scholarly journals The Aromatic Ring of Phenylalanine 334 Is Essential for Oligomerization of Vibrio vulnificus Hemolysin

2009 ◽  
Vol 192 (2) ◽  
pp. 568-574 ◽  
Author(s):  
Takashige Kashimoto ◽  
Shunji Ueno ◽  
Takeshi Koga ◽  
Shinji Fukudome ◽  
Hayato Ehara ◽  
...  
Keyword(s):  
Aromatic Ring ◽  
Vibrio Vulnificus ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Binding Activity ◽  
Gram Negative Bacteria ◽  
Ovary Cells ◽  
In The Wild ◽  
And Function ◽  
Phenylalanine Residue

ABSTRACT Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.

Chinese hamster ovary cell mutants defective in the internalization of ricin

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

scholarly journals Increased sensitivity to oxidative injury in chinese hamster ovary cells stably transfected with rat liver S-adenosylmethionine synthetase cDNA

1996 ◽  
Vol 319 (3) ◽  
pp. 767-773 ◽  
Author(s):  
Estrella SÁNCHEZ-GÓNGORA ◽  
John G. PASTORINO ◽  
Luis ALVAREZ ◽  
María A. PAJARES ◽  
Concepción GARCÍA ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Rat Liver ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Wild Type ◽  
Ovary Cells ◽  
Transfected Cells ◽  
In The Wild ◽  
Adenosylmethionine Synthetase

Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetase cDNA. As a result, S-adenosylmethionine synthetase activity increased 2.3-fold, an effect that was accompanied by increased S-adenosylmethionine, a depletion of ATP and NAD levels, elevation of the S-adenosylmethionine/S-adenosylhomocysteine ratio (the methylation ratio), increased DNA methylation and polyamine levels (spermidine and spermine), and normal GSH levels. By contrast, the transfected cells showed normal growth curves and morphology. Exposure to an oxidative stress by the addition of H2O2 resulted in a greater consumption of ATP and NAD in the transfected cells than in the wild-type cells. In turn, cell killing by H2O2 was greater in the transfected cells than in the wild-type cells. This killing of Chinese hamster ovary cells by H2O2 involved the activation of poly(ADP-ribose) polymerase with the resultant loss of NAD and ATP. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, but not the antioxidant N,N´-diphenylphenylenediamine, prevented the killing of Chinese hamster ovary cells by H2O2 and maintained the contents of NAD and ATP. The results of this study indicate that a moderate activation of the synthesis of S-adenosylmethionine leads to ATP and NAD depletion and to a greater sensitivity to cell killing by oxidative stress.

scholarly journals The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

1977 ◽  
Vol 73 (3) ◽  
pp. 601-615 ◽  
Author(s):  
RR Gould ◽  
GG Borisy
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Carbon Coated ◽  
Pericentriolar Material ◽  
And Function

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.

