PubMed · 6732434
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R T Rubin, R E Poland. 1984. Variability in cortisol level assay methods.. https://doi.org/10.1001/archpsyc.1984.01790180094017
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Our discovery that Herpes virus thymidine kinase (TK) and cellular deoxycytidine kinase lack enantioselectivity, being able to phosphorylate both D- and L-enantiomers of the substrate, suggested the use of unnatural L-nucleoside analogues as antiviral drugs (Herpes, hepatitis and immunodeficiency viruses). Several L-nucleoside analogues have displayed a short-term cytotoxicity much lower than their corresponding D-counterpart. Since the delayed cytotoxicity of a drug often depends on its effects on mitochondrial metabolism, we have investigated the degree of enantioselectivity of human mitochondrial thymidine kinase (mt-TK). We demonstrate that mt-TK does not show an absolute enantioselectivity, being able to recognize, although with lower efficiency, the L-enantiomers of thymidine, deoxycytidine and modified deoxyuridines, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine and 5-iodo-2'-deoxyuridine. Interestingly, the reported negative co-operativity of mt-TK phosphorylating beta-D-2'-deoxythymidine (D-Thd), disappears when the deoxyribose moiety has the inverted configuration, resulting in the preferential phosphorylation of d-Thd even in the presence of high concentrations of the L-enantiomer. This, coupled with the higher Km for beta-L-2'-deoxythymidine (L-Thd), makes mt-TK resistant to high concentrations of L-Thd and L-Thd analogues, minimizing the mitochondria-dependent delayed cytotoxicity that might be caused by the administration of L-nucleoside analogues as antivirals.
The molecular basis for the well known synergistic biological effects of tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) is still poorly understood. This report demonstrates that expression of interferon-regulatory factor 1 (IRF-1), also known as interferon-stimulated-gene factor 2 (ISGF-2), is synergistically induced by these cytokines. The induction is a primary transcriptional response that occurs rapidly without a requirement for new protein synthesis. Synergism is mediated by a novel composite element in the IRF-1 promoter that includes an IFNgamma-activation site (GAS) overlapped by a non-consensus site for nuclear factor kappa B (NFkappaB). These sequences are bound strongly by signal transducer and activator of transcription 1 (STAT-1) and weakly by the p50/p65 heterodimer form of NFkappaB, respectively. However, the binding of STAT-1 and NFkappaB to the GAS/kappaB element in vitro seems to be mutually exclusive and independent. Synergistic induction of IRF-1 is likely to be an important early step in regulatory networks critical to the synergism of TNFalpha and IFNgamma. The GAS/kappaB element may mediate synergistic transcriptional induction of IRF-1 by other pairs of ligands that together activate NFkappaB and STAT family members. Other genes are likely to contain this motif and be regulated similarly.
The neutrophil enzyme myeloperoxidase uses H2O2 to oxidize chloride, bromide, iodide and thiocyanate to their respective hypohalous acids. Chloride is considered to be the physiological substrate. However, a detailed kinetic study of its substrate preference has not been undertaken. Our aim was to establish whether myeloperoxidase oxidizes thiocyanate in the presence of chloride at physiological concentrations of these substrates. We determined this by measuring the rate of H2O2 loss in reactions catalysed by the enzyme at various concentrations of each substrate. The relative specificity constants for chloride, bromide and thiocyanate were 1:60:730 respectively, indicating that thiocyanate is by far the most favoured substrate for myeloperoxidase. In the presence of 100 mM chloride, myeloperoxidase catalysed the production of hypothiocyanite at concentrations of thiocyanate as low as 25 microM. With 100 microM thiocyanate, about 50% of the H2O2 present was converted into hypothiocyanite, and the rate of hypohalous acid production equalled the sum of the individual rates obtained when each of these anions was present alone. The rate of H2O2 loss catalysed by myeloperoxidase in the presence of 100 mM chloride doubled when 100 microM thiocyanate was added, and was maximal with 1mM thiocyanate. This indicates that at plasma concentrations of thiocyanate and chloride, myeloperoxidase is far from saturated. We conclude that thiocyanate is a major physiological substrate of myeloperoxidase, regardless of where the enzyme acts. As a consequence, more consideration should be given to the oxidation products of thiocyanate and to the role they play in host defence and inflammation.