scholarly journals The identification of the major excreted protein (MEP) from a transformed mouse fibroblast cell line as a catalytically active precursor form of cathepsin L

1987 ◽  
Vol 248 (2) ◽  
pp. 449-454 ◽  
Author(s):  
R W Mason ◽  
S Gal ◽  
M M Gottesman
Keyword(s):  
Sequence Data ◽  
Cathepsin L ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Protein Substrates ◽  
3T3 Fibroblasts ◽  
Catalytically Active ◽  
Mouse Fibroblast Cell Line ◽  
Human Cathepsin ◽  
Active Precursor

The major excreted protein (MEP) purified from Kirsten-virus-transformed 3T3 fibroblasts and mature human cathepsin L were compared in respect to a number of catalytic criteria and found to be similar. The Mr of MEP is 39,000, whereas that of mature human cathepsin L is 30,000. Sequence data suggested that MEP could be a pro-form of mouse cathepsin L. Both enzymes acted on the synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide with similar catalytic constants and acted optimally at pH 5.5. Both were rapidly inactivated by the active-site-directed inhibitors benzyloxycarbonyl-Phe-Phe-diazomethane and L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane, and furthermore, 3H-labelled L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-acetamid o)butane, which binds covalently to the heavy chain of mature cathepsin L, also bound to MEP. MEP autolyses rapidly at pH 3.0 to give lower-Mr (35,000 and 30,000) forms, but all forms react with the radiolabelled inhibitor. No autolysis occurred above pH 5.0. MEP hydrolysed azocasein at pH 5.0, demonstrating that it is capable of hydrolysing protein substrates without autolytic activation. Unlike mature forms of cathepsin L, MEP is stable, but not active, at neutral pH. The present work shows that cathepsin L can be secreted as a higher-Mr precursor that is stable in extracellular fluids but only active where local pH values fall below 6.0. These results suggest that the extra N-terminal peptide on MEP is not an activation peptide, but is a regulatory peptide affecting the pH-stability and activity of mouse cathepsin L.

Rosette formation by a mouse fibroblast cell line

Nature ◽  
1974 ◽  
Vol 248 (5448) ◽  
pp. 514-515 ◽  
Author(s):  
E. V. ELLIOTT ◽  
R. S. KERBEL ◽  
B. J. PHILLIPS
Keyword(s):  
Cell Line ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Rosette Formation ◽  
Fibroblast Cell Line ◽  
Mouse Fibroblast Cell ◽  
Mouse Fibroblast Cell Line

Sodium affinity of brain Na(+)-K(+)-ATPase is dependent on isozyme and environment of the pump

1990 ◽  
Vol 258 (5) ◽  
pp. C803-C811 ◽  
Author(s):  
J. L. Brodsky ◽  
G. Guidotti
Keyword(s):  
Plasma Membranes ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Synaptic Plasma Membranes ◽  
Apparent Dissociation Constant ◽  
Low Sodium ◽  
Physiological Relevance ◽  
Mouse Fibroblast Cell Line

The sodium affinities for the two forms of the Na(+)-K(+)-ATPase in brain were characterized. To mimic physiological conditions, synaptosomes, which are pinched off presynaptic nerve termini, were used. Examination of the pump in vitro was performed by preparing synaptic plasma membranes (SPMs). It was first shown that synaptosomes contain the two forms of the Na(+)-K(+)-ATPase, alpha 1 and alpha 2, and that these forms have markedly different affinities for the inhibitory cardiac glycoside ouabain. The apparent dissociation constant (K0.5) of alpha 1 for sodium changed from 12 to 9 mM when going from synaptosomes to membranes. For alpha 2, however, a shift from 36 to 12.5 mM was evident. The conclusion is that in vivo alpha 2 exists as a low sodium affinity species but can be altered to a high-affinity form simply by vesicle disruption. By comparison, the Na(+)-K(+)-ATPase from the mouse fibroblast cell line, 3T3-F442A cells, expressed only the alpha 1-isozyme, as shown by immunoblotting and by measurement of its ouabain and sodium affinities. The physiological relevance of these observations is also presented.

scholarly journals Evaluation of cytotoxicity of 5-n-alkylresorcinol homologs and fraction on mouse fibroblast cell line L929

2016 ◽  
Vol 243 (7) ◽  
pp. 1137-1148 ◽  
Author(s):  
Izabela Biskup ◽  
Ewa Zaczynska ◽  
Miroslawa Krauze-Baranowska ◽  
Izabela Fecka
Keyword(s):  
Cell Line ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Fibroblast Cell Line ◽  
Mouse Fibroblast Cell ◽  
Mouse Fibroblast Cell Line

scholarly journals Development of Bionanocomposites Based on PLA, Collagen and AgNPs and Characterization of Their Stability and In Vitro Biocompatibility

2020 ◽  
Vol 10 (7) ◽  
pp. 2265
Author(s):  
Maria Râpă ◽  
Laura Mihaela Stefan ◽  
Traian Zaharescu ◽  
Ana-Maria Seciu ◽  
Anca Andreea Țurcanu ◽  
...  
Keyword(s):  
Normal Growth ◽  
Fibroblast Cell ◽  
Stability Study ◽  
Mouse Fibroblast ◽  
Poly Lactic Acid ◽  
In Vitro Biocompatibility ◽  
Diphenyltetrazolium Bromide ◽  
Mouse Fibroblast Cell Line ◽  
The Stability

