Abe, "Induction of apoptosis by
bestatin (ubenimex) in human leukemic cell lines," Leukemia, vol.
The samples were homogenized in a bullet blender with zirconium oxide beads in ice-cold buffer (in mmol [L.sup.-1]: Tris Base 100, Sucrose 500, NaF 200, NaCl 100, [Na.sub.4][O.sub.7][P.sub.2] 10, sodium orthovanadate 200, [beta]-glycerophosphate 300, with 5 mL/L Sigma protease inhibitor cocktail P8340 containing AEBSF 104, aprotinin 0.08,
bestatin hydrochloride 4, E-64 1.4, leupeptin hemisulfate salt 2, pepstatin A 1.5) and protein concentrations were determined spectrophotometrically using Bradford protein assays (Bradford 1976).
Total protein from skin tissue was prepared by the addition 1:20 of T-PER Tissue Protein Extraction Reagent (PIERCE, Rockford, IL, USA) (25 mM bicine, 150 mM sodium chloride [pH 7.6]) containing protease inhibitors [100 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 80 mM aprotinin, crystalline, 5 mM
bestatin, 1.5 mM E-64, protease inhibitor, 2 M leupeptin, and 1 mM pepstatin A].
Frozen heart tissues (-80[degrees]C) of placebo- or Rapamycin-treated rats (n = 5 per group) were powdered under liquid nitrogen and lysed with ice cold lysis buffer (Cat#: AA-Lys, RayBiotech, Norcross, GA) supplemented with Okadaic Acid (0.1 [micro]M), sodium orthovanadate (250 [micro]M), sodium pyrophosphate (650 [micro]M),
Bestatin (5 nM), and 1x cOmplete[TM] Protease Inhibitor Cocktail (Roche Life Sciences).
Briefly, cells were washed three times with ice-cold PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (0.01mol/L sodium phosphate, pH7.2, 150 mmol/L NaCl, 2mmol/L EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 2 mmol/L AEBSF, 130mmol/L
bestatin, 14 mmol/L E64,0.3 mmol/L aprotinin, and 1 mmol/L leupeptin).
Obminska- Mrukowicz (2013).The effects of
bestatin on humoral response to sheep erythrocytes in non- treated and cyclophosphamide- immunocompromised mice.ImmunopharmacolImmunotoxicol.
At the end of incubation the cells were washed with phosphate buffered saline and lysed with cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM [Na.sub.4][P.sub.2][O.sub.7], 2 mM [Na.sub.3]V[O.sub.4], 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF and protease inhibitor cocktail (2 mM [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], 14 [micro]M (N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide], 130 [micro]M
bestatin, 0.9 [micro]M leupeptin, 0.3 [micro]M aprotinin, and 1 mM EDTA)).
Effect of protease inhibitors on endogenous or exogenous [beta]-galactosidase in human fibroblasts Inhibitor Protease [beta]- [beta]- Galactosidase Galactosidase (endogenous) * (exogenous) ** None 100 100 E-64 Thiol 430 229 (Cysteine) *** Leupeptin Thiol 460 222 (Cysteine) *** Antipain Thiol 490 224 (Cysteine) *** Chymostatin Thiol 520 188 (Cysteine) *** Elastatinal Elastase 60 103 Pepstatin Pepsin 100 97 Phosphoramidon Metallopeptidase 110 88
Bestatin Aminopeptidase 110 90 * Relative enzyme activity in human fibroblasts with cathepsin A deficiency (galactosialidosis).
This mixture of protease inhibitors contains the serine protease inhibitors AEBSF (1 mmol [l.sup.-1]) and aprotinin (0.8 mmol [l.sup.-1]); the amino-peptidase inhibitor
bestatin (50 [micro] mol [l.sup.-1]); the cysteine protease inhibitor E-64 (15 [micro] mmol [l.sup.-1]); the broad-spectrum protease inhibitor leupeptin (20 [micro] mmol [l.sup.-1]); and the aspartyl protease inhibitor pepstatin A (10 [micro] mmol [l.sup.-1]).
Isoserine derivatives constitute an essential fragment in natural products of high therapeutic value [3], such as Taxol (an anticancer agent), whose side chain is N-acyl-(2R,3S)-phenylisoserine;
bestatin (a dipeptide modifier of the immune response), in which one of the amino acids is (2S,3R)-2-hydroxy-3-amino-3-phenylbutyric acid, and the kinostatins (potent inhibitors of the HIV-1 protease), in which one of the constituent amino acids is (2S,3S)-2-hydroxy-3-amino-3-phenylbutyric acid.
Wang et al., "Leukotriene [A.sub.4] hydrolase in rat and human esophageal adenocarcinomas and inhibitory effects of
bestatin," Journal of the National Cancer Institute, vol.
The cells lysates were prepared using an ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM EDTA, 1% Triton) supplemented with a mixture of protease inhibitors containing antipain,
bestatin, chymostatin, leupeptin, pepstatin, phosphoramidon, pefabloc, EDTA, and aprotinin (Boehringer, Mannheim, Germany).
