scholarly journals Detection by means of cell fusion of macromolecular synthesis involved in the reconstruction of the nuclear envelope in mitosis.

1975 ◽  
Vol 64 (2) ◽  
pp. 378-388 ◽  
Author(s):  
Y Obara ◽  
H Weinfeld ◽  
A A Sandberg
Keyword(s):  
Protein Synthesis ◽  
Nuclear Envelope ◽  
Prolonged Exposure ◽  
Chinese Hamster ◽  
M Cells ◽  
Macromolecular Synthesis ◽  
M Cell ◽  
G2 Cells ◽  
G1 Cells ◽  
Inhibitor Of Protein Synthesis

Using the cultured Chinese hamster cell line Don, G1 or S or a mixture of late-S/G2 cells were prepared by release from metaphase arrest. Metaphase (M) cells were also obtained by mitotic arrest of log-phase cultures with Colcemid and held in metaphase; such M cells remained untreated with any other compound and were termed standard M cells. When interphase (I) cells were fused at pH 8.0 and 37 degrees C with standard cells in the presence of Colcemid by means of UV-inactivated Sendai virus, binucleate interphase-metaphase (I-M) cells were obtained. In a given I-M cell there occurred within 30 min after fusion either prophasing of the I nucleus or formation of a nuclear envelope (NE) around the chromosomes. About 20% of early G1 cells, 35% of cells at the G1/S boundary, 50% of S cells, and 70% of late S/G2 cells could induce NE formation. If, before fusion, cycloheximide (CHE), an inhibitor of protein synthesis, was present during release from M arrest, the cells entered G1 but not S. About 20% of such early G1 cells, like the untreated early G1 cells, had the capacity to induce NE formation during subsequent fusion. If the cells were blocked in S with 5 mM thymidine (TdR), At least 80% of these cells could induce NE formation during subsequent fusion, but in the presence of both TdR and CHE only 35% could do so. It appeared, therefore, that protein synthesis in interphase was required for NE formation. Experiments with actinomycin D indicated that RNA synthesis was also necessary for acquisition of NE-inducing capacity. About 35% of G1 cells from confluent monolayers had the NE-inducing capacity, but prolonged exposure to CHE reduced their number to 8% . Removal of CHE restored the ability while the cells still remained in G1. This result indicated that continuing protein synthesis in the G1 cell was needed for NE formation subsequent to fusion. The fact that macromolecular synthesis must occur in the I cell before fusion if NE formation was to occur in the fused I-M cell lends further support to evidence adduced earlier that this phenomenon is a normal mitotic event. Prophasing of the I nucleus in I-M cells did not appear to be dependent on macromolecular synthesis in the I cell; earlier results from this laboratory showed, however, that protein synthesis in the prior G2 period of the M cell of the I-M pair was required for prophasing.

scholarly journals CONTRAST BETWEEN THE ENVIRONMENTAL pH DEPENDENCIES OF PROPHASING AND NUCLEAR MEMBRANE FORMATION IN INTERPHASE-METAPHASE CELLS

1973 ◽  
Vol 58 (3) ◽  
pp. 608-617 ◽  
Author(s):  
Yoshitaka Obara ◽  
Hiroshi Yoshida ◽  
Lee S. Chai ◽  
Herbert Weinfeld ◽  
Avery A. Sandberg
Keyword(s):  
Structural Changes ◽  
Chinese Hamster ◽  
Binucleate Cell ◽  
M Cells ◽  
M Cell ◽  
Metaphase Chromosomes ◽  
Membrane Formation ◽  
Interphase Cell ◽  
Double Membrane ◽  
Mononucleate Cell

In Chinese hamster Don cells, fusion of an interphase cell with a metaphase cell resulted either in prophasing of the interphase nucleus, including loss of the nuclear envelope (NE), or in the formation of a double membrane around the metaphase chromosomes. Only one of these phenomena occurred in a given interphase-metaphase (I–M) binucleate cell. At pH 7.4, there was about an equal probability that either event could occur amongst the population of I–M cells. The effect of pH changes in the medium containing the fused cells was examined. At pH 6.6, prophasing was the predominant event; at pH 8.0, membrane formation predominated. It was found that the rate of progression of a mononucleate cell from G2 to metaphase was appreciably faster at pH 6.6 than at pH 8.0. Conversely, the progression from metaphase to G1 was faster at pH 8.0 than at pH 6.6. These results with the mononucleate cells strengthen the hypothesis that structural changes in I–M cells are reflections of normal mitotic phenomena. Additional evidence for this hypothesis was produced by electron microscope examination after direct fixation in chrom-osmium. The double membrane around the chromosomes of the I–M cell was indistinguishable from the normal NE. The results obtained by varying the pH of the medium containing the fused cells provide an indication that disruption or formation of the NE of Don cells depends on the balance reached between disruptive and formative processes.

