scholarly journals Isolation and partial characterization of a cage of filaments that surrounds the mammalian mitotic spindle.

1980 ◽  
Vol 87 (1) ◽  
pp. 160-169 ◽  
Author(s):  
G W Zieve ◽  
S R Heidemann ◽  
J R McIntosh
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Intermediate Filaments ◽  
Hela Cells ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Microtubule Assembly ◽  
Polypeptide Composition ◽  
Mitotic Apparatus ◽  
Filamentous Material

Mitotic cells have been detergent extracted under conditions that support microtubule assembly. When HeLa cells are lysed in the presence of brain tubulin, mitotic-arrested cells nucleate large asters and true metaphase cells yield spindles that remain enclosed within a roughly spherical cage of filamentous material. Detergent-extracted mitotic Chinese hamster ovary (CHO) cells show a similar, insoluble cage but the mitotic apparatus is only occasionally stabilized. In later stages of mitosis, HeLa cages are observed in elongated and furrowed configurations. In the terminal stages of cell division, two daughter filamentous networks are connected by the intercellular bridge. When observed in the electron microscope the cages include fibers 7-11 nm in diameter. The polypeptide composition of cages isolated from mitotic HeLa cells is complex, but the major polypeptides are a group with mol wt ranging from 43,000-60,000 daltons and a high molecular weight polypeptide. CHO cells contain a subset of these proteins which includes a major 58,000-dalton and a high molecular weight polypeptide. Two different antisera directed against the vimentin-containing intermediate filaments bind to polypeptides in the electrophoretic profiles of isolated HeLa and CHO cages and stain the cages, as visualized by indirect immunofluorescence. These results suggest that the HeLa and CHO cages include intermediate filaments of the vimentin type. The polypeptide composition of HeLa cages suggests that they also contain tonofilaments. The cages apparently form as the cells enter mitosis. We propose that these filamentous cages maintain the structural continuity of the cytoplasm while the cell is in mitosis.

The mutation Ser511Asn leads to N-glycosylation and increases the cleavage of high molecular weight kininogen in rats genetically susceptible to inflammation

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 2835-2842 ◽  
Author(s):  
Irma Isordia-Salas ◽  
Robin A. Pixley ◽  
Hemant Parekh ◽  
Satya P. Kunapuli ◽  
Fengling Li ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Chronic Inflammation ◽  
Cho Cells ◽  
Single Point ◽  
Consensus Sequence ◽  
Chinese Hamster ◽  
Single Point Mutation ◽  
High Molecular Weight Kininogen ◽  
Molecular Alteration

Abstract Crohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation. In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility. Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma. A single point mutation at nucleotide 1586 was found translating from Ser511 (Buffalo and Fisher) to Asn511 (Lewis). The latter defines an Asn-Xaa-Thr consensus sequence for N-glycosylation. We expressed these domains in Escherichia coli and found no differences in the rate of cleavage by purified kallikrein in the 3 strains in the absence of N-glycosylation. We then expressed these domains in Chinese hamster ovary (CHO) cells, which are capable of glycosylation, and found an increased rate of cleavage of Lewis HK. The Lewis mutation is associated with N-glycosylation as evidenced by a more rapid migration after treatment with N-glycosidase F. When CHO cells were cultured in the presence of tunicamycin, the kallikrein-induced cleavage rate of Lewis HK was not increased. This molecular alteration might be one contributing factor resulting in chronic inflammation in Lewis rats.

scholarly journals Sertoli cell processes have axoplasmic features: an ordered microtubule distribution and an abundant high molecular weight microtubule-associated protein (cytoplasmic dynein).

1988 ◽  
Vol 107 (5) ◽  
pp. 1767-1776 ◽  
Author(s):  
M D Neely ◽  
K Boekelheide
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Sertoli Cell ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cytoplasmic Dynein ◽  
Microtubule Assembly ◽  
Sucrose Density Gradient Centrifugation ◽  
Cell Stage ◽  
Structural And Functional Properties

