scholarly journals FURTHER CHARACTERIZATION OF THE F1-HISTONE PHOSPHOKINASE OF METAPHASE-ARRESTED ANIMAL CELLS

1973 ◽  
Vol 58 (2) ◽  
pp. 317-331 ◽  
Author(s):  
Robert S. Lake
Keyword(s):  
Gel Filtration ◽  
Chinese Hamster ◽  
Adenosine Monophosphate ◽  
Animal Cells ◽  
M Cell ◽  
M Phase ◽  
Comparative Examination

Exponentially growing Chinese hamster cells are found to contain two major phosphokinase activities with specificity for the phosphorylation of F1 (lysine-rich) histone. These two activities, designated KI and KII, were extracted with 0.35 M NaCl and fractionated in 0.2 M NaCl by Sephadex G-200 gel filtration. KI, which is similar to the ubiquitous cyclic 3',5'-adenosine monophosphate (cAMP)-dependent phosphokinase, differs from KII by several criteria. KII is mol wt 90,000, cAMP independent, rapidly turned over in vivo, low Km for ATP, and phosphorylates F1 histone at several unique sites. Comparative examination of metaphase-arrested (M) and counterpart interphase (I) cells for these two activities reveals that KII is responsible for the overall high activity in M-arrested cells. Pulse labeling of cells with 32P during traverse of the G2-M phase of the cell cycle reveals an in vivo tryptic-phosphopeptide pattern in whole unfractionated F1 which is unique to M cells. Seven major phosphopeptides derived by in vitro phosphorylation of F1 with the KII enzyme correspond to these M cell-specific phosphorylation sites observed in vivo. It is suggested that KII activity predominates during the G2-M transition and that F1 is its natural in vivo substrate.

scholarly journals Romidepsin Induces G2/M Phase Arrest and Apoptosis in Cholangiocarcinoma Cells

2020 ◽  
Vol 19 ◽  
pp. 153303382096075
Author(s):  
Pihong Li ◽  
Luguang Liu ◽  
Xiangguo Dang ◽  
Xingsong Tian
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Antitumor Effect ◽  
Caspase 3 ◽  
M Cell ◽  
Cycle Arrest ◽  
Phase Arrest ◽  
M Phase

Background: Cholangiocarcinoma (CCA) is an extremely intractable malignancy since most patients are already in an advanced stage when firstly discovered. CCA needs more effective treatment, especially for advanced cases. Our study aimed to evaluate the effect of romidepsin on CCA cells in vitro and in vivo and explore the underlying mechanisms. Methods: The antitumor effect was determined by cell viability, cell cycle and apoptosis assays. A CCK-8 assay was performed to measure the cytotoxicity of romidepsin on CCA cells, and flow cytometry was used to evaluate the effects of romidepsin on the cell cycle and apoptosis. Moreover, the in vivo effects of romidepsin were measured in a CCA xenograft model. Results: Romidepsin could reduce the viability of CCA cells and induce G2/M cell cycle arrest and apoptosis, indicating that romidepsin has a significant antitumor effect on CCA cells in vitro. Mechanistically, the antitumor effect of romidepsin on the CCA cell lines was mediated by the induction of G2/M cell cycle arrest and promotion of cell apoptosis. The G2/M phase arrest of the CCA cells was associated with the downregulation of cyclinB and upregulation of the p-cdc2 protein, resulting in cell cycle arrest. The apoptosis of the CCA cells induced by romidepsin was attributed to the activation of caspase-3. Furthermore, romidepsin significantly inhibited the growth of the tumor volume of the CCLP-1 xenograft, indicating that romidepsin significantly inhibited the proliferation of CCA cells in vivo. Conclusions: Romidepsin suppressed the proliferation of CCA cells by inducing cell cycle arrest through cdc2/cyclinB and cell apoptosis by targeting caspase-3/PARP both in vitro and in vivo, indicating that romidepsin is a potential therapeutic agent for CCA.

scholarly journals Characterization of Fibroblasts with Son of Sevenless-1 Mutation

2006 ◽  
Vol 85 (11) ◽  
pp. 1050-1055 ◽  
Author(s):  
E.J. Lee ◽  
S.I. Jang ◽  
D. Pallos ◽  
J. Kather ◽  
T.C. Hart
Keyword(s):  
Cell Proliferation ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Histological Assessment ◽  
M Phase ◽  
Son Of Sevenless ◽  
Gingival Fibromatosis