Bionanocomposites including poly(lactic acid) (PLA), collagen, and silver nanoparticles (AgNPs) were prepared as biocompatible and stable films. Thermal properties of the PLA-based bionanocomposites indicated an increase in the crystallinity of PLA plasticized due to a small quantity of AgNPs. The results on the stability study indicate the promising contribution of the AgNPs on the durability of PLA-based bionanocomposites. In vitro biocompatibility conducted on the mouse fibroblast cell line NCTC, clone 929, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed high values of cell viability (>80%) after cell cultivation in the presence of bionanocomposite formulations for 48 h, while the percentages of lactate dehydrogenase (LDH) released in the culture medium were reduced (<15%), indicating no damages of the cell membranes. In addition, cell cycle analysis assessed by flow cytometry indicated that all tested bionanocomposites did not affect cell proliferation and maintained the normal growth rate of cells. The obtained results recommend the potential use of PLA-based bionanocomposites for biomedical coatings.

scholarly journals Hepatic glutathione S-transferases in mice fed on a diet containing the anticarcinogenic antioxidant butylated hydroxyanisole. Isolation of mouse glutathione S-transferase heterodimers by gradient elution of the glutathione-Sepharose affinity matrix

1991 ◽  
Vol 277 (2) ◽  
pp. 501-512 ◽  
Author(s):  
J D Hayes ◽  
L A Kerr ◽  
S D Peacock ◽  
A D Cronshaw ◽  
L I McLellan
Keyword(s):  
Amino Acid ◽  
Mouse Liver ◽  
Sequence Data ◽  
Fibroblast Cell ◽  
Exchange Chromatography ◽  
Mouse Fibroblast ◽  
Cdna Libraries ◽  
Butylated Hydroxyanisole ◽  
N Terminus ◽  
Glutathione S Transferases

Induction of glutathione S-transferases (GSTs) is believed to represent an important mechanism whereby butylated hydroxyanisole inhibits chemical carcinogenesis. The soluble hepatic GSTs expressed by mice fed on normal diets are all homodimers comprising Ya3 (Mr 25,800), Yb1 (Mr 26,400) and Yf (Mr 24,800) subunits. In addition to these constitutively expressed GSTs, we have identified enzymes containing Ya1 (Mr 25,600), Ya2 (Mr 25,600), Yb2 (Mr 26,200) and Yb5 (Mr 26,500) subunits from the livers of Balb/c mice fed on diets containing butylated hydroxyanisole (BHA). Gradient affinity elution of GSH-Sepharose has been used to resolve the mouse liver enzymes into several discrete pools of activity from which GSTs were purified by cation-exchange chromatography. The inducible Mu-class Yb2 and Yb5 subunits were separately isolated as the heterodimers GST Yb1Yb2 and GST Yb1Yb5 and their catalytic properties are described; this showed that 1,2-dichloro-4-nitrobenzene and trans-4-phenylbut-3-en-2-one are marker substrates for the mouse Yb1 and Yb2 subunits respectively, but no discriminating model substrate was found that allows the identification of the Yb5 subunit. Individual GST subunits were resolved by reverse-phase h.p.l.c. and their amino acid compositions were determined. Certain subunits (Yb1, Yb2, Yb5 and Yf) were also subjected to automated amino acid sequence analysis, and this demonstrated that the Yb5 subunit has a blocked N-terminus. The mouse Yb1, Yb2 and Yb5 subunits from the major inducible Mu-class heterodimers were cleaved with CNBr and purified peptides from the Yb2 and Yb5 subunits were sequenced. These data show that the Yb2 subunit is distinct from the GSTs that are encoded by the cDNAs that have been cloned from mouse liver cDNA libraries but possesses identity with the protein that is encoded by pmGT2, a cDNA isolated from a mouse fibroblast cell line by Townsend, Goldsmith, Pickett & Cowan [(1989) J. Biol. Chem. 264. 21582-21590]. The sequence data also show that the cDNA encoding the mouse Yb5 subunit has not, to date, been cloned, and the relationship between this subunit and Mu-class GSTs in other species that possess a blocked N-terminus (e.g. rat GST YoYo) is discussed.

Parasiticidal activity of bovine lactoperoxidase against Toxoplasma gondii

2006 ◽  
Vol 84 (5) ◽  
pp. 774-779 ◽  
Author(s):  
Tetsuya Tanaka ◽  
Shin Murakami ◽  
Haruto Kumura ◽  
Ikuo Igarashi ◽  
Kei-ichi Shimazaki
Keyword(s):  
Toxoplasma Gondii ◽  
Cell Line ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
A Cell ◽  
Mouse Fibroblast Cell Line ◽  
Infected Meat ◽  
Free Environment ◽  
Nih 3T3 ◽  
Mouse Macrophage Cell Line

Toxoplasma gondii is an obligatory intracellular parasitic protozoan transmitted via the ingestion of raw, infected meat that causes congenital infections. In a cell-free environment, virulent Toxoplasma was strikingly resistant to H2O2. The activity of H2O2 or H2O2 generated by glucose – glucose oxidase against the resistant tachyzoite stage of pathogenic T. gondii was enhanced by adding KI and bovine lactoperoxidase (bLPO), referred to here as the bLPO system. Replacing bLPO (heme content, 90%) with recombinant bLPO (heme content, 6%) did not enhance the parasiticidal activity with KI and H2O2. These results indicated that heme contributed to the enzyme activity and resulted in the killing of tachyzoites of T. gondii. Tachyzoites treated with the bLPO system also lost the ability to penetrate the mouse fibroblast cell line (NIH/3T3), and could be killed intracellularly after exposure by bLPO to a mouse macrophage cell line (J774A.1). These findings suggested that toxicity was mediated through small amounts of H2O2 generated by phagocytic events in naive macrophages, and by the peroxidative activity of bLPO. Our observations suggest that the bLPO system could help prevent the development of Toxoplasmosis in humans after ingesting raw, infected meat.

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