Genetic and biochemical distinction among Chinese hamster cell emtA, emtB, and emtC mutants

1983 ◽  
Vol 3 (3) ◽  
pp. 429-438
Author(s):  
S Chang ◽  
J J Wasmuth
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Chinese Hamster ◽  
Cross Resistance ◽  
Protein Synthesis Inhibitor ◽  
Chromosome 2 ◽  
Synthesis Inhibitor ◽  
Chinese Hamster Cells ◽  
Inhibitor Of Protein Synthesis

Genetic and biochemical experiments have enabled us to more clearly distinguish three genetic loci, emtA, emtB, and emtC, all of which can be altered to give rise to resistance to the protein synthesis inhibitor, emetine, in cultured Chinese hamster cells. Genetic experiments have demonstrated that, unlike the emtB locus, neither the emtA locus nor the emtC locus is linked to chromosome 2 in Chinese hamster cells, clearly distinguishing the latter two genes from emtB. emtA mutants can also be distinguished, biochemically, from emtB and emtC mutants based upon different degrees of cross-resistance to another inhibitor of protein synthesis, cryptopleurine. Two-dimensional gel electrophoretic analysis of ribosomal proteins failed to detect any electrophoretic alterations in ribosomal proteins from emtA or emtC mutants that could be correlated with emetine resistance. However, a distinct electrophoretic alteration in ribosomal protein S14 was observed in an emtB mutant. In addition, the parental Chinese hamster peritoneal cell line of an emtC mutant, and the emtC mutant itself, are apparently heterozygous for an electrophoretic alteration in ribosomal protein L9.

scholarly journals Genetic and biochemical distinction among Chinese hamster cell emtA, emtB, and emtC mutants.

1983 ◽  
Vol 3 (3) ◽  
pp. 429-438 ◽  
Author(s):  
S Chang ◽  
J J Wasmuth
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Chinese Hamster ◽  
Cross Resistance ◽  
Protein Synthesis Inhibitor ◽  
Chromosome 2 ◽  
Synthesis Inhibitor ◽  
Chinese Hamster Cells ◽  
Inhibitor Of Protein Synthesis

Genetic and biochemical experiments have enabled us to more clearly distinguish three genetic loci, emtA, emtB, and emtC, all of which can be altered to give rise to resistance to the protein synthesis inhibitor, emetine, in cultured Chinese hamster cells. Genetic experiments have demonstrated that, unlike the emtB locus, neither the emtA locus nor the emtC locus is linked to chromosome 2 in Chinese hamster cells, clearly distinguishing the latter two genes from emtB. emtA mutants can also be distinguished, biochemically, from emtB and emtC mutants based upon different degrees of cross-resistance to another inhibitor of protein synthesis, cryptopleurine. Two-dimensional gel electrophoretic analysis of ribosomal proteins failed to detect any electrophoretic alterations in ribosomal proteins from emtA or emtC mutants that could be correlated with emetine resistance. However, a distinct electrophoretic alteration in ribosomal protein S14 was observed in an emtB mutant. In addition, the parental Chinese hamster peritoneal cell line of an emtC mutant, and the emtC mutant itself, are apparently heterozygous for an electrophoretic alteration in ribosomal protein L9.

scholarly journals Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

1985 ◽  
Vol 101 (3) ◽  
pp. 1086-1093 ◽  
Author(s):  
S F Preston ◽  
C S Regula ◽  
P R Sager ◽  
C B Pearson ◽  
L S Daniels ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Biochemical Analysis ◽  
Core Protein ◽  
Chinese Hamster Ovary Cells ◽  
Membrane Vesicle ◽  
Chinese Hamster ◽  
Glycosaminoglycan Synthesis ◽  
Ovary Cells ◽  
Interphase Cells ◽  
G1 Cells

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two- to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate. Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.

scholarly journals Cytosolic deglycosylation process of newly synthesized glycoproteins generates oligomannosides possessing one GlcNAc residue at the reducing end

1998 ◽  
Vol 335 (2) ◽  
pp. 389-396 ◽  
Author(s):  
Sandrine DUVET ◽  
Odette LABIAU ◽  
Anne-Marie MIR ◽  
Daniel KMIÉCIK ◽  
Sharon S. KRAG ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Rough Endoplasmic Reticulum ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Glcnac Residue ◽  
Chase Period ◽  
Mechanism Of Degradation ◽  
Inhibitor Of Protein Synthesis

