scholarly journals Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family.

1990 ◽  
Vol 10 (10) ◽  
pp. 5160-5165 ◽  
Author(s):  
S Ahmad ◽  
R Ahuja ◽  
T J Venner ◽  
R S Gupta
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Immunofluorescent Staining ◽  
Mutant Form ◽  
Two Dimensional ◽  
Wild Type ◽  
Cho Cell ◽  
Microtubule Inhibitors

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.

Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family

1990 ◽  
Vol 10 (10) ◽  
pp. 5160-5165
Author(s):  
S Ahmad ◽  
R Ahuja ◽  
T J Venner ◽  
R S Gupta
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Immunofluorescent Staining ◽  
Mutant Form ◽  
Two Dimensional ◽  
Wild Type ◽  
Cho Cell ◽  
Microtubule Inhibitors

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.

A stress-inducible 40 kDa protein (hsp40): purification by modified two-dimensional gel electrophoresis and co-localization with hsc70(p73) in heat-shocked HeLa cells

1993 ◽  
Vol 104 (3) ◽  
pp. 629-638 ◽  
Author(s):  
H. Hattori ◽  
T. Kaneda ◽  
B. Lokeshwar ◽  
A. Laszlo ◽  
K. Ohtsuka
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Hela Cells ◽  
Gel Electrophoresis ◽  
Recovery Period ◽  
Chinese Hamster ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Terminal Amino ◽  
Kinetics Of

We have previously reported that a novel 40 kDa protein is induced by heat shock and several environmental stresses in mammalian and avian cells and that the N-terminal amino acid sequence of this 40 kDa protein has homology with the bacterial DnaJ heat-shock protein. We have purified this protein (40 kDa heat-shock protein, hsp40) from HeLa cells by modified two-dimensional gel electrophoresis and generated a polyclonal antibody against hsp40. This antibody was highly specific for human hsp40 and cross-reacted weakly with rat and Chinese hamster hsp40. Indirect immunofluorescence revealed that the hsp40 in HeLa cells accumulates in the nucleus, especially in the nucleolus, during heat shock and returns to the cytoplasm during the recovery period. The kinetics of the accumulation in the nucleoli and subsequent return to the cytoplasm of hsp40 was similar to that of hsp70. In addition, hsp40 was co-localized with hsc70(p73) in heat-shocked HeLa cells as demonstrated by double immunofluorescence staining. These results suggest that hsp40 (a DnaJ homologue) and hsp70 (a DnaK homologue) may act in concert to repair (refold) denatured proteins and protein aggregates in the nuclei and nucleoli of heat-shocked HeLa cells.

Application of heat shock protein assay and proteome assay to water from wastewater treatment plant

2008 ◽  
Vol 57 (8) ◽  
pp. 1183-1189 ◽  
Author(s):  
Naoyuki Funamizu ◽  
Mikako Takenaka ◽  
Junkyu Han ◽  
Hiroko Isoda
Keyword(s):  
Wastewater Treatment ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Cho Cells ◽  
Wastewater Treatment Plants ◽  
Treatment Plant ◽  
Chinese Hamster ◽  
Decay Process ◽  
Aeration Tank ◽  
Sludge Treatment

In this study we applied bioassay using Chinese hamster ovary (CHO) cells with a heat shock protein (HSP) 47 promoter to the effluent of the wastewater treatment plants in Sapporo and we observed the statistically significant HSP production. This implied the effluent contained some organic matter which can stress the CHO cells. To investigate the possible causes of the toxicity of the effluent, we applied the assay to the rejected water from the sludge treatment plant, the mixtures of sewage and rejected water. The evolution of HSP production during the aerobic decay process and thickening process of sludge was also examined. These assay results showed that dissolved microbial products generated and/or released from activated sludge during its decay process in the aeration tank and during thickening and dewatering process in the sludge treatment train contributed to develop HSP production. The proteomics analysis was also applied to the effluent and detected the production of elongation factor 1β. This result implies that the effluent from wastewater treatment plants may cause changes in cell proteins involved in allergic reaction.

scholarly journals Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27.

1995 ◽  
Vol 15 (1) ◽  
pp. 505-516 ◽  
Author(s):  
J N Lavoie ◽  
H Lambert ◽  
E Hickey ◽  
L A Weber ◽  
J Landry
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Actin Filament ◽  
Cellular Localization ◽  
Chinese Hamster ◽  
Heat Shock Protein 27 ◽  
Cytochalasin D ◽  
Wild Type ◽  
Protective Function ◽  
Hsp27 Phosphorylation

Phosphorylation of heat shock protein 27 (HSP27) can modulate actin filament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells) or a nonphosphorylatable form of human HSP27 (HU27pm3 cells) were developed. In contrast to HU27 cells, which showed increased survival after heat shock, HU27pm3 cells showed only slightly enhanced survival. Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-type HSP27 caused an increased stability of these structures in HU27 cells. HU27 but not HU27pm3 cells were highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localization of the protein, indicating that phosphorylation was not involved in these processes. However, induction of HSP27 phosphorylation by stressing agents or mitogens caused a reduction in the multimeric size of the wild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilament dynamics, resulting in both enhanced stability and accelerated recovery of the filaments. The level of protection provided by HSP27 during heat shock may thus represent the contribution of better maintenance of actin filament integrity to overall cell survival.

