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41.
Kelem Kassahun Pei Hu Mark P. Grillo Margaret R. Davis Lixia Jin Thomas A. Baillie 《Chemico-biological interactions》1994,90(3):253-275
The ability of 2-n-propyl-4-pentenoic acid (Δ4-VPA) and 2-n-propyl-2(E)-pentenoic acid ([E]-Δ2-VPA), two unsaturated metabolites of valproic acid (VPA), to form reactive intermediates, deplete hepatic glutathione (GSH) and cause accumulation of liver triglycerides was investigated in the rat. With the aid of ionspray liquid chromatography-tandem mass spectrometry (LC-MS/MS), three GSH adducts were detected in the bile of Δ4-VPA-treated animals and were identified as 4-hydroxy-5-glutathion-S-yl-VPA-γ-lactone, 5-glutathion-S-yl-(E)-Δ3-VPA and 3-oxo-5-glutathion-S-yl-VPA. A fourth conjugate was identified tentatively as 4-glutathion-S-yl-5-hydroxy-VPA. Quantitative analysis of the corresponding N-acetylcysteine (NAC) conjugates in urine indicated that metabolism of Δ4-VPA via the GSH-dependent pathways accounted for approximately 20% of an acute dose (100 mg kg−1 i.p.). In contrast, when rats were given an equivalent dose of (E)-Δ2-VPA, only one GSH adduct (5-glutathion-S-yl-(E)-Δ3-VPA) was detected at low concentrations in bile. In vitro experiments with rat liver mitochondria demonstrated that Δ4-VPA undergoes coenzyme A- and ATP-dependent metabolic activation in this organelle via the β-oxidation pathway to intermediates which bind covalently to proteins. When liver homogenates and hepatic mitochondria from rats injected with Δ4-VPA, (E)-Δ2-VPA or VPA were analyzed for GSH content, it was found that only Δ4-VPA depleted GSH pools significantly. Treatment of rats with Δ4-VPA and (to a lesser extent) VPA led to an accumulation of liver triglycerides, whereas (E)-Δ2-VPA had no measurable effect. It is concluded that Δ4-VPA undergoes metabolic activation by both microsomal cytochrome P-450-dependent and mitochondrial coenzyme A-dependent processes, and that the resulting electrophilic intermediates, which are trapped in part by GSH, may mediate the hepatotoxic effects of this compound. In contrast, (E)-Δ2-VPA is not transformed to any appreciable extent to reactive metabolites, which thus accounts for the apparent lack of hepatotoxicity of this positional isomer in the rat. 相似文献
42.
Abstract: The radiolabeled progesterone (PG) analogue progesterone-11α-hemisuccinate-(2-[125 I]iodohistamine) was used to label PG binding proteins in brain membranes from mouse cerebellum. Photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified specific PG binding protein bands 1–4 of 64−29 kDa. Bands 1 and 4 were well resolved on the gel and easily quantified. Preincubation with PG inhibited photolabeling in a dose-dependent manner. The labeling was specific with respect to steroid structure. For band 1, the extent of inhibition of labeling by PG and 3α,5α-pregnanolone (3α) was pronounced. Other steroids such as testosterone (Tes), estradiol (Est), and corticosterone (Cor) were less effective, whereas pregnenolone sulfate (PS) and cholesterol (Cho) were ineffective. With respect to band 4, Est was the most effective; PG, 3α, and Tes were intermediate; and PS, Cho, and Cor were ineffective. The results describe specific membrane proteins that bind PG (band 1) and Est (band 4). 相似文献
43.
Emmanuel Sivvas Georgia Voukelatou Dionissios Papaioannou Alexios J. Aletras Elias D. Kouvelas 《Journal of neurochemistry》1994,63(4):1544-1550
Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [3 H]CNQX and [3 H]kainic acid binding on chick cerebellar membranes. [3 H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [3 H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [3 H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate. 相似文献
44.
Graźyna Zimowska Paul David Shirk Donald LeRoy Silhacek Eli Shaaya 《Development genes and evolution》1994,203(4):215-226
We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium. 相似文献
45.
Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA). 相似文献
46.
D.Allan Butterfield Bin Sun Srithal Bellary Warwick A. Arden Kimberly Ward Anderson 《生物化学与生物物理学报:疾病的分子基础》1994,1225(2):231-234
Electron paramagnetic resonance employing a lipid-specific spin label has been used to investigate the molecular effects of endotoxin on the physical state of bilayer lipids in rat erythrocyte membranes. When added at a concentration as low as 40 μg/ml to whole blood (plasma plus leukocytes present), decreased membrane lipid motion was found in subsequently washed and spin-labeled intact erythrocytes (P < 0.02). However, if endotoxin were added to washed, plasma plus leukocyte-free intact erythrocytes, no change in the motion of the spin label was found, suggesting that plasma-soluble substances and/or leukocytes are required to produce the change in the physical state of lipids. The decreased lipid motion found in these studies is discussed with reference to the known decreased deformability of endotoxin-treated red cells and to the pathogenesis of sepsis. 相似文献
47.
Bruce F. Giffin Kuixiong Gao Randal E. Morris Robert R. Cardell 《Biotechnic & histochemistry》1993,68(6):309-315
We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy. 相似文献
48.
PCR-mediated screening and labeling of DNA from clones 总被引:1,自引:0,他引:1
Yun Hai Lu Sylvie Nègre Philippe Leroy Michel Bernard 《Plant Molecular Biology Reporter》1993,11(4):345-349
A simplified and economical protocol for DNA library screening and nonradioactive labeling is described. Bacterial clones
are lysed in 1% of Triton X-100 and subjected to polymerase chain reaction in the presence of digoxigenin-11-dUTP to screen
and simultaneously to label the DNA inserts. Bacteriallysates are stable in storage at −20°C and can be used repeatedly for
PCR-mediated labeling. In this protocol, very low concentrations of dNTP, digoxigenin-dUTP, and primers are used in combination
with a reduced reaction volume. This will considerably reduce the expense of screening and labeling bacterial clones and facilitate
the exchange of DNA probes among laboratories. 相似文献
49.
David S. Waugh 《Journal of biomolecular NMR》1996,8(2):184-192
Summary A collection of genetic tools that can be used to manipulate amino acid metabolism in Escherichia coli is described. The set comprises 21 strains of bacteria, each containing a different genetic defect that is closely linked to a selectable transposon marker. These tools can be used to construct strains of E. coli with ideal genotypes for residue-specific, selective labeling of proteins with nearly any 15N-amino acid. By using strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis, dilution of the isotope by endogenous amino acid biosynthesis and scrambling of the label to other types of residues can be avoided.Abbreviations
15N-amino acid
-15N-amino acid
- CamR
chloramphenicol-resistant
- DPA
diaminopimelic acid
- Hfr
high-frequency recombinant
- LB
Luria broth
- KanR
kanamycin resistant
- P1
bacteriophage P1
- pfu
plaque-forming units
- StrR
streptomycin-resistant
- TetR
tetracycline-resistant 相似文献
50.
Ingmar Sethson Ulf Edlund Tadeusz A. Holak Alfred Ross Bengt-Harald Jonsson 《Journal of biomolecular NMR》1996,8(4):417-428
Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific 15N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A. 相似文献