Removing the Covalent Links between GP Ibα and GP Ibβ Hampers Binding of von Willebrand Factor to GP Ib-IX Complex in CHO Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3641-3641
Author(s):  
Xi Mo ◽  
Shi-Zhong Luo ◽  
Yuandong Peng ◽  
Adam D. Munday ◽  
Carol Sun ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Cell Model ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Binding Activity ◽  
Ovary Cells ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract In the glycoprotein (GP) Ib-IX complex, GP Ibα contains the binding region for its ligand von Willebrand factor (vWF). How the other subunits in the complex, GP Ibβ and GP IX, regulate the GP Ibα-vWF interaction is not clear. Since GP Ibα connects with two GP Ibβ subunits via disulfide bonds, these inter-subunit covalent links may be important to the proper vWF-binding activity of the GP Ib-IX complex. To test this idea, we have obtained Chinese hamster ovary cells stably expressing the mutant GP Ib-IX complex (CHOαSSβIX) in which both residues C484 and C485 of GP Ibα were changed to Ser. As expected, the mutant GP Ibα did not form any disulfide bonds with GP Ibβ in these cells. Nonetheless, co-immunoprecipitation experiments in the digitonin-containing buffer demonstrated that association of GP Ibα with GP Ibβ and GP IX was retained in CHOαSSβIX cells. The expression level of GP Ibα in CHOαSSβIX cells, detected by its binding to WM23, a conformation-insensitive antibody, was comparable to that in the transfected cells stably expressing the wild type GP Ib-IX complex (CHOαβIX). In contrast, binding of CHOαSSβIX cells to AK2, SZ2 or AN51, all of which are GP Ibα-specific conformation-sensitive antibodies, was significantly different from that of CHOαβIX cells. Compared to CHOαβIX cells, ristocetin-induced binding of CHOαSSβIX cells to vWF under static conditions was reduced by about 50% as determined by flow cytometry. Consistently, rolling of CHOαSSβIX cells on the vWF-coated glass slide was significantly faster than CHOαβIX cells under various flow conditions. Thus, in a transfected cell model, removing the disulfide links between GP Ibα and GP Ibβ in the GP Ib-IX complex leads to an alteration in the conformation of the ligand-binding domain in GP Ibα and as a consequence hampers the complex binding to vWF. Our finding suggests that the relative position of GP Ibα and GP Ibβ in the receptor complex is crucial to its vWF-binding activity, which may be exploited to modulate the GP Ibα-vWF interaction.

scholarly journals Chinese hamster ovary cell mutants defective in the internalization of ricin.

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544 ◽  
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

scholarly journals Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced by Vibrio tubiashii

2001 ◽  
Vol 67 (8) ◽  
pp. 3707-3711 ◽  
Author(s):  
Mahendra H. Kothary ◽  
Rachel B. Delston ◽  
Sherill K. Curtis ◽  
Barbara A. McCardell ◽  
Ben D. Tall
Keyword(s):  
Vibrio Vulnificus ◽  
Chinese Hamster Ovary Cells ◽  
Gel Filtration ◽  
Chinese Hamster ◽  
Hydrophobic Interaction Chromatography ◽  
Atlantic Menhaden ◽  
Amino Acid Residues ◽  
Ovary Cells ◽  
Vibrio Tubiashii

ABSTRACT An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.

scholarly journals Direct biochemical measurements of microtubule assembly and disassembly in Chinese hamster ovary cells. The effect of intercellular contact, cold, D2O, and N6,O2'-dibutyryl cyclic adenosine monophosphate.

1975 ◽  
Vol 64 (1) ◽  
pp. 42-53 ◽  
Author(s):  
R W Rubin ◽  
G D Weiss
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Binding Activity ◽  
Microtubule Assembly ◽  
Thin Sections ◽  
Ovary Cells ◽  
Significant Difference

A study was undertaken to develop a means of quantitating the amount of tubulin present as a soluble pool and as intact microtubules in cultured Chinese hamster ovary cells. A procedure was developed in which these cells grown on monolayer culture in Petri dishes were placed in a "microtubule stabilizing medium" (MTM) consisting of 50% glycerol, 10% dimethylsulfoxide and sodium phosphate magnesium buffer, as described previously by Filner and Behnke. These cells then were homogenized and the homogenate was spun in the ultracentrifuge. Colchicine binding activity was then determined in the supernates and the pellets. The values, when compared with total colchicine binding activity present in replicate homogenates, were used to determine the percentage of tubulin present as intact microtubules. A statistical analysis of thin sections of cells treated with MTM revealed no statistically significant difference between MTM-treated cells and untreated controls. It was further discovered that the relative amount of colchicine binding activity recovered in the high speed pellet varied dramatically, depending upon the cell number of the culture being studied. Preconfluent cultures showed very low colchicine binding activity averaging less than 5%, while confluent and postconfluent cultures often possessed as high as 25% of their total colchicine binding activity in pelletable material. Although cold and D2O treatment had little or no effect on these values, N6,O2'-dibutyryl cyclic adenosine monophosphate increased them. It is hoped that this study will serve as the basis for a reliable quantitative procedure for measuring microtubule polymerization and depolymerization in vivo.

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