BACKGROUND: Scavenging of superoxide radical by salicylate-iron complex was studied to determine whether or not the salicylate-iron complex was able to catalyze the dismutation of superoxide radicals, the result perhaps yielding an explanation of the antioxidant and anti-inflammatory properties of the drug. METHODS: The scavenging was studied with an assay that generates O2.- without the intervention of metal ions. RESULTS: Results indicated that, in the presence of iron, salicylate was able to bring about the catalytic dismutation of the superoxide radical. The rate of superoxide removal was dependent on both the concentration of iron and the salicylate:iron molar ratio. CONCLUSIONS: These results may help to explain the interaction of nonsteroidal anti-inflammatory drugs with free radicals and the anti-inflammatory properties of these agents, inasmuch as accumulating evidence indicates that much of the injury observed during inflammatory disorders may be mediated by oxidative stress frequently induced by iron-dependent reactions.

Results of a survey conducted in the summer of 1985 of beneficiaries of the Arizona Health Care Cost Containment System and a matched group of Medicaid beneficiaries concerning their access to and satisfaction with medical care services are described in this article. The Arizona Health Care Cost Containment System is an alternative to Medicaid's acute medical care coverage. The results of the study indicate few differences in access and satisfaction between the two groups of beneficiaries on access to care, reported use of services, or satisfaction with the care received.

Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.

The ribosome binding site (RBS) of prokaryotic mRNA is divided into 5' and 3' portions by the translation initiation codon. Although it is well known that the presence of an appropriate RBS containing only the 5' portion is sufficient to direct the initiation of protein synthesis, the 3' portion appears to play a significant role in modulating the initiation process as well. Here we examine the influence of adenine-rich motifs frequently found in the 3' portion of highly expressed prokaryotic mRNAs. Two synthetic DNA fragments, GAGAAAAAAATC (corresponding to the first 12 nucleotides following the initiation codon of the chloramphenicol acetyltransferase gene), and AAAAAAATTAA were used to modify the beginning of the coding region of the human immune interferon-gamma (IFN-gamma) gene. The level of the protein synthesis in Escherichia coli directed by plasmids containing these constructs was quantitated. We found that placing either adenine-rich motif in the 3' portion of the RBS strongly enhanced gene expression, probably through an effect on translation initiation. We have also compared the protein expression levels of these gene constructs containing different series of 5'-RBSs with varying precistronic lengths and Shine-Dalgarno sequence lengths. The results suggest a positive functional role for the 3' adenine-rich motif. A possible mechanism for these effects is discussed.

With an assay that generates superoxide anion radicals without the intervention of metal ions we investigated the antioxidant properties of captopril, an angiotensin-converting enzyme inhibitor with a sulfhydryl group. Under these conditions, increasing concentrations of the drug were seen not to scavenge O.-2 directly. However, a combination of captopril and iron could bring about the breakdown of the superoxide anion; a result that may help to understand the free radical-scavenging properties of captopril.

The prokaryotic mRNA ribosome binding site (RBS) usually contains part or all of a polypurine domain UAAGGAGGU known as the Shine-Dalgarno (SD) sequence found just 5' to the translation initiation codon. It is now clear that the SD sequence is important for identification of the translation initiation site on the mRNA by the ribosome, and that as a result, the spacing between the SD and the initiation codon strongly affects translational efficiency (1). It is not as clear, however, whether there is a unique optimal spacing. Complications involving the definition of the spacing as well as secondary structures have obscured matters. We thus undertook a systematic study by inserting two series of synthetic RBSs of varying spacing and SD sequence into a plasmid vector containing the chloramphenicol acetyltransferase gene. Care was taken not to introduce any secondary structure. Measurements of protein expression demonstrated an optimal aligned spacing of 5 nt for both series. Since aligned spacing corresponds naturally to the spacing between the 3'-end of the 16S rRNA and the P-site, we conclude that there is a unique optimal aligned SD-AUG spacing in the absence of other complicating issues.

An icosadeoxyribonucleotide containing the several features found in prokaryotic mRNA ribosome binding sites has been synthesized. This sequence can stimulate the binding of initiator fMet-tRNAf to the ribosome to form a stable 71 S initiation complex identical with those induced by natural messengers. The binding of this synthetic ribosome binding site is absolutely dependent upon initiation factor IF3, and the bound fMet-tRNAf is sensitive to puromycin indicating the formation of a functional initiation complex. A heptadecadeoxyribonucleotide, identical with the icosanucleotide but lacking the terminal A-T-G codon, can also stimulate the stable binding of fMet-tRNAf to the ribosome, suggesting that the selection of the proper A-U-G initiation codon by fMet-tRNAf is subsequent to and a result of the recognition and binding of the fMet-tRNAf . 30 S ribosome complex to the initiation site. The prospect of ligating a similar synthetic ribosome binding site in front of a eukaryotic gene for cloning in an appropriate prokaryotic vector to assure the expresion of the protein is discussed.