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S J Fey

Publications and source records attributed to S J Fey.

At least 55 records · Page 3Linked to original sources
PubMed1984

Expression of the transformation-sensitive protein "cyclin" in normal human epidermal basal cells and simian virus 40-transformed keratinocytes.

A cell population highly enriched in human epidermal basal cells has been obtained and characterized by using antibodies specific for various cell types in the epidermis. Quantitative two-dimensional gel electrophoretic analysis (isoelectric focusing) of [35S]methionine-labeled polypeptides from basal cells and simian virus 40-transformed keratinocytes showed that the basal cells synthesize very low amounts (less than 0.02% of the total protein) of the nuclear, transformation-sensitive protein cyclin as compared to the transformed cells, which synthesize this protein constitutively (0.15% of the total protein). Very low levels of cyclin were observed in total human epidermis, and preliminary studies of two basaliomas have shown a significant synthesis of this protein in these tumors. Immunofluorescence studies using antibodies to proliferating cell nuclear antigen that immunoprecipitate cyclin confirmed the above observations at least in the case of the cultured cells. Taken together, these results support the notion that cyclin may be a central component of the pathway(s) that controls cell proliferation.

Antigens, Neoplasm↗
PubMed1984

Intermediate filaments in monkey kidney TC7 cells: focal centers and interrelationship with other cytoskeletal systems.

Two-dimensional gel electrophoresis of intermediate-sized filament-enriched cytoskeletons of epithelial monkey kidney TC7 cells has shown that they are composed of at least two keratins (isoelectric focusing 36, Mr = 48,500; IEF 46, Mr = 43,500; HeLa protein catalogue number) and vimentin. Indirect immunofluorescence as well as immunoelectron microscopy using antibodies directed against specific polypeptides sometimes revealed a discontinuous staining of keratin-containing filaments. Indirect immunofluorescence analysis of cells stained with keratin or vimentin antibodies also revealed a bright perinuclear staining in 58% of the cells in interphase. Of particular interest were focal centers from which filaments radiated. Double-label immunofluorescence using tubulin and keratin antibodies showed that these centers codistributed with focal arrays of microtubules (most likely centrosomes) in interphase cells but were not colocalized with centrioles in mitosis or, in many cases, with the microtubule organizing centers seen after release from nocodazole treatment. Treatment of TC7 cells with demecolcine (10 micrograms/ml, 20 hr) resulted in a drastic rearrangement of the keratin and vimentin filaments. Likewise, treatment with cytochalasin B (10 micrograms/ml, 1 hr) produced a star-like arrangement of the keratin and vimentin filaments and, in most cases, these codistributed with patches of actin. The results provide evidence for the interaction of intermediate filaments (keratins and vimentin) with both microtubules and microfilaments.

Animals↗
PubMed1983

Evidence for coordinated phosphorylation of keratins and vimentin during mitosis in transformed human amnion cells. Phosphate turnover of modified proteins.

Two-dimensional gel electrophoresis (IEF) analysis of short-term [32P]orthophosphate-labelled intermediate-sized filament proteins (keratins and vimentin) from transformed mitotic amnion cells (AMA), have shown that these proteins are modified coordinately and that the half life of the phosphate is about 13 min for the keratins and 11 min for vimentin. These results support the notion that the preferential modification of intermediate-sized filament proteins during mitosis may play a role in modulating filament associations with organelles and other cellular structures.

Amnion↗
PubMed1983

Differential immunological crossreactivity of HeLa keratin antibodies with human epidermal keratins.

HeLa cells contain four keratin-like proteins having molecular weights of 50,000 (IEF 31), 48,500 (IEF 36), 44,000 (IEF 44), and 43,500 (IEF 46), respectively. Mouse polyclonal antibodies prepared against two of these keratins (IEF 31 and 46) have been used in this study to identify human epidermal keratins with common antigenic determinants. Using a sensitive immunoprecipitation procedure we show that the IEF 31 antibody crossreacts with three human acidic epidermal keratins, termed K1, K2, and K3, having molecular weights of 44,000, 47,500, and 54,000, respectively. One of these keratins (K1) comigrated with HeLa keratin IEF 44 and exhibited an identical one-dimensional peptide map. This protein is also abundant in basaliomas. In contrast to these results, the IEF 46 antibody showed no crossreactivity with any of the human acidic or basic [35S]methionine-labeled epidermal proteins. The lack of crossreactivity of this antibody was further confirmed by indirect immunofluorescence staining of cryostat sections from human split skin. These results emphasize both the similarity and diversity of antigenic determinants among HeLa and epidermal keratins.