Although non-syndromic hereditary gingival fibromatosis (HGF) is genetically heterogeneous, etiologic mutations have been identified only in the Son of Sevenless-1 gene ( SOS1). To test evidence of increased cell proliferation, we studied histological, morphological, and proliferation characteristics in monolayer and three-dimensional cultures of fibroblasts with the SOS1 g.126,142–126,143insC mutation. Histological assessment of HGF gingiva indicated increased numbers of fibroblasts (30%) and increased collagen (10%). Cell proliferation studies demonstrated increased growth rates and 5-bromo-2-deoxyuridine incorporation for HGF fibroblasts. Flow cytometry showed greater proportions of HGF fibroblasts in the G2/M phase. Attachment of HGF fibroblasts to different extracellular matrix surfaces demonstrated increased formation of protrusions with lamellipodia. HGF fibroblasts in three-dimensional culture showed greater cell proliferation, higher cell density, and alteration of surrounding collagen matrix. These findings revealed that increased fibroblast numbers and collagen matrix changes are associated with mutation of the SOS1 gene in vitro and in vivo.

Activity and stability of centrosomes in Chinese hamster ovary cells in nucleation of microtubules in vitro

1984 ◽  
Vol 66 (1) ◽  
pp. 277-295
Author(s):  
R. Kuriyama
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Ovary Cells ◽  
Protease Digestion ◽  
The Difference

Mitotic centrosomes were prepared from Chinese hamster ovary cells and their capacity to nucleate microtubules in vitro was demonstrated by incubation with exogenous brain microtubule protein. The number of microtubules polymerized onto centrosomes was directly counted by electron microscopy of whole-mount preparations. This simple and accurate quantitative assay has permitted characterization of the microtubule nucleating activity of centrosomes in vitro. The number of microtubules polymerized onto centrosomes varied according to the structure of the centrosome. The activity was roughly proportional to the centriole number. The number and length of microtubules nucleated by centrosomes depended both on the concentration of tubulin and the incubation time with tubulin. Under saturating conditions, an average of 200–250 microtubules were initiated by single centrosomes. Centrosomal activity is unstable (t 1/2 = 8 h) and could easily be irreversibly disrupted by a medium of high ionic strength. The activity is stabilized by the addition of glycerol. Centrosomes can be stored at −80 degrees C. The optimum pH for microtubule nucleation is 6.8. Activity is sensitive to protease digestion, but neither DNase or RNase affected the nucleating activity of centrosomes. The activity is temperature-sensitive, but addition of phenylmethylsulphonyl fluoride (PMSF) induces thermal stability. At an optimal concentration of 0.5 mg/ml, this drug increased the half-life of the activity (t 1/2 = 95 h) and made it resistant to salt extraction. Protease inhibitors other than PMSF or dansyl fluoride did not have any stabilizing effect on the activity. The difference between the centrosomal structure of polymerized microtubules in vivo and in vitro is discussed.

Characterization of an Endogenous Transcription Inhibitor from Physarum polycephalum

1979 ◽  
Vol 34 (1-2) ◽  
pp. 76-86 ◽  
Author(s):  
Armin Hildebrandt ◽  
Rudolf Mengel
Keyword(s):  
Rna Polymerase ◽  
Physarum Polycephalum ◽  
Gel Filtration ◽  
In Vitro Transcription ◽  
Exchange Chromatography ◽  
Heat Stable ◽  
Transcription Inhibitor

Abstract A substance has been purified from isolated nuclei of Physarum polycephalum by equilibrium and velocity gradient centrifugations, ion exchange chromatography and gel filtration which has a high molecular weight, can be labeled in vivo with 32P, is heat stable and resistant to amylases, proteases, nucleases and phosphodiesterase but is sensitive to phosphatases or hydrolysis. This material consists of phospate and glycerol. It selectively inhibits in vitro transcription of RNA polymerases, predominantly the homologous enzyme A by binding to the enzyme. In the presence of this inhibitor of transcription a stable RNA polymerase-template complex cannot be formed. Binding to and inactivation of RNA polymerase is reversible at high ionic strength.

scholarly journals Specific association of an M-phase kinase with isolated mitotic spindles and identification of two of its substrates as MAP4 and MAP1B.

