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31.
Simultaneous determination of ascorbic acid and dehydroascorbic acid in cultures of C3H/10T1/2 cells
Luminita L. V. Ibric William F. Benedict Andrew R. Peterson 《In vitro cellular & developmental biology. Plant》1988,24(7):669-676
Summary A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing
reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations
with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied
to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments
with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both
ascorbic acid and dehydroascorbic acid to 10 to 20 pmol.
This research was supported by grant CA 09320 and CA 31574 from the National Cancer Institute, Bethesda, MD, and grant BC441
from The American Cancer Society. 相似文献
32.
T. J. Budd C. D. Dolman A. M. Lawson W. Chai J. Saxton F. W. Hemming 《Glycoconjugate journal》1992,9(5):274-278
N-Acetylneuraminic acid (Neu5Ac) andN-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.Abbreviations Neu5Ac
N-acetylneuraminic acid
- Neu5Gc
N-glycoloylneuraminic acid
- RPM
rat promegakaryoblast
- MEG-01
human megakaryoblastic leukaemia cell line
- PAD
pulsed amperometric detection
- WGA
wheat germ agglutinin
- FCS
foetal calf serum
- PPEADF
phosphatidylethanolamine dipalmitoyl
- LSIMS
liquid secondary ion mass spectrometry
- HPAEC
high performance anion exchange chromatography
- TBA
thiobarbituric acid 相似文献
33.
Radiolabelled calmodulin has previously been used to screen cDNA expression libraries to isolate calmodulin-binding proteins.
We have modified this technique for the isolation of plant calmodulin-binding proteins. [35S]-methionine was used instead of the inorganic [35S]-sulfate, or125I used in previous methods. In addition, theE. coli pET expression system was chosen to obtain high levels of recombinant calmodulin at the time of labelling. The procedure
thus takes into account both the specific activity of the probe and the amount of protein necessary for screening a large
number of filters. Here we describe in detail a procedure for the production and purification of [35S]-recombinant calmodulin and the use of the radiolabelled protein as a probe to screen plant cDNA expression libraries. The
[35S]-labeled calmodulin probe easily detects the λICM-1 phage encoding a partial mouse calmodulin-dependent protein kinase II
that was previously isolated using a [125I]-calmodulin probe (Sikela and Hahn, 1987). Subsequently, a tobacco root cDNA expression library was screened and a positive
clone encoding a calcium-dependent calmodulin-binding protein was isolated. 相似文献
34.
Five strains of Bradyrhizobium japonicum (USDA 6, 110, 122, 138, and 143) were screened in cell culture for tolerance to acidity (pH 4.2, 4.4, and 4.6) and Al (0, 3, 4, 5, and 6 mg L–1) under low P conditions. Each strain was later grown in association with seven soybean [Glycine max. (L) Merr.] cultivars which were also screened for tolerance to the same stresses in nutrient culture to determine which soybean-Bradyrhizobium combinations would establish the most effective symbiotic N2 fixing relationships. Results indicated that strains USDA 110 and 6 were more tolerant than USDA 122, 138 and 143 with USDA 110 being the most tolerant. Acidity appeared to be the more severe stress; but even when strains showed tolerance to the stresses, cell numbers were significantly reduced. This suggests that colonization of soils and soybean roots can be adversely affected under similar conditions in the field which may result in reduced nodulation. The strains found to be more tolerant to the stresses were more effective N2 fixers in symbiosis with all soybean cultivars, with USDA 110 being definitely superior. The association between the more tolerant strains and cultivars had the largest nitrogenase activity. Further studies on the inclusion of tolerant Bradyrhizobium strains in inoculum used on tolerant soybean cultivars in the field are warranted. 相似文献
35.
E. Friar G. Kochert 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1991,82(6):697-703
Summary Bamboo species are difficult to identify because flowering material is seldom available and taxonomy is of necessity based on vegetative characters. To evaluate the utility of restriction fragment length polymorphism (RFLP) analysis in bamboo systematics and germplasm screening, a library of random genomic probes from a Phyllostachys nigra PstI library was constructed. Probes from the library were used to screen bamboo germplasm consisting mostly of temperate bamboos of the genus Phyllostachys. RFLP variation was abundant, and species-specific patterns were readily obtained. Chloroplast DNA showed little variation among the bamboo accessions analyzed. 相似文献
36.
A. A. Serikov 《Journal of biological physics》1991,18(1):65-77
Model equations for the kinetics of the synthesis and decay of molecular aggregates are used to show the high sensitivity of equilibrium concentrations of high-molecular aggregates to external radiation. This phenomenon is used to explain the effects of low-intensity microwave fields on the functioning of biological systems. The experimental results on the influence of SHF-radiation on ferricyanide reduction by erythrocytes are interpreted in detail. 相似文献
37.
High-affinity binding of3H-folate in Triton X-100 solubilized membranes of human liver displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. Ultrogel® AcA 44 chromatography of solubilized membranes saturated with3H-folate revealed a major peak of 100 kDa and a minor peak of 25 kDa. The 100 kDa peak could represent a hydrophobic membrane associated molecular form of the protein. This notion was supported by the fact that the two peaks had identical molecular weights as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis with immunoblotting. 相似文献
38.
39.
Addition of calpain II (EC 3.4.22.17) to soluble proteins from 10-day-old rat lens caused an increase in turbidity and production of water-insoluble protein. The insolubilization increased with higher concentrations of both lens protein and calpain II, it could be prevented by the cysteine protease inhibitor E-64; it required at least 0.5 mM Ca2+, it was limited to 6% of the soluble protein present and resulted from precipitation β-crystallin polypeptides. When compared by two-dimensional electrophoresis, the insoluble β-crystallin polypeptides produced by calpain II were similar to insoluble β-crystallin polypeptides found incataractous lenses. Trypsin also caused insolubilization of β-crystallin polypeptides, but these polypeptides were unlike polypeptides produced during cataract formation. These data suggested that the loss of solubility was due to a specific removal of N/or C-terminal extensions from β-crystallin polypeptides by calpain II, and that a similar process may occur in vivo during cataract formation. It is hypothesized that the insoluble protein produced by calpain II causes cataract by increasing light scatter in the lens. 相似文献
40.
Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport. 总被引:3,自引:3,他引:0 下载免费PDF全文
R. A. Binnie H. Zhang S. Mowbray M. A. Hermodson 《Protein science : a publication of the Protein Society》1992,1(12):1642-1651
Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically. 相似文献