Antibody Specificity↗
PubMed1983

Proteins IEF (isoelectric focusing) 31 and IEF 46 are keratin-type components of the intermediate-sized filaments: keratins of various human cultured epithelial cells.

Mouse polyclonal antibodies have been raised against two human proteins (IEF [isoelectric focusing] 31, Mr = 50,000; IEF 46, Mr = 43,500) that have previously been shown to be present in HeLa cytoskeletons enriched in intermediate-sized filaments. Immunoprecipitation studies show that both proteins share common antigenic determinants with each other and with the putative human keratins IEF 36 and 44, also present in HeLa cytoskeletons. Indirect immunofluorescence studies showed that both antibodies revealed similar filamentous networks in various cultured epithelial cells of human origin. These included AMA (transformed amnion), HeLa (cervical carcinoma), normal amnion cells, Fl-amnion (transformed amnion), WISH-amnion (transformed amnion), Chang liver (liver), and Detroid-98 (sternal marrow). Human cells that did not react with both antibodies included skin fibroblasts, lung fibroblasts (WI-38), SV40-transformed lung fibroblasts, Molt 4 (leukemia), lymphocytes, and monocytes. These results were in complete agreement with the presence or absence of both proteins in two-dimensional gels of the different cell types. Exposure of AMA cells to demecolcine (24 h; 10 micrograms/ml) caused the total collapse of vimentin filaments but, as seen by indirect immunofluorescence, caused only a partial redistribution of the IEF 31 and 46 filaments. These results are taken to suggest that both proteins are components of the intermediate-sized filaments of the "keratin" type. The antibodies could be clearly differentiated by staining human bladder carcinoma EJ 19 cells, as only the IEF 46 antibody stained a filamentous network in these cells The occurrence of keratins IEF 31, 36, 44, and 46 in different cultured human epithelial cells has been studied using two-dimensional gel electrophoresis.

Antibody Specificity↗
PubMed1983

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis.

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.

Amnion↗
PubMed1982

Putative association of mitochondria with a subpopulation of intermediate-sized filaments in cultured human skin fibroblasts.

Cultured cell Triton cytoskeletons enriched in intermediate filaments by extraction in high and low salt show, in addition to known intermediate-filament proteins and actin, a few other tenaciously bound polypeptides. An antibody raised against one of these polypeptides (IEF 24) is shown to react specifically with mitochondria of different cell types. With methanol-acetone fixation, required for the antibody reaction, the characteristically long mitochondria of human skin fibroblasts fragment into strings of beads, as identified by antibody staining in the light microscope and whole-mount electron microscopy. From the colinear organization of these beads, their codistribution with the intermediate-filament network and their retention in salt-extracted cytoskeletons, it is suggested that a subpopulation of the intermediate filaments can serve as an anchorage site for mitochondria.

Amnion↗
PubMed1982

More than one-third of the discernible mouse polypeptides are not expressed in a Chinese hamster-mouse embryo fibroblast hybrid that retains all mouse chromosomes.

Gene expression at the level of polypeptide synthesis has been investigated in a somatic cell hybrid (20 BW-4) isolated after fusion of spontaneously transformed, tumorigenic Chinese hamster lung fibroblasts with mouse embryo fibroblasts. This hybrid exhibited suppression of tumorigenicity and retained--in addition to the parental Chinese hamster genome--copies of all mouse chromosomes as demonstrated by direct karyotype analysis and confirmed for 18 different mouse chromosomes by analysis of 18 different mouse isozymes. Two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides from hybrid 20 BW-4 showed that the overall polypeptide pattern corresponded to that of the hamster parent. All polypeptides detected in the hamster parental cells were also expressed in the hybrid although some of them were expressed in altered amounts. Of approximately 1200 labeled polypeptides revealed in the parental cells, 115 mouse polypeptides could be clearly distinguished from the hamster polypeptides due to their different electrophoretic mobilities. Forty-two of these (i.e., 37%) were not expressed in the hybrid 20 BW-4. These observations were confirmed by analysis of another independently isolated hybrid (2W 23) of the same parental cells that also exhibited suppression of malignancy and that retained copies of all mouse chromosomes except no. 5. The results suggest that the genome of the tumorigenic cell after hybridization can suppress expression of more than one-third of the normal parental genome. The suppressed mouse genetic information is probably located on many, if not all, different mouse chromosomes. Even if the level of genetic suppression is high the mouse genome is able to reduce the tumorigenicity of the hamster parental cell.

Animals↗
PubMed1981

Coexistence of three major isoactins in a single sarcoma 180 cell.

