Purification and biologic characterization of a specific tumor necrosis factor alpha inhibitor

The purpose of the present investigation was to purify a urine-derived tumor necrosis factor alpha inhibitor (TNF alpha INH) and to characterize its mechanism of action. For the purification procedure, urine was concentrated and TNF alpha INH purified by ion-exchange chromatographies, gel filtration, TNF alpha affinity column, and reverse-phase chromatography. The TNF alpha INH migrates with an apparent Mr of approximately 33,000 when estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under both reducing and nonreducing conditions. Elution of TNF alpha INH activity from the gel yields also a approximately 33,000-Da inhibitory fraction. Besides inhibiting TNF alpha-induced cytotoxicity in L929 cells in the presence of actinomycin D, the TNF alpha INH impeded in a dose-dependent manner prostaglandin E2 production and expression of cell-associated interleukin-1 by human dermal fibroblasts. Therefore, TNF alpha INH is active on both actinomycin D-treated and untreated cells. In contrast to TNF alpha, TNF beta-induced cytotoxicity was only slightly affected by the inhibitor. This specificity was confirmed by the fact that it affected neither interleukin-1 alpha nor interleukin-1 beta biologic activities. The mechanism of action of TNF alpha INH involves blocking of 125I-TNF alpha binding to the promonocytic cell line U937. Moreover, preincubation of 125I-TNF alpha with TNF alpha INH increased binding inhibition, suggesting an interaction between TNF alpha and the inhibitor.     

Effect of interferon-gamma and 1 alpha,25-dihydroxyvitamin D3 on superoxide anion, prostaglandins E2, and mononuclear cell factor production by U937 cells

Both interferon-gamma (IFN-gamma) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) induce changes in the human monocytic cell line U937 that may reflect cellular differentiation. The effects of recombinant IFN-gamma and 1 alpha,25(OH)2D3 on U937 cells with regard to the release of superoxide anion (O2-), prostaglandin E2 (PGE2), and mononuclear cell factor (MCF) after stimulation with phorbol myristate acetate (PMA) were examined. PMA did not induce O2- production in untreated cells. A 3-day preincubation with IFN-gamma or 1 alpha,25(OH)2D3 resulted in a 5- to 10-fold increase in PMA-stimulated production of O2- as compared to cells preincubated in medium alone. The response was related to IFN-gamma and 1 alpha,25(OH)2D3 concentrations. In contrast, the PMA-induced production of PGE2 and MCF does not require preincubation with either IFN-gamma or 1 alpha,25(OH)2D3. These results suggest that O2- production and cytokine production (i.e., PGE2 and MCF) are modulated by different signals related to maturation processes.     

Biologically active interleukin 1 in human eccrine sweat: site-dependent variations in alpha/beta ratios and stress-induced increased excretion

Human eccrine sweat devoid of epidermal protein contamination was collected from palms, soles, and different sites on the trunk. Interleukin 1 alpha (IL 1 alpha) and interleukin 1 beta (IL 1 beta) content were analyzed for immunoreactivity by enzymo-immunoassay and immunoblotting and for bioactivity by the stimulation of prostaglandin E2 (PGE2) production in human dermal fibroblasts. The bioassay was validated by using blocking antibodies against IL 1 alpha and beta. All sweat samples were found to contain significant amounts of immunoreactive and biologically active IL 1. The immunoreactive forms were at 17 kDa as shown by immunoblotting analysis, indicating that they were mature (secreted), undegraded IL 1 peptides. Whereas IL 1 alpha was detectable in sweat samples obtained from both truncal and palmo-plantar regions, IL 1 beta was only detectable in the sweat of palms and soles (IL 1 alpha/beta ratio greater than 700 in trunk and 5.4 in palms and soles) indicating a site-dependent difference in the excretion of the two IL 1 molecules. IL 1 concentration was high in spontaneous (IL 1 alpha, 3.7; IL 1 beta, 0.3 ng/mL) and pilocarpine induced sweat (IL 1 alpha, 3.9; IL 1 beta, 1.2 ng/mL), and it was much increased during jogging and sauna (IL 1 alpha, 22.6; IL 1 beta, 3.3 ng/mL). This does not appear to represent an excretory process aimed at clearing blood IL 1, but rather a stress-induced increased production of IL 1 by sweat gland cells.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.