scholarly journals Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia

2021 ◽  
Vol 22 (10) ◽  
pp. 5218
Author(s):  
Tomu Kamijo ◽  
Takahiro Kaido ◽  
Masahiro Yoda ◽  
Shinpei Arai ◽  
Kazuyoshi Yamauchi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cho Cells ◽  
Fibrinogen Level ◽  
Culture Media ◽  
Chinese Hamster ◽  
Plasma Fibrinogen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cho Cell ◽  
Accelerated Degradation ◽  
Heterozygous Patient

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients’ plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and “D:D” interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient’s hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

scholarly journals Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180 ◽  
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

Expression of human Mn SOD in Chinese hamster ovary cells confers protection from oxidant injury

1993 ◽  
Vol 264 (6) ◽  
pp. L598-L605
Author(s):  
B. Warner ◽  
R. Papes ◽  
M. Heile ◽  
D. Spitz ◽  
J. Wispe
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Lethal Dose ◽  
Chinese Hamster ◽  
Eukaryotic Expression ◽  
Wild Type ◽  
Oxidant Injury ◽  
Sod Activity ◽  
Recombinant Cho Cells

Manganese superoxide dismutase (Mn SOD) is an important component of antioxidant defense in aerobic cells because of its location in the mitochondria, a significant source of oxygen radicals and an important target of oxidant injury. To test the hypothesis that increased mitochondrial Mn SOD protects from oxidant injury, Chinese hamster ovary (CHO) cells were transfected with a eukaryotic expression vector containing the human Mn SOD cDNA. In recombinant CHO cells, Mn SOD activity was increased threefold over wild-type controls. Acute survival during paraquat exposure (0–500 microM) was significantly improved in CHO cells expressing human Mn SOD, with 71% of recombinant CHO cells surviving at the 50% lethal dose (LD50) for wild-type CHO controls. Cell growth following exposure to paraquat (100 microM) was also significantly improved in recombinant CHO cells. CHO cells expressing human Mn SOD continued to grow and divide after paraquat exposure, whereas growth of wild-type CHO cells was negligible. Protection against oxidant-induced injury was directly related to increased Mn SOD, occurring in the absence of changes in other antioxidant enzymes including catalase, Cu,Zn SOD, and glutathione associated cellular antioxidant mechanisms. We conclude that increased expression of human Mn SOD in vitro directly confers protection against oxidant injury.

scholarly journals The Carboxyl-Terminal Region Is a Determinant for the Intracellular Behavior of the Chorionic Gonadotropin β Subunit: Effects on the Processing of the Asn-Linked Oligosaccharides

1998 ◽  
Vol 12 (5) ◽  
pp. 766-772
Author(s):  
Mesut Muyan ◽  
Irving Boime
Keyword(s):  
Intramolecular Interaction ◽  
Chinese Hamster ◽  
Glycoprotein Hormones ◽  
Wild Type ◽  
Cho Cell ◽  
Α Subunit ◽  
Glycosylation Sites ◽  
Carboxyl Terminal ◽  
Complex Forms ◽  
Placental Hormone

Abstract The placental hormone human CG (hCG) consists of two noncovalently linked α- and β-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common α subunit but differ in their structurally distinct β subunits. The CGβ subunit is distinguished among the β subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGβ subunit (CGβ114) with the α-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGβ and CGβ114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.

Effect of heat shock on ribosome structure: appearance of a new ribosome-associated protein

1986 ◽  
Vol 6 (7) ◽  
pp. 2527-2535
Author(s):  
T W McMullin ◽  
R L Hallberg
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Ribosomal Proteins ◽  
Tetrahymena Thermophila ◽  
Small Subunit ◽  
Stoichiometric Ratio ◽  
Two Dimensional ◽  
Hyperthermic Treatment ◽  
Stressed Cells ◽  
Ribosome Association

After a nonlethal but heat shock protein-inducing hyperthermic treatment, ribosomes isolated from Tetrahymena thermophila contained an additional 22-kilodalton protein (p22). When maximally ribosome associated, this protein was found to be on the small subunit in a 1:1 stoichiometric ratio with other ribosomal proteins. Using an antiserum directed against the purified 22-kilodalton protein, we found that non-heat-shocked and heat-shocked cells contain identical amounts of this protein, the only difference being that in the stressed cells p22 is entirely ribosome bound, whereas in the unstressed cells p22 has little or no detectable ribosome association. Because the two-dimensional electrophoretic properties of p22 showed no alterations after heat shock, this change in state of ribosome-p22 interaction does not appear to be caused by a chemical modification of p22. When not strongly ribosome associated, p22 is not found free in the cytoplasm. During that time in heat shock when p22 is first becoming ribosome associated, it is found preferentially on polysomal ribosomes. Subsequently, all ribosomes, whether polysome bound or not, obtain a bound p22. The functional significance of this association is discussed.

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