single cell type to the extent that differences in cell function can be observed. The proliferative crypt cells are separated from the digestive‐absorptive villus cells. A third subpopulation with a distinctive morphology is also obtained. Quantitation of DNA recoveries from the gradients indicates that this population constitutes approximately one‐third of the epithelial cell population. These carrot‐shaped cells are found adjacent to the digestive‐absorptive columnar epithelial cells on the villus. The two types of villus cells differ in glycolipid or glycoprotein components of the brush border as shown by lectin binding experiments with the isolated cells. The gradient data also suggest that only one‐third of the intestinal epithelial cell population is responsible for most monosaccharide absorption in hamster smal

ISSN:0021-9541    

DOI:10.1002/jcp.1041110111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY