Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast.
(1/1610)
Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate. (+info)
Determination of the anomeric configurations of Corbicula ceramide di- and trihexoside by chromium trioxide oxidation.
(2/1610)
The anomeric configurations of Corbicula ceramide dihexoside and ceramide trihexoside were determined by chromium trioxide oxidation and the structures of these lipids were shown to be Man-beta(1 leads to 4)-Glc-beta(1 leads to 1)-ceramide and Man-alpha(1 leads to 4)-Man-beta(1 leads to 4)-Glc-beta(1 leads to 1)-ceramide. These results are compatible with those obtained by enzymic hydrolysis reported previously. (+info)
Antioxidative and chelating activities of phenylpropanoid glycosides from Pedicularis striata.
(3/1610)
AIM: To study the antioxidative and iron chelating activities of phenylpropanoid glycosides (PPG) isolated from a Chinese herb Pedicularis striata. METHODS: Antioxidative effects of PPG on lipid peroxidation induced by FeSO4-edetic acid in linoleic acid were measured by thiobarbituric acid method. Chelating activities of PPG for Fe2+ were tested by differential spectrum method. RESULTS: The reaction rates (A532.min-1) of lipid peroxidation were 0.0046 in the control, 0.0021 in verbascoside group, and 0.0008 in isoverbascoside group. The chelating activity of isoverbascoside was 2-fold stronger than that of verbascoside. Permethyl verbascoside showed neither antioxidative nor chelating activities. CONCLUSION: The inhibitory effects of PPG with phenolic hydroxy groups on lipid peroxidation are owing to their chelating properties. Under physiological condition PPG-Fe2+ chelates are sufficiently stable. Thus PPG are able to inhibit the Fe(2+)-dependent lipid peroxidation in vivo through chelating Fe2+ and exhibit their therapeutic potential by the same mechanism in vitro. (+info)
Reduction of serum cholesterol and hypercholesterolemic atherosclerosis in rabbits by secoisolariciresinol diglucoside isolated from flaxseed.
(4/1610)
BACKGROUND: Secoisolariciresinol diglucoside (SDG) is a plant lignan isolated from flaxseed. Lignans are platelet-activating factor-receptor antagonists that would inhibit the production of oxygen radicals by polymorphonuclear leukocytes. SDG is an antioxidant. Antioxidants studied thus far are known to reduce hypercholesterolemic atherosclerosis. The objective of this study was to determine the effect of SDG on various blood lipid and aortic tissue oxidative stress parameters and on the development of atherosclerosis in rabbits fed a high-cholesterol diet. METHODS AND RESULTS: Rabbits were assigned to 4 groups: group 1, control; group 2, SDG control (15 mg. kg body wt-1. d-1 PO); group 3, 1% cholesterol diet; and group 4, same as group 3 but with added SDG (15 mg. kg body wt-1. d-1 PO). Blood samples were collected before (time 0) and after 4 and 8 weeks of experimental diets for measurement of serum triglycerides, total cholesterol (TC), and LDL, HDL, and VLDL cholesterol (LDL-C, HDL-C, and VLDL-C). The aorta was removed at the end of the protocol for assessment of atherosclerotic plaques; malondialdehyde, an aortic tissue lipid peroxidation product; and aortic tissue chemiluminescence, a marker for antioxidant reserve. Serum TC, LDL-C, and the ratios LDL-C/HDL-C and TC/HDL-C increased in groups 3 and 4 compared with time 0, the increase being smaller in group 4 than in group 3. Serum HDL-C decreased in group 3 and increased in group 4 compared with time 0, but changes were lower in group 3 than in group 4. SDG reduced TC and LDL-C by 33% and 35%, respectively, at week 8 but increased HDL-C significantly, by>140%, as early as week 4. It also decreased TC/LDL-C and LDL-C/HDL-C ratios by approximately 64%. There was an increase in aortic malondialdehyde and chemiluminescence in group 3, and they were lower in group 4 than in group 3. SDG reduced hypercholesterolemic atherosclerosis by 73%. CONCLUSIONS: These results suggest that SDG reduced hypercholesterolemic atherosclerosis and that this effect was associated with a decrease in serum cholesterol, LDL-C, and lipid peroxidation product and an increase in HDL-C and antioxidant reserve. (+info)
Comparative study of carbohydrate-protein complexes. II. Determination of hydroxylysine and its glycosides in human skin and scar collagens by an improved method.
