Biochemical engineering of the N-acyl side chain of sialic acid: biological implications.
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N-Acetylneuraminic acid is the most prominent sialic acid in eukaryotes. The structural diversity of sialic acid is exploited by viruses, bacteria, and toxins and by the sialoglycoproteins and sialoglycolipids involved in cell-cell recognition in their highly specific recognition and binding to cellular receptors. The physiological precursor of all sialic acids is N-acetyl D-mannosamine (ManNAc). By recent findings it could be shown that synthetic N-acyl-modified D-mannosamines can be taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acids in vitro and in vivo. Successfully employed D-mannosamines with modified N-acyl side chains include N-propanoyl- (ManNProp), N-butanoyl- (ManNBut)-, N-pentanoyl- (ManNPent), N-hexanoyl- (ManNHex), N-crotonoyl- (ManNCrot), N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc), and N-azidoacetyl D-mannosamine (ManNAc-azido). All of these compounds are metabolized by the promiscuous sialic acid biosynthetic pathway and are incorporated into cell surface sialoglycoconjugates replacing in a cell type-specific manner 10-85% of normal sialic acids. Application of these compounds to different biological systems has revealed important and unexpected functions of the N-acyl side chain of sialic acids, including its crucial role for the interaction of different viruses with their sialylated host cell receptors. Also, treatment with ManNProp, which contains only one additional methylene group compared to the physiological precursor ManNAc, induced proliferation of astrocytes, microglia, and peripheral T-lymphocytes. Unique, chemically reactive ketone and azido groups can be introduced biosynthetically into cell surface sialoglycans using N-acyl-modified sialic acid precursors, a process offering a variety of applications including the generation of artificial cellular receptors for viral gene delivery. This group of novel sialic acid precursors enabled studies on sialic acid modifications on the surface of living cells and has improved our understanding of carbohydrate receptors in their native environment. The biochemical engineering of the side chain of sialic acid offers new tools to study its biological relevance and to exploit it as a tag for therapeutic and diagnostic applications. (+info)
The cathepsin B inhibitor z-FA.fmk inhibits cytokine production in macrophages stimulated by lipopolysaccharide.
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Cathepsin B has previously been shown to proteolytically activate the proinflammatory caspase-11 in vitro. Here we show that cathepsin B is not involved in activation of caspase-11 induced by lipopolysaccharide (LPS) and subsequent maturation of interleukin (IL)-1beta in macrophages. Nevertheless, we found that the cathepsin B inhibitor benzyloxycarbonyl-Phe-Ala-fluoromethylketone (z-FA.fmk) prevents LPS-induced production of IL-1alpha, IL-1beta, and tumor necrosis factor at the transcriptional level. The latter was not because of cathepsin B inhibition, but was mediated by inhibition of the transactivation potential of the nuclear factor kappaB (NF-kappaB). z-FA.fmk did not prevent LPS-induced activation of p38 mitogen-activated protein kinase, which was shown to be involved in NF-kappaB transactivation in response to LPS. These results suggest that the previously described therapeutic effect of z-FA.fmk in the treatment of rheumatoid arthritis might not only result from inhibition of cathepsin B but also implicates an important contribution from the inhibition of NF-kappaB-dependent gene expression. (+info)
Key regulatory oxysterols in liver: analysis as delta4-3-ketone derivatives by HPLC and response to physiological perturbations.