Microtubules in the cytoplasm of rat Sertoli cell stage VI-VIII testicular seminiferous epithelium were studied morphometrically by electron microscopy. The Sertoli cell microtubules demonstrated axonal features, being largely parallel in orientation and predominantly spaced one to two microtubule diameters apart, suggesting the presence of microtubule-bound spacer molecules. Testis microtubule-associated proteins (MAPs) were isolated by a taxol, salt elution procedure. Testis MAPs promoted microtubule assembly, but to a lesser degree than brain MAPs. High molecular weight MAPs, similar in electrophoretic mobilities to brain MAP-1 and MAP-2, were prominent components of total testis MAPs, though no shared immunoreactivity was detected between testis and brain high molecular weight MAPs using both polyclonal and monoclonal antibodies. Unlike brain high molecular weight MAPs, testis high molecular weight MAPs were not heat stable. Testis MAP composition, studied on postnatal days 5, 10, 15, and 24 and in the adult, changed dramatically during ontogeny. However, the expression of the major testis high molecular weight MAP, called HMW-2, was constitutive and independent of the development of mature germ cells. The Sertoli cell origin of HMW-2 was confirmed by identifying this protein as the major MAP found in an enriched Sertoli cell preparation and in two rat models of testicular injury characterized by germ cell depletion. HMW-2 was selectively released from testis microtubules by ATP and co-purified by sucrose density gradient centrifugation with MAP-1C, a neuronal cytoplasmic dynein. The inhibition of the microtubule-activated ATPase activity of HMW-2 by vanadate and erythro-(2-hydroxy-3-nonyl)adenine and its proteolytic breakdown by vanadate-dependent UV photocleavage confirmed the dynein-like nature of HMW-2. As demonstrated by this study, the neuronal and Sertoli cell cytoskeletons share morphological, structural and functional properties.

scholarly journals Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions.

1987 ◽  
Vol 104 (3) ◽  
pp. 537-546 ◽  
Author(s):  
N Carlson ◽  
M Rechsteiner
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Size Distribution ◽  
High Molecular Weight ◽  
Hela Cells ◽  
Insoluble Fraction ◽  
Nuclear Fraction ◽  
Molecular Weights ◽  
Differential Sedimentation ◽  
Triton X

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.

scholarly journals A silencer element for the lipoprotein lipase gene promoter and cognate double- and single-stranded DNA-binding proteins.

1995 ◽  
Vol 15 (1) ◽  
pp. 517-523 ◽  
Author(s):  
Y Tanuma ◽  
H Nakabayashi ◽  
M Esumi ◽  
H Endo
Keyword(s):  
Dna Binding ◽  
Lipoprotein Lipase ◽  
Reporter Gene ◽  
Hela Cells ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Binding Factor ◽  
Lpl Gene ◽  
Silencer Element

Transfection experiments with constructs containing various 5'-deleted fragments of the human lipoprotein lipase (LPL) promoter and the chloramphenicol acetyltransferase reporter gene revealed an LPL silencer element (LSE) in the region of nucleotides -225 to -81 of the LPL gene that functioned in Chinese hamster ovary (CHO) and HeLa cells. Gel retardation competition analysis showed the presence of a nuclear factor(s) capable of binding to the sequence of nucleotides -169 to -152 of LSE (LSE-6) in a single-stranded (opposite-strand) and double-stranded specific fashion, the binding affinity being almost the same in the two binding forms. Site-directed mutagenesis indicated that almost the entire sequence of LSE-6 was necessary to form the complexes and also critical for silencing activity in CHO cells. The amounts of this binding factor(s) in CHO and HeLa cells were closely associated with transcriptional silencing activity. Photochemical cross-linking experiments indicated that the single- and double-stranded elements recognized the same binding factor(s) with molecular masses of 54 to 63 kDa and 109 to 124 kDa. The 109- to 124-kDa DNA binding factor(s) was found to be a doublet of that of the 54- to 63-kDa factor by isoelectric focusing or by increasing the time of exposure to UV irradiation. When inserted upstream of another gene such as that of the simian virus 40 enhancer/promoter of pSV2CAT, the sequence of nucleotides -190 to -143 (LSE-1) also suppressed transcription of the reporter gene in CHO cells. These results strongly suggest that the LSE plays a role in regulation of LPL gene expression by suppressing its transcription.

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells

1987 ◽  
Vol 7 (8) ◽  
pp. 2700-2707
Author(s):  
B Boggs ◽  
F Cabral
Keyword(s):  
Molecular Weight ◽  
Gene Product ◽  
Cho Cells ◽  
Peptide Mapping ◽  
Chinese Hamster ◽  
Apparent Molecular Weight ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Beta Tubulin Gene

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.

