Fluctuations in the production of specific cellular peptides during the growth of animal cells.

Lee, G T; Engelhardt, Dean

doi:10.1083/jcb.78.2.r28

Cited by 11 publications

(6 citation statements)

References 14 publications

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“…Spot intensities on two gels containing two different samples exposed for the same period of time (72 h) were compared, and the obvious differences in the intensity of spots were observed visually. To confirm the results obtained by visualization of gels, we exposed two gels containing two different samples for different periods of time by analyzing the length of exposure required before a peptide spot became visible, as previously described (15). The spots differing in intensity according to the first technique were found to be the same and to differ by at least 8-fold by the second technique.…”

Section: Double Gel Electrophoresismentioning

confidence: 87%

“…The synthesis of these 14 new peptides may reflect a direct effect of the growth factors on the pattern of newly synthesized proteins once cultures become confluent. Previous studies done with bovine vascular endothelial cells (22) and Vero cells (15) have indicated that cells will synthesize more peptides in their logarithmic growth phase than when confluent and resting. In contrast, the bovine granulosa cells derived from smallsized follicles synthesized more peptides at confluence than when actively growing.…”

Section: Discussionmentioning

confidence: 99%

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Patterns of Cellular Peptide Synthesis by Cultured Bovine Granulosa Cells*

Savion

Gospodarowicz

1980

Endocrinology

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The electrophoretic distribution of the polypeptides synthesized by bovine granulosa cell cultures after metabolic labeling with [35S]methionine has been analyzed by double gel electrophoresis. The fluctuations of 35 polypeptides have been followed as a function of the size of the follicles from which cultures originated, as a function of the cultures' proliferative stage (sparce, actively growing vs. confluent, resting cultures), and finally as a function of whether cells were exposed to either epidermal or fibroblast growth factor. When the patterns of protein synthesis in sparce vs. confluent granulosa cell cultures derived from small-sized follicles were compared, only a few differences were observed. In confluent cultures, 6 new peptides appeared, while 1 peptide present in sparce cultures disappeared. Cultures maintained in the presence of fibroblast or epidermal growth factor synthesized 20 new peptides upon reaching confluence. Among these were the 6 new peptides present in confluent but not in sparse granulosa cell cultures maintained in the absence of growth factors. The changes in protein synthesis observed in cultures grown in the presence of growth factors may reflect their direct effect on the cellular metabolism. A comparison between the protein distribution in cells derived from small- vs. large-sized follicles showed that fewer proteins were ultimately produced at confluence in cells derived from large-size follicles than in cells derived from small-sized follicles. This could be related to the process of cellular differentiation taking place within granulosa cells. The patterns of [35S]methionine-labeled proteins secreted by the granulosa cells into the incubation medium were analyzed and found to be similar regardless of the size of the follicle from which the culture originated. Little similarity between the proteins present in the follicular fluid and the pattern of the labeled proteins secreted into the incubation medium by cultured granulosa cells was observed. Only three proteins were identified which comigrate with proteins present in the follicular fluid. One of these was identified as fibronectin. This raises the possibility that the fibronectin present in the follicular fluid originated from granulosa cells and is not derived from plasma.

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Section: Double Gel Electrophoresismentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Patterns of Cellular Peptide Synthesis by Cultured Bovine Granulosa Cells*

Savion

Gospodarowicz

1980

Endocrinology

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“…After this time, the labeled medium was removed and the cells were rinsed three times with 2 ml of ice cold phosphate buffered saline (PBS). After the last wash, the cells were solubilized by the addition of 0.3 ml of lysis buffer (9.5 M urea, 2% w/v NP 40,1.6% ampholines, pH 3.5-10, and 5% p-mercaptoethanol) to the culture plates (Lee and Engelhardt, 1978). The lysate was then used directly for two-dimensional polyacrylamide gel electrophoresis, using the procedure described by O'Farrell(1975) as modified by Lee and Engelhardt (1978).…”

Section: Chemicalsmentioning

confidence: 99%

“…After the last wash, the cells were solubilized by the addition of 0.3 ml of lysis buffer (9.5 M urea, 2% w/v NP 40,1.6% ampholines, pH 3.5-10, and 5% p-mercaptoethanol) to the culture plates (Lee and Engelhardt, 1978). The lysate was then used directly for two-dimensional polyacrylamide gel electrophoresis, using the procedure described by O'Farrell(1975) as modified by Lee and Engelhardt (1978). Ampholines, pH 5-8 and pH 3.5-10, were used for isoelectric focusing in the first dimension and 10% polyacrylamide gels were used in the second dimension.…”

Section: Chemicalsmentioning

confidence: 99%

Specific protein production during melanogenesis in B16/C3 melanoma cells

Laskin

Piccinini

Engelhardt

et al. 1983

Journal Cellular Physiology

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The mouse melanoma cell line B16/C3 offers an excellent in vitro model for studying melanocyte differentiation. Melanogenesis can be induced by serum, a hormone-supplemented serum-free medium, melanocyte stimulating hormone, and dibutyryl cAMP. The tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, 5-bromodeoxyuridine, and acidic pH inhibit this process. Using two-dimensional polyacrylamide gel electrophoresis, we have identified four cellular proteins whose production is modulated during melanogenesis, a process which includes concomitant increases in levels of tyrosinase, the rate limiting enzyme for melanin biosynthesis, melanization, and ultimately, cell death. The production of these proteins are coordinately expressed or inhibited in response to the diverse inducers and inhibitors of melanogenesis. We conclude from these studies that these specific proteins are intimately involved in the differentiation of B16/C3 melanoma cells.

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“…This system separates polypeptides in the first dimension by charge and in the second dimension by subunit molecular size. It can resolve several hundred polypeptides from proteins present in biological samples and has been used by many workers to study complex cellular phenomena, such as changes in polypeptide synthesis during cell growth (Lee and Engelhardt, 1978) or following various events, such as neoplastic transformation (Hirsch et aL, 1978;Takami and Busch, 1979), viral transformation (Strand and August, 1977), hormonal treatment (Ivarie and O'Farrell, 1978;Garrels and Schubert, 1979) and cell differentiation (Vermorken and Bloemendal, 1978).…”

mentioning

confidence: 99%

Two‐dimensional polyacrylamide gel analysis of fibroblast polypeptides: Discussion of its Relevance for inherited diseases

Choo

Cotton

Danks

et al. 1980

J of Inher Metab Disea

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Two-dimensional polyacrylamide gel electrophoresis has been used to resolve several hundred polypeptides from six different human fibroblast cell lines. Two of the cell lines were from normals while the other four were from patients with diseases of the type known to be due to single gene mutations. Less than 2% of the polypeptides from the different cell lines were found to differ in their electrophoretic mobility. The existence of only a small degree of polypeptide variability in pairwise comparison between cell lines suggests the possibility of employing two-dimensional gel electrophoretic comparison as a direct approach for the recognition of pathogenetically important polypeptide alterations in genetic diseases.

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Fluctuations in the production of specific cellular peptides during the growth of animal cells.

Cited by 11 publications

References 14 publications

Patterns of Cellular Peptide Synthesis by Cultured Bovine Granulosa Cells*

Patterns of Cellular Peptide Synthesis by Cultured Bovine Granulosa Cells*

Specific protein production during melanogenesis in B16/C3 melanoma cells

Two‐dimensional polyacrylamide gel analysis of fibroblast polypeptides: Discussion of its Relevance for inherited diseases

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