Sequence analysis of heparan sulphate and heparin oligosaccharides.
(1/153)
The biological activity of heparan sulphate (HS) and heparin largely depends on internal oligosaccharide sequences that provide specific binding sites for an extensive range of proteins. Identification of such structures is crucial for the complete understanding of glycosaminoglycan (GAG)-protein interactions. We describe here a simple method of sequence analysis relying on the specific tagging of the sugar reducing end by 3H radiolabelling, the combination of chemical scission and specific enzymic digestion to generate intermediate fragments, and the analysis of the generated products by strong-anion-exchange HPLC. We present full sequence data on microgram quantities of four unknown oligosaccharides (three HS-derived hexasaccharides and one heparin-derived octasaccharide) which illustrate the utility and relative simplicity of the technique. The results clearly show that it is also possible to read sequences of inhomogeneous preparations. Application of this technique to biologically active oligosaccharides should accelerate progress in the understanding of HS and heparin structure-function relationships and provide new insights into the primary structure of these polysaccharides. (+info)
Sequencing complex polysaccharides.
(2/153)
Although rapid sequencing of polynucleotides and polypeptides has become commonplace, it has not been possible to rapidly sequence femto- to picomole amounts of tissue-derived complex polysaccharides. Heparin-like glycosaminoglycans (HLGAGs) were readily sequenced by a combination of matrix-assisted laser desorption ionization mass spectrometry and a notation system for representation of polysaccharide sequences. This will enable identification of sequences that are critical to HLGAG biological activities in anticoagulation, cell growth, and differentiation. (+info)
Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid: mechanism and intermediates.
(3/153)
The reaction mechanism for the formation of 2'-deoxy-oxanosine from 2'-deoxyguanosine by nitrous acid was explored using methyl derivatives of guanosine and an isolated intermediate of the reaction. When 1-methylguanosine was incubated with NaNO(2)under acidic conditions, N (5) -methyloxanosine and 1-methylxanthosine were generated, whereas the same treatment of N (2), N (2)-dimethylguanosine generated no product. In a similar experiment without NO(2)(-), participation of a Dimroth rearrangement was ruled out. In the guanosine-HNO(2)reaction system, an intermediate with a half-life of 5.6 min (pH 7.0, 20 degrees C) was isolated and tentatively identified as a diazoate derivative of guanosine. The diazoate intermediate was converted into oxanosine and xanthosine at a molar ratio (oxanosine:xanthosine) of 0.26 at pH 7.0 and 20 degrees C. The ratio was not affected by the incubation pH between 2 and 10, but increased linearly with temperature from 0.22 (0 degrees C) to 0.32 (50 degrees C). The addition of acetone also increased the ratio up to 0.85 (98% acetone). Based on these results, a con-ceivable pathway for the formation of 2'-deoxyoxanosine from 2'-deoxyguanosine by HNO(2)is proposed. (+info)
The nitration of platelet cytosolic proteins during agonist-induced activation of platelets.
(4/153)
The nitration of protein tyrosine residues by peroxynitrous acid has been associated with pathological conditions. Here it is shown, using a sensitive competitive enzyme-linked immunosorbent assay and immunoblotting for nitrotyrosine, that spontaneous nitration of specific proteins occurs during a physiological process, the activation of platelets by collagen. One of the main proteins nitrated is vasodilator-stimulated phosphoprotein. Endogenous synthesis of nitric oxide and activity of cyclo-oxygenase were required for the nitration of tyrosine. The nitration was mimicked by addition of peroxynitrite to unstimulated platelets, although the level of nitrotyrosine formation was greater and its distribution among the proteins was less specific. (+info)
Positive selection of mutants with deletions of the gal-chl region of the Salmonella chromosome as a screening procedure for mutagens that cause deletions.