A probe for flagellar dynein in the mammalian mitotic apparatus

1981 ◽  
Vol 48 (1) ◽  
pp. 241-257
Author(s):  
G.W. Zieve ◽  
J.R. NcIntosh
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Indirect Immunofluorescence ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Structural Studies ◽  
Mitotic Apparatus ◽  
Specific Staining ◽  
Bovine Sperm ◽  
Sperm Proteins

An anti-serum has been prepared in rabbits that precipitates high-molecular-weight bovine sperm proteins, including the dyneins. The activity of the serum against the dyneins is demonstrated by the recognition of dynein polypeptides in stained electrophoretic profiles of sperm proteins and in immunoprecipitates of radiolabelled sperm proteins. In addition, the serum stains the sperm flagella when used in indirect immunofluorescence and quantitatively inhibits the motility of demembranated sperm reactivated with ATP. However, the serum has additional anti-sperm activities besides those directed against the dyneins as demonstrated by the staining of the acrosome in indirect immunofluorescence. When used to immunoprecipitate proteins from extracts of cultured cells, the serum precipitates 2 polypeptides; one has a molecular weight higher than the flagellar dyneins, one lower. No specific staining of cultured cells is observed when an affinity-purified anti-dynein fraction IgG is used to stain a variety of cultured cells including bovine fibroblasts. We interpret these data to suggest that flagellar dynein is not a component of the mammalian mitotic spindle and discuss how this conclusion is consistent with recent genetic and structural studies on the mitotic spindle.

Effect of altered oligosaccharide structure on the cell surface number, distribution and turnover of the high molecular weight acidic glycoproteins of CHO cells

1984 ◽  
Vol 67 (1) ◽  
pp. 1-23
Author(s):  
L.A. Fitzgerald ◽  
J.B. Denny ◽  
G.A. Baumbach ◽  
C.M. Ketcham ◽  
R.M. Roberts
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
High Molecular Weight ◽  
Cho Cells ◽  
Carbohydrate Structure ◽  
Membrane Glycoproteins ◽  
Number Distribution ◽  
Mutant Lines ◽  
Mutant Cells ◽  
Surface Glycoproteins

The influence of altered carbohydrate structure on the surface number, distribution and turnover of plasma membrane glycoproteins has been studied in Chinese hamster ovary (CHO) cells by comparing three lines that are resistant to the cytotoxic effects of wheat germ agglutinin (WGA) with parental CHO cells. The glycoproteins investigated were members of a group of high molecular weight acidic glycoproteins (HMWAG). On parental cells these represent the major surface components that become labelled by lactoperoxidase-catalysed iodination. They are the only plasma membrane glycoproteins that bind WGA. The mutant lines also possess iodinatable surface polypeptides of high molecular weight, but these were less acidic and electrophoretically less diffuse than those from parental cells. These polypeptides in general did not bind [125I]WGA when two-dimensional polyacrylamide gels were overlaid with iodinated lectin. Mutant cells treated with fluorescein-conjugated WGA showed low surface fluorescence. However, the nuclear envelope and a small region in the perinuclear zone fluoresced strongly. Together, these results confirm that the surface glycoproteins of mutant cells had altered carbohydrate structure. Mouse antiserum prepared against the HMWAG, however, bound equally effectively to the mutant lines as to the parental lines. Indirect immunofluorescence experiments showed that the HMWAG had a fairly uniform distribution over the surface, and that internalization induced by second antibody occurred at a similar rate and in a similar manner in all lines, including the mutants. Electron microscopic observations using immunoperoxidase procedures confirmed the similarities in glycoprotein distribution on mutant and parental cells. Two mouse monoclonal antibodies raised against the HMWAG also revealed no difference in the number or topography of surface glycoproteins. Finally, the half-lives of several HMWAG in a parental and a mutant line (15B) maintained on low-serum medium were compared by means of a 125I/131I double-label technique. Half-lives of HMWAG from the former averaged 12 h and from the latter 11 h. It is concluded that the lack of complex termini on oligosaccharides of this particular group of CHO plasma membrane glycoproteins has no effect on their number, distribution or turnover.

Presence of histone mRNA sequences in high molecular weight RNA of HeLa cells

Cell ◽  
1977 ◽  
Vol 11 (3) ◽  
pp. 651-661 ◽  
Author(s):  
Marialuisa Melli ◽  
Giovanni Spinelli ◽  
Hilda Wyssling ◽  
Eva Arnold
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Hela Cells ◽  
Histone Mrna
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