Conversion of dihomo-gamma-linolenic acid to mono- and dihydroxy acids by potato lipoxygenase: evidence for the formation of 8,9-leukotriene A3

Evidence for the formation of a positional isomer of leukotriene (LT) C3 (8,9-LTC3) from dihomo-gamma-linolenic acid has been published (Hammarstr?m, S. J. Biol. Chem. 256, 7712-7714, 1981). This report describes the conversion of dihomo-gamma-linolenic acid to a postulated intermediate in former reaction, 8,9-LTA3, by purified lipoxygenase from potato tubers. 8(S)-Hydroperoxyeicosatrienoic acid (8(S)-HPETrE) was the most abundant dioxygenation product formed followed by 11-, 15-, and 12-HPETrEs (in decreasing order of abundance). In addition, 8(S),15(S)- plus 8(S), 15(R)-dihydroperoxyeicosatetraenoic acid (DiHPE-TrE) (EZE), and 8(S),15(S)- plus 8(S),15(R)-dihydroxy-eicosatetraenoic acid (DiHETrE) (EEE) were generated. Under anaerobic conditions only the latter two isomers of 8,15-DiHETrE (EEE) were obtained from 8-HPETrE. The results suggest that 8,9-LTA3 is synthesized by the sequential action of 8- and 11-lipoxygenase activities associated with the potato enzyme.     

The primary nucleotide sequence of U4 RNA

U4 RNA is one of the "capped" nuclear snRNAs recently found to be precipitable by anti-Sm antibodies as ribonucleoprotein particles. U4 RNA, along with other snRNAs, has been implicated in hnRNA processing, mRNA transport, or both (Lerner, M. R., Boyle, J., Mount, S., Wolin, S., and Steitz, J. A. (1980) Nature 283, 220-224). Since the proteins bound to different snRNAs appear to be the same, the functions of different snRNPs might be dependent on the RNA components. To help understand the function of U4 RNP, the nucleotide sequence of U4 RNA was determined. The sequence is (formula see text) In addition to the modified nucleotides in the "cap," U4 RNA contains Am at position 63 and m6A at position 98. It also exhibited A-C microheterogeneity at position 97.     

Rapid return of cyclo-oxygenase active platelets in dogs after a single oral dose of aspirin

A single dose of oral aspirin in human subjects inhibits the aggregation response of platelets to arachidonate and other agents for approximately one week after ingestion. In the present study we have evaluated the rate at which cyclo-oxygenase active platelets return to the circulation in humans and dogs and compared the response curves obtained to improvements in cyclo-oxygenase activity produced by the aspirin platelets. After a single dose of aspirin, dog platelet function was compromised for several days. Normal responses to arachidonate and other aggregating agents were restored six days after aspirin, and the pattern of recovery was the same for dogs and human subjects. However, cyclo-oxygenase active platelets returned to the circulation in dogs more rapidly than in humans and chemical competence was restored in both species well before correction of the defective response to aggregating agents. The delay of 1-3 days before return of significant numbers of cyclo-oxygenase active platelets most likely reflects acetylation of bone marrow megakaryocytes by the drug. More rapid return of chemically competent cells in dogs than humans probably relates to the more rapid turnover and shorter life span of canine platelets. The basis for the discrepancy in return of chemical integrity compared to functional activity after aspirin in vivo compared to simultaneous correction of chemistry and function when 10% normal platelets are added to aspirin platelets in vitro remains unresolved.     

A combination of two Brugia malayi filarial vaccine candidate antigens (BmALT-2 and BmVAH) enhances immune responses and protection in jirds

In this study filarial recombinant protein or DNA vaccine constructs encoding BmALT-2 and BmVAH as single or as cocktail antigens were evaluated. Male jirds were immunized intramuscularly with DNA vaccine constructs or were immunized intraperitoneally with protein vaccine. The single and bicistronic DNA constructs induced substantial interferon-γ responses in spleen cells; antigen-specific responses were higher following immunization with the bicistronic cocktail construct and evoked a significant protective response of 57% in jirds challenged with Brugia malayi that was similar in the antibody-dependent cellular cytotoxicity (ADCC) assay and micropore chamber experiment. The cocktail protein vaccines induced a mixture of IgG2a (Th1) and IgG1 (Th2) responses with 80% protective response when challenged with B. malayi infective larvae. However, the single protein vaccine rALT-2 induced Th2 (IgG1/IgG3) with a 70% protective response and rVAH induced Th1 (IgG2a) with a lower proliferative response with 60% protection following challenge with B. malayi infective larvae. These results suggest that filarial cocktail protein vaccines are able to elicit substantial immune and protective responses when compared with single antigen vaccination in suitably vaccinated jirds.     

The sites for catalysis and activation of ribulosebisphosphate carboxylase share a common domain

The complexation of ribulosebiphosphate carboxylase with CO2, Mg2+, and carboxyarabinitol bisphosphate (CABP) to produce the quaternary enzyme-carbamate-Mg2+-CABP complex closely mimics the formation of the catalytically competent enzyme-carbamate-Mg2+-3-keto-CABP form during enzymatic catalysis. Quaternary complexes were prepared with various metals (Mg2+, Cd2+, Mn2+, Co2+, and Ni2+) and with specifically 13C-enriched ligands. 31P and 13C NMR studies of these complexes demonstrate that the activator CO2 site (carbamate site), the metal binding site, and the substrate binding site are contiguous. It follows that both the carboxylase and oxygenase activities of this bifunctional enzyme are influenced by the structures of the catalytic and activation sites.     

Rapid resolution of phenylthiohydantoin amino acids by thin-layer chromatography on silica gel plates impregnated with transition metal ions

The resolution of a 15-component mixture of phenylthiohydantoin (PTH) amino acids using metal ion impregnated silica gel plates is reported. The spots are located by exposing the chromatograms to an iodine chamber. The method provides a rapid, simple, and less expensive chromatographic system, provides resolution for certain difficult combinations, and leaves the PTH amino acids unaltered chemically.