[6] Preparation of microsomes with calcium

Schenkman, John B.; Cinti, Dominick L.

doi:10.1016/s0076-6879(78)52008-9

Cited by 267 publications

(127 citation statements)

References 23 publications

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“…For whole-lysosome planar patch-clamp recordings, isolated intact lysosomes from HEK293 cells or MEFs were prepared by differential centrifugation as described previously 21,22,55 . HEK293 cells stably expressing murine TPC2 or the TPC2 mutant TPC2(N257A), both N-terminally fused to green fluorescent protein and subcloned into pcDNA5FRT vector with hygromycin resistance (Invitrogen) were used.…”

Section: Methodsmentioning

confidence: 99%

“…To investigate STX7 effects on TPC2, a double stable HEK293 cell line was generated coexpressing murine STX7, N-terminally fused to mCherry (in pcDNA3 vector with geneticin (G418) resistance) and TPC2 as described above. The planar patchclamp technology combined with a fast internal perfusion system (Port-a-Patch, Nanion Technologies) was applied as described previously 21,22,55 . Currents were recorded using an EPC-10 patch-clamp amplifier and PatchMaster acquisition software (HEKA).…”

Section: Methodsmentioning

confidence: 99%

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High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

et al. 2014

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Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

et al. 2014

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“…Liver microsome was measured as described by previously (18) . Briefly, 100 mg liver was excised and homogenized with a loosefitting Teflon pestle in 900 mL of 50 mM L -1 Tris-HC1buffer, pH 7.4, containing 0.3 M L -1 sucrose, 10 mM L -1 DTT, and 10 mM L -1 EDTA.…”

Section: Preparation Of Mice Of Liver Microsomementioning

confidence: 99%

Hepatic hyperplasia and damages induces by zearalenone Fusarium mycotoxins in BALB/c mice

Chatopadhyay

Pandey

Chaurasia

et al. 2012

Arq. Gastroenterol.

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-Context -Zearalenone is a mycoestrogen and considered a mycotoxin. Objective -To establish whether zearalenone produced hepatotoxicity via oral administration. Methods -Zearalenone was orally administered at a dose of 50 μg, 100 μg and 200 μg ZEN/ body weight/daily, respectively, for 14 days to three groups of BALB/c mice. Diagnostic modalities used to evaluate hepatic damage and impaired hepatic function pre-and post zearalenone administration included hepatic marker enzyme activity, pentobarbital sleeping time, cytochrome P-450 activities and histopathologic evaluation of liver. Results -Significant histopathologic changes viz. sinusoidal congestion, cytoplasmic vacuolization, hepatocellular necrosis and neutrophil infiltration were observed after evaluating of liver section from each group after accumulated zearalenone exposure. Further, zearalenone exposure increased activities of alanine transaminase, aspartate transaminase and lipid peroxides whereas activities of tissue glutathione and cytochrome P 450 were decreased as compared to control mice. Zearalenone also increased the sleeping time and decreased sleeping latency after pentobarbital through intraperitoneal route as compared to control mice which indicates that the impairment of hepatic metabolizing enzymes by zearalenone. Conclusion -Zearalenone is a potential hepatotoxin by oral route. Headings -Zearalenone. Hyperplasia. Liver, pathology. Mice.

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“…Step 1 : preparation and solubilization of microsomes Guinea-pig liver microsomes were prepared by the method of Schenkman and Cinti [25] and solubilized with 1 % (w\v) saponin as described by Hosokawa et al [22], in 0.1 M Tris\HCl buffer, pH 8.5, at a final protein concentration of 3.5 mg\ml. The mixture was stirred for 1 h and then centrifuged at 100 000 g for 1 h.…”

Section: Purification Of Gha Deacetylasementioning

confidence: 99%

Purification and characterization of guinea-pig liver microsomal deacetylase involved in the deacetylation of the O-glucoside of N-hydroxyacetanilide

Suzuki-Kurasaki

Yoshioka

Uematsu

1997

Biochemical Journal

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A microsomal deacetylase that catalyses the deacetylation of the O-glucoside of N-hydroxyacetanilide (GHA) was purified from guinea-pig liver. The activity was located exclusively in the microsomes and not detected in the cytosol. The purified GHA deacetylase was a trimeric protein with a molecular mass of 160±10 (S.D.) kDa composed of subunits of 53±2 kDa; its pI was 4.7. The N-terminal amino acid sequence of GHA deacetylase was similar to those reported for guinea-pig and rat liver microsomal carboxylesterases. The GHA deacetylase showed a comparable hydrolytic activity towards p-nitrophenyl acetate (PNPA), although the activities towards N-hydroxyacetanilide, acetanilide and some endogenous acylated compounds were very low or not detectable. The deacetylase activity towards GHA was inhibited by organophosphates but not by p-chloromercuribenzoate, suggesting that GHA deacetylase can be classified as a B-esterase. The enzyme exhibited a positive homotropic co-operativity towards GHA. The values of the Hill coefficient, the half-saturating concentration ([S]0.5) for GHA, and Vmax were 1.59±0.03, 5.51±0.07 mM and 32.5±1.4 μmol/min per mg respectively, at the optimum pH of 8.5. The bell-shaped pH dependence of the Vmax/[S]0.5 profile indicated pKa values attributed to histidine and lysine residues. The study of stoichiometric inhibition by di-isopropyl fluorophosphate and kinetic analysis with the Monod–Wyman–Changeux model suggests that GHA deacetylase has six substrate binding sites and three catalytically essential serine residues per enzyme molecule.

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[6] Preparation of microsomes with calcium

Cited by 267 publications

References 23 publications

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

Hepatic hyperplasia and damages induces by zearalenone Fusarium mycotoxins in BALB/c mice

Purification and characterization of guinea-pig liver microsomal deacetylase involved in the deacetylation of the O-glucoside of N-hydroxyacetanilide

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