(5/1610)
A modification of the existing methods for measuring hydroxylysine, galactosylhydroxylysine, and glucosylgalactosylhydroxylysine is described. The method is based on analysis with an automated amino acid analyzer using a conventional separation system for basic amino acids. The prior removal of acidic and neutral amino acids was necessary. This was achieved by passing an alkaline hydrolysate of collagen through a column of Amberlite CG-120, Type II (H+) and washing the column with 8% aqueous pyridine. A basic fraction containing the hydroxylysine compounds was then recovered from the column by elution with 3 M NH4OH. Model experiments showed that hydroxylysine and its glycosides could be analyzed with an hour and that recoveries exceeded 90%. This method was applied to human tissues to investigate whether the dermal scar is different in collagen composition from normal skin. With the limited number of samples analyzed, the data suggested that long-standing scar tissues reverted to a composition similar to that of normal skin. The composition of hydroxylysine-linked carbohydrate units is also discussed on the basis of the age-related change. (+info)
Latency of some glycosidases of rat liver lysosomes.
(6/1610)
The latency of the alpha-glucosidase activity of intact rat liver lysosomes was studied by using four substrates (glycogen, maltose, p-nitrophenyl, alpha-glucoside, alpha-fluoroglucoside) at a range of substrate concentrations. The results indicate that the entire lysosome population is impermeable to glycogen and maltose, but a proportion of lysosomes are permeable to alpha-fluoroglucoside and a still higher proportion permeable to p-nitrophenyl alpha-glucoside. Incubation at 37 degrees C in an osmotically protected buffer of of pH 5.0 caused lysosomes to become permeable to previously impermeant substrates and ultimately to release their alpha-glucosidase into the medium. The latencies of lysosomal beta-glucosidase and beta-galactosidase were examined by using p-nitrophenyl beta-glucoside and beta-galactoside as substrates. The results indicate permeability properties to these substrates similar to that to p-nitrophenyl alpha-glucoside. On incubation in an osmotically protected buffer of pH 5, lysosomes progressively released their beta-galactosidase in soluble form, but beta-glucosidase remained attached to sedimentable material. Lysosomal beta-glucosidase was inhibited by 0.1% Triton X-100; alpha-glucosidase and beta-galactosidase were not inhibited. (+info)
Elucidation of a PTS-carbohydrate chemotactic signal pathway in Escherichia coli using a time-resolved behavioral assay.
(7/1610)
Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)-carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates D-glucose and its nonmetabolizable analog methyl alpha-D-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 +/- 3.1 s-1. The response threshold was <10 nM for glucose. Responses to methyl alpha-D-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP-CheW-CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption. (+info)
Effects of endotoxin on surfactant protein A and D stimulation of NO production by alveolar macrophages.
(8/1610)
Surfactant protein (SP) A and SP-D affect numerous functions of immune cells including enhancing phagocytosis of bacteria and production of reactive species. Previous studies have shown that SP-A and SP-D bind to a variety of bacteria and to the lipopolysaccharide (LPS) components of their cell walls. In addition, purified preparations of SPs often contain endotoxin. The goals of this study were 1) to evaluate the effects of SP-A and SP-D and complexes of SPs and LPS on the production of nitric oxide metabolites by rat alveolar macrophages and 2) to evaluate methods for the removal of endotoxin with optimal recovery of SP. Incubation of SP-A or SP-D with polymyxin, 100 mM N-octyl-beta-D-glucopyranoside, and 2 mM EDTA followed by dialysis was the most effective method of those tested for reducing endotoxin levels. Commonly used storage buffers for SP-D, but not for SP-A, inhibited the detection of endotoxin. There was a correlation between the endotoxin content of the SP-A and SP-D preparations and their ability to stimulate production of nitrite by alveolar macrophages. SP-A and SP-D treated as described above to remove endotoxin did not stimulate nitrite production. These studies suggest that the functions of SP-A and SP-D are affected by endotoxin and illustrate the importance of monitoring SP preparations for endotoxin contamination. (+info)