(51/1302)
A number of oxysterols have been implicated in metabolic regulation. Key among these are (24S),25-epoxycholesterol and (24S)-hydroxycholesterol, high affinity ligands for the nuclear transcription factor liver X receptor alpha; 27-hydroxycholesterol, a bile acid synthetic intermediate; and 25-hydroxycholesterol, which has been used to study regulation of lipid metabolism by the sterol regulatory element-binding protein family of transcription factors. Investigation of the physiological importance of these compounds in vivo has been hampered by lack of analytical methods to reproducibly and accurately determine their concentrations in tissues. This article describes a method designed to determine quantitatively the amounts of these important side-chain oxysterols by derivatization to the Delta4-3-ketones followed by high performance liquid chromatography. The method was validated with known standards and then was used to determine the concentrations of these oxysterols in rodent liver under various physiological conditions. All four oxysterols were present in the picogram per milligram protein range and have distinct subcellular distributions and responses to physiological perturbations in vivo. (+info)
Ab initio molecular orbital study of reactivity of active alkyl groups. IV. Nitrosation of acyclic carbonyl compound with methyl nitrite via "open-chain" transition state.
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The mechanism of the nitrosation of enolate anion of acetone [CH3COCH2]- (1) with methyl nitrite CH3ONO (2) via an "open-chain" transition state without Na+ in the C-N bond formation process was studied by the ab initio MO method. The complex [CH3COCH2NO(OCH3)]- (C-II) was first formed from the adduct (C-I) of 1 and 2 through the transition state (TSI). Finally, E-1-hydroxyimino-2-oxo-propane CH3COCH=NOH (3E), together with Z-form (3Z), was obtained by way of the elimination process. It has become apparent that 3E is formed when C-II-A is produced in the C-N bond formation process. (+info)
Acute, chronic and terminal toxicity to 4'-demethylepipodophyllotoxin thenylidene glucoside (VM26) in mice.
(53/1302)
The development of toxicity to 4'-demethylepipodophyllotoxin-9-(4,6,-O-thenylidene-beta-glucopyranoside) an epipodophyllotoxin with oncolytic activity, was characterized in mice treated three times at 3-day intervals with 10 mg of drug i.p. per kg of body weight. Changes in organ function and general metabolism were determined by measuring 18 constituents of blood for up to 10 weeks after drug administration. The results indicate three distinct phases of toxicity to 4'-demethylepipodophyllotoxin 9-(4,6-O-2-thenylidene-beta-glucopyranoside). Acute toxicity developed within the first 10 days and was expressed by a depressed hematocrit and elevated plasma levels of glutamate-pyruvate transaminase, glutamate-oxaloacetate transaminase, lactic dehydrogenase, amylase, lipase, and uric acid. By 4 weeks, levels ahd returned to normal. The acute phase was followed by a chronic phase, which was characterized by progressive decreases in plasma levels of glucose, cholesterol, albumin, and total protein. Finally, about 7 weeks after treatment, a terminal phase indicated by correlated increases in glutamate-pyruvate transaminase, glutamate-oxaloacetate transaminase, lactic dehydrogenase, and blood urea nitrogen became apparent. Plasma levels of creatine phosphokinase, calcium, inorganic phosphate, total bilirubin, ketones, and alkaline phosphatase did not change. Although the pancreas liver and marrow were all affected during acute toxicity, boserved changes in blood components during the chronic and terminal phases correlate best with continued hepatotoxicity. The present evidence on delayed toxicity to 4'-demethylepipodophyllotoxin 9-(4,6-o-2-thenylidene-beta-D-glucopyranoside) is most compatible with irreversible hepatotoxocity which leads to metabolic deficiencies and terminates in death of mice. (+info)
Effect of ketone infusions on amino acid and nitrogen metabolism in man.
