A 226-nucleotide fragment was derived from alfalfa mosaic virus RNA 4 (ALMV RNA 4), the subgenomic messenger for viral coat protein, and its sequence was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. The fragment contains the 3'-terminal 45 nucleotides of the coat protein cistron and the complete 3'-noncoding region of 182 nucleotides. The total length of RNA 4 was calculated to be 881 nucleotides. AlMV RNAs 1, 2 and 3 were elongated with a 3'-terminal poly(A) stretch and subjected to sequence analysis by using a specific primer, reverse transcriptase and chain terminators. This revealed and extensive homology between the 3'-terminal 140 to 150 nucleotides of all four ALMV RNAs. Despite a number of base substitutions, the secondary structure of the homologous region is highly conserved. The observed homology indicates that, as with RNA 4, the sites with a high affinity for the viral coat protein are located at the 3'-termini of the genomic RNAs.
Human granulocytes isolated from peripheral blood have been described to synthesize both LTB4 and LTC4 from arachidonic acid. We have observed that the amount of LTC4 produced by human granulocyte preparations is strongly dependent on the relative amount of eosmophils. To investigate a possibly significant difference in leukotriene synthesis of the eosinophilic and neutrophilic granulocytes, we developed a purification method to isolate both cell types from granulocytes obtained from the blood of healthy donors. Leukotrlenes were generated by incubation of the purified cells with arachidonic acid, calcium lonophore A23187, calcium-chlonde and reduced glutatbione. Surprisingly, eosinophils were found to produce almost exclusively the spasmogenic LTC4. In contrast, neutropbils produce almost exclusively the chemotactic LT&, its w-hydroxylated metabolite 20-hydroxy-LTB4 and two nonenzymically formed LTB4 isomers.
Leukotnene Granulocyte Eosmophil
In this study we present evidence for the existence of an intrinsic 12-lipoxygenase in the bovine polymorphonuclear leukocyte which differs from the well-known platelet 12-lipoxygenase. Intact bovine polymorphonuclear leukocytes synthesize predominantly 5-lipoxygenase products. However, this 5-lipoxygenase activity disappears completely upon sonication of the cells, whereas a 12-lipoxygenase activity then becomes apparent. This 12-lipoxygenase resembles the platelet 12-lipoxygenase in metabolizing arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and in being independent of Ca2+ as well as of ATP. The most striking difference between the two 12-lipoxygenases is their behaviour towards linoleic acid. While the platelet 12-lipoxygenase does not convert linoleic acid, the 12-lipoxygenase from bovine polymorphonuclear leukocytes, apparent only in the cell-free system, converts linoleic acid into 13-hydroxyoctadecadienoic acid as efficiently as it converts arachidonic acid into 12-hydroxyeicosatetraenoic acid. This provides a convenient method to distinguish both 12-lipoxygenase activities. The fact that this new 12-lipoxygenase is able to metabolize linoleic acid into 13-hydroxyoctadecadienoic acid suggests that this enzyme, in contrast to platelet 12-lipoxygenase, resembles 5-lipoxygenases in showing a preference for hydrogen abstraction at a position which is determined by the distance to the carboxylic end of the fatty acid.
The leukotriene production by bovine polymorphonuclear leukocytes isolated from peripheral blood has been studied. Cells were incubated in the presence of arachidonic acid, glutathione, calcium ionophore A23187 and Ca2+. The leukotrienes then formed are leukotriene C4, leukotriene B4, two all-trans isomers of leukotriene B4 and the double dioxygenation product 12-epi-6-trans-8-cis-leukotriene B4. Leukotriene C4 is formed in such a large quantity by the bovine polymorphonuclear leukocyte that it might constitute an excellent and inexpensive source for the biosynthetic preparation of this spasmogenic leukotriene.
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