Isolation of a crustaceann-acetyl-d-glucosamine-1-phosphate transferase and its activation by phospholipids

Summary Then-acetyl-d-glucosamine-1-phosphate: dolichol phosphate transferase fromArtemia has been partially purified and characterized. The enzyme is solubilized from crude microsomes using Triton X-100, and after detergent removal appears to be associated with phospholipids. Using dolichol phosphate and UDP-n-acetyl-d-glucosamine as substrates, the enzyme catalyzes the formation of dolichol-pyrophosphate-n-acetyl-d-glucosamine. the product identity has been verified by TLC and paper chromatography following mild acid hydrolysis. Under the incubation conditions used only one product is made, i.e., Dol-p-p-GlcNAc. The formation of product is linear with increasing amounts of added protein and with time of incubation. The enzyme requires magnesium ions for activity. Activity of the enzyme is stimulated 6-fold by exogenous dolichol phosphate and is also stimulated by added phospholipids, with optimal activity being obtained in the presence of mixtures of phosphatidylcholine and phosphatidylglycerol. Enzymatic activity is not increased upon addition of GDP-mannose or dolichol phosphate mannose. The enzyme is rapidly inactivated by exposure to several detergents, including Triton X-100 and deoxycholate. The activity is inhibited by tunicamycin and by the purified B₂ homologue of this antibiotic. Other antibiotic inhibitors such as diumycin and polyoxin D have little effect on the enzyme. Both the microsomal and solubilized enzyme preparations are inactivated by 70% upon treatment with phospholipase A₂; activity may be restored by addition of phospholipids. Following hydrophobic interaction chromatography on Phenyl Sepharose, gel filtration chromatography on Sepharose CL-4B indicated that the enzyme, purified 81-fold, contained phophatidylcholine and phosphatidyl-ethanolamine.Abbreviations SDS sodium dodecyl sulfate - PMSF phenyl methanesulfonylfluoride - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - GlcNAc N-acetyl-d-glucosamine - Dol-PP-GlcNAc dolichol pyrophosphate N-acetyl-d-glucosamine - Dol-P-man dolichol-phosphate-mannose - Dol-PP- (GlcNAc)₂ dolichol-pyrophosphate-di-N- acetylchitobiose - DMSO dimethylsulfoxide - C:M (2:1) chloroform:methanol (2:1) - C:M:W (10:10:3) chloroform:methanol:water (10:10:3) - GlcNAc-1-P N-acetyl-d-glucosamine-1-phosphate - Dol-P dolichol phosphate - EGTA ethylene glycol bis (b-aminoethyl ether)-NNNN tetraacetic acid     

Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of -tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR--tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.     

Nefiracetam (DM-9384) reverses apomorphine-induced amnesia of a passive avoidance response: Delayed emergence of the memory retention effects

Nefiracetam is a novel pyrrolidone derivative which attenuates scopolamine-induced learning and post-training consolidation deficits. Given that apomorphine inhibits passive avoidance retention when given during training or in a defined 10–12h post-training period, we evaluated the ability of nefiracetam to attenuate amnesia induced by dopaminergic agonism. A step-down passive avoidance paradigm was employed and nefiracetam (3 mg/kg) and apomorphine (0.5 mg/kg) were given alone or in combination during training and at the 10–12h post-training period of consolidation. Co-administration of nefiracetam and apomorphine during training or 10h thereafter produced no significant anti-amnesic effect. However, administration of nefiracetam during training completely reversed the amnesia induced by apomorphine at the 10h post-training time and the converse was also true. These effects were not mediated by a dopaminergic mechanism as nefiracetam, at millimolar concentrations, failed to displace either [³H]SCH 23390 or [³H]spiperone binding from D₁ or D₂ dopamine receptor subtypes, respectively. It is suggested that nefiracetam augments molecular processes in the early stages of events which ultimately lead to consolidation of memory.     

Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector

Abstract The comparative chromosomal locations of polymeric β-fructosidase SUC genes have been determined by Southern blot hybridization with the SUC2 probe in 91 different strains of Saccharomyces cerevisiae . Most of the strains exhibited a single SUC2 gene, but in some strains two or three SUC genes were found. All Suc⁻ strains carried a silent suc2⁰ sequence. The accumulation of SUC genes was observed in populations derived from sources containing sucrose and seems to be absent in strains from sources promoting the MEL gene.     

Impact of sar and agr on methicillin resistance in Staphylococcus aureus

Abstract The global regulators agr and sar control expression of cell wall and extracellular proteins. Inactivation of either sar and/or agr in a typical heterogeneously methicillin-resistant Staphylococcus aureus resulted in a small but reproducible decrease in the number of cells in the subpopulation expressing high methicillin resistance. The amount of low affinity penicillin-binding protein PBP2', the prerequisite for methicillin resistance, was apparently not affected, however, a reduction in PBP1 and PBP3 production was observed, suggesting that these resident PBPs of the cells might be involved somehow together with PBP2' in high level methicillin resistance.     

Association of RFLP markers with loci conferring broad-based resistance to the soybean cyst nematode (Heterodera glycines)

Soybean cyst nematode (SCN) is a major soybean yield-limiting pest. The present study was conducted to map broad-based SCN resistance loci from the cultivar Hartwig. Two-hundred F₂₃ lines derived from the cross Williams 82 x Hartwig were screened with a fourth-generation SCN inbred and 56 polymorphic molecular markers. Allele states and phenotypes were analyzed using stepwise regression and the model selection was made at P 0.01. Four unlinked RFLP markers (A006, A567, A487, A112) were associated with SCN resistance and the partial coefficient of determinations (R²) were 91%, 1%, 1%, and 1%. We have mapped a new, major SCN resistance locus (A006) and three minor loci (A567, A487, A112). This complete mapping will accelerate the transfer of broad-based resistance without linkage drag and aid in the determination of relationships among various SCN-resistant germplasm sources.