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To evaluate the role of hyperketonemia in the hypoalaninemia and decreased protein catabolism of prolonged starvation, Na dl-beta-hydroxybutyrate was administered as a primed continuous 3-6-h infusion in nonobese subjects and in obese subjects in the postabsorptive state and after 3 days and 3-5 1/2 wk of starvation. An additional obese group received 12-h ketone infusions on 2 consecutive days after 5-10 wk of fasting. The ketone infusion in nonobese and obese subjects studied in the postabsorptive state resulted in total blood ketone acid levels of 1.1-1.2 mM, a 5-15 mg/100 ml decrease in plasma glucose, and unchanged levels of insulin, glucagon, lactate, and pyruvate. Plasma alanine fell by 21% (P smaller than 0.001) in 3 h. In contrast, other amino acids were stable or varied by less than 10%. Infusions lasting 6 h reduced plasma alanine by 37%, reaching levels comparable to those observed in prolonged starvation. Equimolar infusions of NaC1 and/or administration of NaHCO3 failed to alter plasma alanine levels. During prolonged fasting, plasma alanine, which had fallen by 40% below prefast levels, fell an additional 30% in response to the ketone infusion. In association with repeated prolonged (12 h) infusions in subjects fasted 5-10 wk, urinary nitrogen excretion fell by 30%, returning to base line after cessation of theinfusions and paralleling the changes in plasma alanine. Ketone infusins resulted in two- to fourfold greater increments in blood ketone acids in fasted as compared to postabsorptive subjects. It is concluded that increased blood ketone acid levels induced by infusions of Na DL-beta-hydroxybutyrate result in hypoalaninemia and in nitrogen conservation in starvation. These data suggest that hyperketonemia may be a contributory factor in the decreased availability or circulating alanine and reduction in protein catabolism characteristic of prolonged fastings9 (+info)
Cyclopentenone prostaglandins of the J series inhibit the ubiquitin isopeptidase activity of the proteasome pathway.
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Electrophilic eicosanoids of the J series, with their distinctive cross-conjugated alpha,beta-unsaturated ketone, inactivate genetically wild type tumor suppressor p53 in a manner analogous to prostaglandins of the A series. Like the prostaglandins of the A series, prostaglandins of the J series have a structural determinant (endocyclic cyclopentenone) that confers the ability to impair the conformation, the phosphorylation, and the transcriptional activity of the p53 tumor suppressor with equivalent potency and efficacy. However, J series prostaglandins have a unique structural determinant (exocyclic alpha,beta-unsaturated ketone) that confers unique efficacy as an apoptotic agonist. In seeking to understand how J series prostaglandins cause apoptosis despite their inactivation of p53, we discovered that they inhibit the ubiquitin isopeptidase activity of the proteasome pathway. In this regard, J series prostaglandins were more efficacious inhibitors than representative members of the A, B, or E series prostaglandins. Disruption of the proteasome pathway with proteasome inhibitors can cause apoptosis independently of p53. Therefore, this finding helps reconcile the p53 transcriptional independence of apoptosis caused by Delta12-prostaglandin J(2). This discovery represents a novel mechanism for proteasome pathway inhibition in intact cells. Furthermore, it identifies isopeptidases as novel targets for the development of antineoplastic agents. (+info)
3alpha-hydroxysteroid dehydrogenase messenger RNA transcription in the immature rat ovary in response to an ovulatory dose of gonadotropin.
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The ovulatory process in mammals involves a substantial increase in the metabolism of steroids and eicosanoids in response to a surge in LH or to an injection of hCG into experimental animals. This study provides evidence that the ovulatory stimulus causes induction of the gene for 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), an enzyme that belongs to several oxidoreductase superfamilies that affect steroid and eicosanoid metabolism. Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction (PCR) differential display to detect gene expression in the stimulated ovarian tissue. One of the PCR primer sets differentially amplified a cDNA fragment that is 52.3% homologous with a 3alpha-HSD gene in rat liver. Northern analyses revealed that maximum transcription was at 8 h after the animals had been treated with hCG. The Northerns also indicated that the 3alpha-HSD cDNA probe cross-hybridized with as many as six different bands of mRNA on the blots. In situ hybridization localized 3alpha-HSD mRNA in the granulosa and thecal layers of mature follicles and in newly formed corpora lutea at 24 h after the ovulatory stimulus. In conclusion, gene(s) for 3alpha-HSD are transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin. A possible function of the oxidoreductase enzyme that is translated from the 3alpha-HSD mRNA may be to reduce the toxic aldehyde and ketone components of the steroids and eicosanoids that accumulate in the mammalian ovary at the time of ovulation. (+info)