(5/153)
We have developed a convenient and specific positive selection for long deletions through the gal region of the chromosomes of Salmonella typhimurium and Escherichia coli. Through simultaneous selection for mutations in the two closely linked genes, gal and chlA, a variety of deletions of varying length, some extending through as much as 1 min of the chromosome, could be readily obtained. Many of these deletions resulted in the loss of a gene, which we named dhb, concerned with the ability of the bacterium to synthesize the iron chelating agent enterobactin. The selection was adapted for the screening of mutagens for their ability to generate long deletions in the bacterial deoxyribonucleic acid. Forty agents were screened for this capability. Nitrous acid, previously reported to be an efficient mutagen for this purpose, increased the frequency of deletion mutations 50-fold in our system. Three others, nitrogen mustard, mitomycin C, and fast neutrons, were shown to increase the frequency of long deletions between five- and eightfold. The remainder were found to be incapable of generating these deletions. (+info)
Genetic analysis of adenovirus type 2. I. Isolation and genetic characterization of temperature-sensitive mutants.
(6/153)
Temperature-sensitive mutants which replicate normally at 33 C but poorly at 39 C were isolated from nitrosoguanidine- or nitrous acid-mutagenized adenovirus 2 by (i) testing the cytopathic effect or inclusion body-forming capacity of random plaque isolates, or (ii) reduced plaque enlargement upon shifting from 33 to 39 C. Thirty-six mutants were isolated with 33 C/39 C plaque ratios varying from 20 to 10-5. Some of these mutants could be arranged into 13 groups by the complementation test. By means of recombination analysis a provisional linear genetic map was constructed. (+info)
Plasmodium falciparum glycosylphosphatidylinositol-induced TNF-alpha secretion by macrophages is mediated without membrane insertion or endocytosis.
(7/153)
The glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum are believed to contribute to the pathogenesis of malaria by inducing the secretion of proinflammatory cytokines by macrophages. Previous studies have shown that P. falciparum GPIs elicit toxic immune responses by protein tyrosine kinase (PTK)- and protein kinase C (PKC)-mediated cell signaling pathways, which are activated by the carbohydrate and acyl moieties of the intact GPIs, respectively. In this study, we show that induction of TNF-alpha by P. falciparum GPIs in macrophages is mediated by the recognition of the distal fourth mannose residue. This event is critical but not sufficient for the productive cell signaling; interaction by the acylglycerol moiety of GPIs is also required. These novel interactions are coupled to previously demonstrated PTK and PKC pathways, since the specific inhibitors of these kinases effectively blocked the GPI-induced TNF-alpha production. Surprisingly, sn-2 lyso-GPIs were also able to elicit TNF-alpha secretion. Contrary to the prevailing notion, GPIs are neither inserted to the plasma membranes nor endocytosized. Thus, this study defines the GPI structural requirements and reveals a novel mechanism for the outside-in activation of cell signaling by P. falciparum GPIs in inducing proinflammatory responses. (+info)
A direct method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels.
(8/153)
BACKGROUND: In vertebrates, two types of cholinesterases exist, acetylcholinesterase and butyrylcholinesterase. The function of acetylcholinesterase is to hydrolyse acetylcholine, thereby terminating the neurotransmission at cholinergic synapse, while the precise physiological function of butyrylcholinesterase has not been identified. The presence of cholinesterases in tissues that are not cholinergically innervated indicate that cholinesterases may have functions unrelated to neurotransmission. Furthermore, cholinesterases display a genuine aryl acylamidase activity apart from their predominant acylcholine hydrolase activity. The physiological significance of this aryl acylamidase activity is also not known. The study on the aryl acylamidase has been, in part hampered by the lack of a specific method to visualise this activity. We have developed a method to visualise the aryl acylamidase activity on cholinesterase in polyacrylamide gels. RESULTS: The o-nitroaniline liberated from o-nitroacetanilide by the action of aryl acylamidase activity on cholinesterases, in the presence of nitrous acid formed a diazonium compound. This compound gave an azo dye complex with N-(1-napthyl)-ethylenediamine, which appeared as purple bands in polyacrylamide gels. Treating the stained gels with trichloroacetic acid followed by Tris-HCl buffer helped in fixation of the stain in the gels. By using specific inhibitors for acetylcholinesterase and butyrylcholinesterase, respectively, differential staining for the aryl acylamidase activities on butyrylcholinesterase and acetylcholinesterase in a sample containing both these enzymes has been demonstrated. A linear relationship between the intensity of colour developed and activity of the enzyme was obtained. CONCLUSIONS: A novel method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels has been developed. (+info)