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21.
Preparation and structural characterization of large heparin-derived oligosaccharides 总被引:4,自引:2,他引:2
Pervin Azra; Gallo Cindy; Jandik Kenneth A; Han Xue-Jun; Linhardt Robert J. 《Glycobiology》1995,5(1):83-95
Porcine mucosal heparin was partially depolymerized with heparinlyase I and then fractionated into low-molecularweight (<5000)and high-molecular-weight (>5000) oligosaccharides by pressurefiltration. The high-molecular-weight oligosaccharide mixture({small tilde}50 wt% of the starting heparin) also containedintact heparin. This intact polymer complicates oligosacsharidepurification. Thus, the low-molecular-weight fraction was usedto prepare homogeneous oligosaccharides for structural characterization.The low-molecular-weight oligosaccharide mixture was first fractionatedby low pressure gel permeation chromatography into size-uniformmixtures of disaccharides, tetrasaccharides, hexasaccharides,octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharidesand higher oligosaccharides. Each size-fractionated mixturewas then purified on the basis of charge by repetitive semi-preparativestrong-anion-exchange high-performance liquid chromatography.This approach has led to the isolation of 14 homogeneous oligosaccharidesfrom disaccharide to tetradecasaccharide. The purity of theseheparin-derived oligosaccharides was determined by gradientpolyacrylamide gel electrophoresis, analytical strong-anion-exchangehigh-performance liquid chromatography, capillary electrophoresisand one-dimensional nuclear resonance spectroscopy. The structureof these oligosaccharides was established using 600 MHz two-dimensionalnuclear resonance spectroscopy . The spectral methods used includedhomonuclear correlation spectroscopy, nuclear Overhauser effectspectroscopy and heteronuclear multiple quantum coherence spech-clscopy.The 1H/1H connectivities of the protons of each sugar residuein an oligosaccharide were established by two-dimensional homonuclearcorrelation spectroscopy, while 1H/13C assignments were madeusing 1H inverse detection. One- and two-dimensional nuclearresonance spectroscopic analysis of these heparin oligosaccharidesshowed two closely related groups of heparin-oligosaccharidesare afforded by enzymatic depolymerization of heparin. One groupis fully sulphated, having the structures 相似文献
22.
A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum 总被引:8,自引:0,他引:8
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In vitro mutagenic techniques have generated an asp→glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C→A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn2+.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn2+.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site. 相似文献
23.
Cindy Lloyd John R. Kennedy Joseph Mendicino 《In vitro cellular & developmental biology. Plant》1984,20(5):416-432
Summary Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability
of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the
active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin
glycoprotein was about 0.035 mg per cm2 per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with
radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal
incorporation was observed at 200 μM glucosamine. A higher concentration of35SO4, 1000 μM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 μg/ml increased the rate of secretion twofold,
whereas 0.1 to 100 μg/ml of hydrocortisone and 0.1 to 100 μg/ml of epinephrine significantly decreased the rate of secretion.
Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10−5
M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10−9
M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats.
Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified.
The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified
by chromatography on Sepharose CL-6B columns under dissociating conditions in 2M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually
indistingushable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified
glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More
than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same
molar ratio ofN-acetylgalactosamine,N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations.
This investigation was supported by U.S. Public Health Service Grants HL 20868, HL 24688, and HL 24718 from the National Heart,
Lung and Blood Institute, Bethesda, MD, and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and
Kidney Diseases, Bethesda, MD. 相似文献
24.
Ronald S. Oremland Cindy Umberger Charles W. Culbertson Richard L. Smith 《Applied microbiology》1984,47(5):1106-1112
The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO3− + NO2− concentrations (≤26 μM) were below the apparent Km (50 μM) for nitrate. During the rainy season, when ambient NO3− + NO2− concentrations were higher (37 to 89 μM), an accurate estimate of the Km could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N2O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N2O in the presence of C2H2 was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N2O loss was caused by incomplete blockage of N2O reductase by C2H2 at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 μmol of N2 m−2 h−1 (for undisturbed sediments) to 17 to 280 μmol of N2 m−2 h−1 (for shaken sediment slurries). 相似文献
25.
26.
A patient with severe aortic insufficiency due to fenestration of the non-coronary aortic valve leaflet is described. A preoperative echocardiogram demonstrated early closure of the mitral valve and early diastolic separation of the aortic valve leaflets. These findings disappeared after partial surgical correction and subsequent hemodynamic improvement. Premature opening of the aortic valve is common in severe aortic insufficiency. 相似文献
27.
The oxidation of 3-hydroxy[3-14C]butyrate to CO2 and its incorporation into cerebral lipids by cortex slices from one-week old rats were markedly inhibited by methylmalonate. However, methylmalonate had no effect on the metabolism of labelled aceto- acetate, glucose and acetate by brain slices. Addition of propionate in the incubation medium reduced cerebral lipogenesis from labelled 3-hydroxybutyrate and acetate. Acute methylmalonic acidemia induced in one-week old pups by injecting 3% methylmalonate solution caused a reduction in the incorporation of labelled 3-hydroxybutyrate into cerebral lipids. However, acute methylmalonic acidemia had no effect on cerebral lipogensis from labelled acetate. These findings show (i) the conversion of 3-hydroxybutyrate to acetoacetate by 3-hydroxybutyrate dehydrogenase in the brain is inhibited by methylmalonate, and (ii) an inhibition of cerebral lipid synthesis by propionate, which also accumulates in patients with methylmalonic aciduria. 相似文献
28.
29.
V Marigliano A Musca C Cordova I Cammarella 《Bollettino della Società italiana di biologia sperimentale》1976,52(15):1131-1136
30.
Tadeusz J. Kawecki Berra Erkosar Cindy Dupuis Brian Hollis R. Craig Stillwell Martin Kapun 《Molecular biology and evolution》2021,38(7):2732
Periods of nutrient shortage impose strong selection on animal populations. Experimental studies of genetic adaptation to nutrient shortage largely focus on resistance to acute starvation at adult stage; it is not clear how conclusions drawn from these studies extrapolate to other forms of nutritional stress. We studied the genomic signature of adaptation to chronic juvenile malnutrition in six populations of Drosophila melanogaster evolved for 150 generations on an extremely nutrient-poor larval diet. Comparison with control populations evolved on standard food revealed repeatable genomic differentiation between the two set of population, involving >3,000 candidate SNPs forming >100 independently evolving clusters. The candidate genomic regions were enriched in genes implicated in hormone, carbohydrate, and lipid metabolism, including some with known effects on fitness-related life-history traits. Rather than being close to fixation, a substantial fraction of candidate SNPs segregated at intermediate allele frequencies in all malnutrition-adapted populations. This, together with patterns of among-population variation in allele frequencies and estimates of Tajima’s D, suggests that the poor diet results in balancing selection on some genomic regions. Our candidate genes for tolerance to larval malnutrition showed a high overlap with genes previously implicated in acute starvation resistance. However, adaptation to larval malnutrition in our study was associated with reduced tolerance to acute adult starvation. Thus, rather than reflecting synergy, the shared genomic architecture appears to mediate an evolutionary trade-off between tolerances to these two forms of nutritional stress. 相似文献