1991 ◽  
Vol 2 (11) ◽  
pp. 861-874 ◽  
Author(s):  
R M Tombes ◽  
J G Peloquin ◽  
G G Borisy
Keyword(s):  
Mitotic Cycle ◽  
Chinese Hamster ◽  
Mitotic Spindles ◽  
Microtubule Associated Proteins ◽  
M Phase ◽  
Associated Proteins ◽  
Specific Association ◽  
Cycle Dependent

Isolated mammalian (Chinese hamster ovary [CHO]) metaphase spindles were found to be enriched in a histone H1 kinase whose activity was mitotic-cycle dependent. Two substrates for the kinase were identified as MAP1B and MAP4. Partially purified spindle kinase retained activity for the spindle microtubule-associated proteins (MAPs) as well as brain and other tissue culture MAPs; on phosphorylation, spindle MAPs exhibited increased immunoreactivity with MPM-2, a monoclonal antibody specific for a subset of mitotic phosphoproteins. Immunofluorescence using an anti-thiophosphoprotein antibody localized in vitro phosphorylated spindle proteins to microtubule fibers, centrosomes, kinetochores, and midbodies. The fractionated spindle kinase was reactive with anti-human p34cdc2 antibodies and with an anti-human cyclin B but not an anti-human cyclin A antibody. We conclude that spindle MAPs undergo mitotic cycle-dependent phosphorylations in vivo and associate with a kinase that remains active on spindle isolation and may be related to p34cdc2.

scholarly journals Cell-cycle-dependent phosphorylation and activity of Chinese-hamster ovary topoisomerase II

1993 ◽  
Vol 293 (1) ◽  
pp. 297-304 ◽  
Author(s):  
D A Burden ◽  
L J Goldsmith ◽  
D M Sullivan
Keyword(s):  
Cell Cycle ◽  
Topoisomerase Ii ◽  
Chinese Hamster ◽  
Dna Topoisomerase ◽  
Protein Levels ◽  
Cleavage Activity ◽  
M Phase ◽  
Cycle Dependent

Cell-cycle-dependent protein levels and phosphorylation of DNA topoisomerase II in relation to its catalytic and cleavage activities were studied in Chinese-hamster ovary cells. Immunoreactive topoisomerase II protein levels were maximal in G2-phase cells, intermediate in S- and M-phase cells, and minimal in a predominantly G1-phase population. When the phosphorylation of topoisomerase II in vivo was corrected for differences in specific radioactivity of intracellular ATP, the apparent phosphorylation of S- and M-phase topoisomerase II was altered significantly. Relative phosphorylation in vivo was found to be greatest in M-phase cells and decreased in the other populations in the order: S > G2 > asynchronous. Phosphoserine was detected in every phase of the cell cycle, with a minor contribution of phosphothreonine demonstrated in M-phase cells. Topoisomerase II activity measured in vivo as 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4′-demethylepipodophylloto xin (VP-16)-induced DNA double-strand breaks (determined by neutral filter elution) increased in the order: asynchronous < S < G2 < M. Topoisomerase II cleavage activity, assayed in vitro as the formation of covalent enzyme-DNA complexes, was lowest in S phase, intermediate in asynchronous and G2-phase cells, and maximal in M phase. Topoisomerase II decatenation activity was 1.6-1.8-fold greater in S-, G2- and M-phase populations relative to asynchronous cells. Therefore DNA topoisomerase II activity measured both in vivo and in vitro is maximal in M phase, that phase of the cell cycle with an intermediate level of immunoreactive topoisomerase II but the highest level of enzyme phosphorylation. The discordance between immunoreactive topoisomerase II protein levels, adjusted relative phosphorylation, catalytic activity, cleavage activity and amino acid residue(s) modified, suggests that the site of phosphorylation may be cell-cycle-dependent and critical in determining catalytic and cleavage activity.

scholarly journals Isolation and partial chemical characterization of macrophage-derived neutrophil chemotactic factor

1995 ◽  
Vol 4 (4) ◽  
pp. 257-262 ◽  
Author(s):  
M. Dias-Baruffi ◽  
M. C. Roque-Barreira ◽  
F. Q. Cunha ◽  
S. H. Ferreira
Keyword(s):  
Neutrophil Migration ◽  
Gel Filtration ◽  
Chemical Characterization ◽  
Chemotactic Factor ◽  
Sds Page ◽  
Neutrophil Chemotactic Factor ◽  
Partial Chemical

Macrophages stimulated with lipopolysaccharide (LPS) release a factor (MNCF; macrophage-derived neutrophil chemotactic factor) which induces neutrophil migrationin vivoandin vitro. Thein vivochemotactic activity of crude MNCF is not affected by pretreating the animals with dexamethasone, an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. We purified MNCF by affinity chromatography of the supernatant from LPS-stimulated macrophages on immobilized D-galactose, followed by gel filtration of the sugar-binding material on Superdex 75. The activity was eluted in the volume corresponding to a MW of 54 kDa. SDS–PAGE of this preparation revealed a single band, also corresponding to a 54 kDa protein. MNCF is an acidic protein (pI < 4) as shown by chromatofocussing. Like the crude MNCF, the homogeneous protein induced neutrophil migrationin vitroas well asin vivo. This was not modified by dexamethasone pretreatment.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

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