Recent studies on the mechanism of degradation of newly synthesized glycoproteins suggest the involvement of a retrotranslocation of the glycoprotein from the lumen of the rough endoplasmic reticulum into the cytosol, where a deglycosylation process takes place. In the studies reported here, we used a glycosylation mutant of Chinese hamster ovary cells that does not synthesize mannosylphosphoryldolichol and has an increased level of soluble oligomannosides originating from glycoprotein degradation. In the presence of anisomycin, an inhibitor of protein synthesis, we observed an accumulation of glucosylated oligosaccharide-lipid donors (Glc3Man5GlcNAc2-PP-Dol), which are the precursors of the soluble neutral oligosaccharide material. Inhibition of rough endoplasmic reticulum glucosidase(s) by castanospermine led to the formation of Glc3Man5GlcNAc2(OSGn2) (in which OSGn2 is an oligomannoside possessing two GlcNAc residues at its reducing end), which was then retained in the lumen of intracellular vesicles. Thus they were protected during an 8 h chase period from the action of cytosolic chitobiase, which is responsible for the conversion of OSGn2 to oligomannosides possessing one GlcNAc residue at the reducing end (OSGn1). In contrast, when protein synthesis was maintained in the presence of castanospermine, glucosylated oligomannosides (Glc1–3Man5GlcNAc1) were recovered in cytosol. Except for monoglucosylated Man5 species, which are potential substrates for luminal calnexin and calreticulin, the pattern of oligomannosides was similar to that observed on glycoproteins. The occurrence in the cytosol of glucosylated species with one GlcNAc residue at the reducing end implies that the deglycosylation process that generates glucosylated OSGn1 from glycoproteins occurs in the cytosol.

scholarly journals Ectopic Overexpression of Wild-Type and Mutant hipA Genes in Escherichia coli: Effects on Macromolecular Synthesis and Persister Formation

2006 ◽  
Vol 188 (11) ◽  
pp. 3826-3836 ◽  
Author(s):  
Shaleen B. Korch ◽  
Thomas M. Hill
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Prolonged Exposure ◽  
Physiological Role ◽  
In Vitro Translation ◽  
Translation System ◽  
Wild Type ◽  
Macromolecular Synthesis ◽  
Dormant State

ABSTRACT Persistence is an epigenetic trait that allows a small fraction of bacteria, approximately one in a million, to survive prolonged exposure to antibiotics. In Escherichia coli an increased frequency of persisters, called “high persistence,” is conferred by mutations in the hipA gene, which encodes the toxin entity of the toxin-antitoxin module hipBA. The high-persistence allele hipA7 was originally identified because of its ability to confer high persistence, but little is known about the physiological role of the wild-type hipA gene. We report here that the expression of wild-type hipA in excess of hipB inhibits protein, RNA, and DNA synthesis in vivo. However, unlike the RelE and MazF toxins, HipA had no effect on protein synthesis in an in vitro translation system. Moreover, the expression of wild-type hipA conferred a transient dormant state (persistence) to a sizable fraction of cells, whereas the rest of the cells remained in a prolonged dormant state that, under appropriate conditions, could be fully reversed by expression of the cognate antitoxin gene hipB. In contrast, expression of the mutant hipA7 gene in excess of hipB did not markedly inhibit protein synthesis as did wild-type hipA and yet still conferred persistence to ca. 10% of cells. We propose that wild-type HipA, upon release from HipB, is able to inhibit macromolecular synthesis and induces a bacteriostatic state that can be reversed by expression of the hipB gene. However, the ability of the wild-type hipA gene to generate a high frequency of persisters, equal to that conferred by the hipA7 allele, may be distinct from the ability to block macromolecular synthesis.

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

1994 ◽  
Vol 92 (4) ◽  
pp. 585-594 ◽  
Author(s):  
T. J. Bouma ◽  
R. De Visser ◽  
J. H. J. A. Janssen ◽  
M. J. De Kock ◽  
P H. Van Leeuwen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Energy Requirements ◽  
Inhibitor Of Protein Synthesis

scholarly journals Characterization of the lectin from the bulbs of Eranthis hyemalis (winter aconite) as an inhibitor of protein synthesis.

1993 ◽  
Vol 268 (33) ◽  
pp. 25176-25183
Author(s):  
M A Kumar ◽  
D E Timm ◽  
K E Neet ◽  
W G Owen ◽  
W J Peumans ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Inhibitor Of Protein Synthesis
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