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61.
62.
Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations 总被引:2,自引:2,他引:0
1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The Km for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the Km for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the Ki being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, Km values for the amino alcohol and NAD+ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol administered, is complete within 24hr., but represents less than 0·1% of the dose given. 8. The possible metabolic role of amino alcohol dehydrogenases is discussed. 相似文献
63.
ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT
chloramphenicol acetyl transferase
- GUS
ß-glucuronidase
- CaMV
cauliflower mosaic virus
- NOS
nopaline synthase
- CCC
covalently closed circular DNA
- L
linear DNA
- PEG
polyethylene glycol
- HS
herring sperm DNA
- P
protoplasts
- PCM
protoplast culture medium
- MES
morpholinoethane-sulfonic acid
- Cm
chloramphenicol
- Ac
acetylated
- MUG
4-methyl umbelliferyl ß-D-glucuronide
- TLC
thin layer chromatography 相似文献
64.
Reduced thioredoxin was subjected to chemical modification studies employing organoarsenical reagents specific for "spatially close" thiols. Modification was monitored by the loss in the free thiol content, by the percent incorporation of radiolabelled organoarsenical reagents, and by observing the changes in the amounts of the various thioredoxins by size exclusion chromatography. The rate of modification depends upon the polarity, rigidity, and size of the reagents. Small nonpolar organoarsenical reagents readily modified reduced thioredoxin, whereas polar and large reagents do not. Modifications resulted in the formation of stable 15-membered cyclodithioarsenite ring structures with no apparent changes in the secondary structure of the protein. Modification was reversed by the extrusion of the arsenical moiety by addition of 2,3-dimercaptopropanol. We have further characterized the oxidized, reduced, and modified thioredoxins by size exclusion chromatography and fluorescence anisotropy decay measurements. Both techniques show an increase in the hydrated volume of the protein upon reduction. Upon modification, the hydrodynamic volume of the protein further swells. Fluorescence anisotropy decay reveals that with modification there is loosening of the protein so that a "domain" containing the fluorophores can relax independently of the whole protein structure. 相似文献
65.
A technique for microwave fixation of inflated rat lung is described. Conventional intratracheal fixation with instillation of fixative into the airways at a constant pressure results in pressure artifacts as well as flushing and disruption of cells and exudates. Microwave fixation fixes these elements in situ without disruption and thus is valuable when evaluating the distribution of inflammatory infiltrates. Exudative pneumonitis was produced in the rat using intratracheal instillations of either endotoxin or silica and comparisons were made between histologic sections fixed using either standard formalin fixation or microwave fixation. 相似文献
66.
Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration. 相似文献
67.
The protein Id: a negative regulator of helix-loop-helix DNA binding proteins 总被引:261,自引:0,他引:261
We have isolated a cDNA clone encoding a novel helix-loop-helix (HLH) protein, Id. Id is missing the basic region adjacent to the HLH domain that is essential for specific DNA binding in another HLH protein, MyoD. An in vitro translation product of Id can associate specifically with at least three HLH proteins (MyoD, E12, and E47) and attenuate their ability to bind DNA as homodimeric or heterodimeric complexes. Id is expressed at varying levels in all cell lines tested. In three cell lines that can be induced to undergo terminal differentiation, Id RNA levels decrease upon induction. Transfection experiments indicate that over-expression of Id inhibits the trans-activation of the muscle creatine kinase enhancer by MyoD. Based on these findings, we propose that HLH proteins lacking a basic region may negatively regulate other HLH proteins through the formation of nonfunctional heterodimeric complexes. 相似文献
68.
We have developed a simple method for the quantitative determination of tellurite in biological media. This assay is suitable for studying tellurite uptake in bacteria and overcomes the problems of older techniques which are time consuming and labor intensive. In earlier protocols diethyldithiocarbamate was reacted with tellurite and the resulting complex was extracted into organic solvents before spectrophotometric determination. In this study, diethyldithiocarbamate was incubated with tellurite at neutral pH to form a yellow colloidal solution. The absorbance of the aqueous yellow sol was used to determine tellurite concentrations in the range of 1 to 50 micrograms/ml (4 to 200 microM) without the need for solvent extraction. 相似文献
69.
Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel
Timothy Turner Howard A. Bern Peter Young Gerald R. Cunha 《In vitro cellular & developmental biology. Plant》1990,26(7):722-730
Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved
in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with
bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol,
putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells
occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture.
Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics.
Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated
and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male
mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal
or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258,
epithelial cells of mouse origin were distinguishable from stromal cells of rat origin.
Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship
GM08730 to T. T. 相似文献
70.
Graham W. Burton Keith U. Ingold Kevin H. Cheeseman Trevor F. Slater 《Free radical research》1990,11(1):99-107
-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of -tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR--tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process. 相似文献