Actin is transformed sarcoma 180 cells is composed of the nonmuscle beta and gamma species and of a third, more acidic stable variant termed zeta. Two-dimensional peptide analysis shows that zeta is similar to beta actin, differing in the mobility of only one tryptic peptide. Several lines of evidence indicate that zeta is not a modified beta-actin species. This third actin species comprises 20% of the total labeled actin, has the same molecular weight as the beta and gamma actins and has a different mobility in isoelectric focusing gels from that of the known alpha actins from skeletal, cardiac and vascular smooth muscle. Like beta and gamma actin, zeta can be extracted with the actin depolymerizing factor from slime mold. Two-dimensional gel electrophoresis (isoelectric focusing) of the 35S-methionine-labeled polypeptides synthesized by a single sarcoma 180 cell showed that all three major actin species coexist within the same cell. This analysis also showed for the first time the coexistence and alpha and beta tubulin, vimentin, alpha actinin and three other polypeptides present in intermediate-filament-enriched cytoplast cytoskeletons (spots 12, 24 and 31). Determination of the ratio of gamma plus beta to zeta actin in different cytoskeletal preparations of intact and enucleated sarcoma 180 cells indicated that this actin species is not localized specifically to any of the major actin-containing structures preserved in the cytoskeletons.

Actinin↗
PubMed1981

[35S]-methionine labelled polypeptides from secondary mouse kidney fibroblasts: coordinates and one dimensional peptide maps of some major polypeptides.

A total of 1147 [35S]-methionine labelled polypeptides (826 acidic (IEF) and 321 basic (NEPHGE)) from asynchronous secondary mouse kidney fibroblasts have been separated using high resolution two dimensional gel electrophoresis. Beside numbering the spots, we give for the major polypeptides their coordinates (M.W. and relative mobility respect to beta-actin (IEF) or NEPHGE polypeptide 9 (NEPHGE)). By using one dimensional peptide mapping it has been possible to find homologies between several major mouse and HeLa cell polypeptides.

Animals↗
PubMed1981

Gene expression in murine hybrids exhibiting different morphologies and tumorigenic properties.

[35S]Methionine labelled polypeptides from mouse CLID, hamster ovary cells (CHO, and 7 derived somatic cell hybrids which have segregated CHO chromosomes, were analysed by means of high resolution two-dimensional gel electrophoresis under conditions in which the position of 600 polypeptides could be reproducibly assessed. As judged from the two-dimensional gel electrophoretic patterns (isoelectric focussing (IEF) and non-equilibrium pH gradient electrophoresis (NEPHGE)) gene expression in all the hybrids resembled the mouse CLID parent and with only one exception they all expressed different numbers and intensities of CHO specific polypeptides. Even though some of the hybrids expressed as much as 50% of the total number of CHO specific polypeptides that could be clearly differentiated from those of the mouse parent we failed to find a direct correlation between the expression of any given CHO polypeptide and the morphological or tumorigenic properties of the hybrids. Most CHO specific polypeptides, however, were expressed at lower levels in the hybrids as compared to the parent CHO cells, a fact that may be due to the chromosomal constitution of the hybrids, regulation or both. Similarly, the quantitation of the major cytoskeletal polypeptides present in the hybrids, such as alpha-and beta-tubulin, vimentin, total actin and 3 polypeptides (IEF 12, 24 and 31) present in intermediate filament enriched cytoskeletons, indicate that changes in the relative proportion of any of these proteins is not sufficient to account for the morphology, actin microfilament pattern or tumorigenicity of the hybrids. Co-expression of cytoskeletal proteins in the hybrids could only be demonstrated in the case of the related mouse IEF 24 and hamster IEF 7 polypeptides. In all other cases the cytoskeletal polypeptides co-migrated and presented similar one-dimensional peptide maps. Some principles are emerging concerning the possibility of using somatic cell hybridization in combination with two-dimensional gel electrophoresis to locate genes coding for particular polypeptides on a given chromosome.

Animals↗
PubMed1979

Pathway-dependent reconstitution of chromatin structure from separated constituents.

Chicken reticulocyte chromatin can be reassembled from its separated constituents, viz. DNA, H1 plus H5, core histones, and non-histone proteins, to yield a product resembling the native starting material by a series of structural criteria. In particular, it possesses nucleosomes separated by spacer regions; the particles contain DNA with a unit length of approximately 200 base pairs. The recovery of the correctly reassembled product depends critically on the annealing conditions: the components are initially mixed in 2 M NaCl and 5 M urea, and it seems to be important to remove urea at a relatively high salt concentration. The results suggest that the characteristic chromatin structure is formed only when core histones bind to DNA in their native conformation and are followed by the addition of H1 and H5 to the spacer regions.

Animals↗