The effect of pertussis toxin on mediator release from human basophils

We have found that basophils (n = 9) treated with pertussis toxin (1.0 microgram/ml) fail to respond to a subsequent challenge with either 1.0 microM f-Met peptide (p less than 0.0005) or 0.24 microgram/ml of C5a (p less than 0.0005) although their responses to anti-IgE (0.1 microgram/ml) and A23187 (1.0 microgram/ml) were unaltered. These results were confirmed in purified (average purity = 89 +/- 3%) basophils (n = 4). Leukotriene C4 release was also reduced to 15 +/- 5% of control (p less than 0.005) when pertussis toxin-treated basophils were exposed to 1.0 microM f-Met peptide, although no inhibition was noted when anti-IgE or A23187 were used as the stimuli. The effect of pertussis toxin on basophils appears to be independent of the presence of contaminating mononuclear cells. We found that pertussis toxin inhibited f-Met peptide-induced histamine release regardless of the magnitude of the stimulus (0.01 microM to 1.0 microM f-Met peptide), although anti-IgE-induced release was unaffected over a dose-response curve. The effect of pertussis toxin was found to be both time- and concentration-dependent. The maximum effects were obtained after a 3-hr incubation with 1 microgram/ml of toxin. Lower (0.01 to 0.05 microgram/ml) concentrations of toxin or shorter (30 to 60 min) incubation periods did not significantly (p greater than 0.05) inhibit mediator release.     

C5a induction of human interleukin 1. Synergistic effect with endotoxin or interferon-gamma

Interleukin-1 (IL-1) is a potent cytokine which possesses the ability to mediate systemic acute phase responses as well as local tissue inflammation. In these studies, we have examined the ability of C5a and C5a des Arg to induce IL-1 production in vitro. Human C5a and C5a des Arg were purified to homogeneity and were found to stimulate IL-1 release from freshly obtained human mononuclear cells into the extracellular medium. Only 2 hr of exposure to the purified complement components were necessary in order to stimulate IL-1 production. The minimal concentration of C5a required was 25 ng/ml, whereas 125 ng/ml of C5a des Arg induced comparable amounts of IL-1. This dose relationship was maintained at higher concentrations (150 ng/ml vs 750 ng/ml, respectively). That the effect was due to the anaphylatoxins themselves, and not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and employing preincubation of C5a/C5a des Arg with polymyxin B. The latter blocked a wide dose range of endotoxin-stimulated IL-1 production. However, when endotoxin was added to C5a or C5a des Arg, significant synergism in the stimulation of IL-1 production was observed, occurring at various concentrations of either agent. A similar synergism with C5a/C5a des Arg was seen with interferon-gamma. In these studies, IL-1 production was measured by bioassay employing cloned D . 10 . G4 . 1 murine T cells and by radioimmunoassay for human IL-1 beta; using C5a/C5a des Arg as stimulants, there was a high degree of correlation (r = 0.82) between the two assays. Since traumatic, infectious, and inflammatory diseases may result in the simultaneous appearance of these stimuli, the synergism described herein is likely to be clinically relevant.     

Variation in adhesion and cell surface hydrophobicity in Candida albicans white and opaque phenotypes

A previous study had established that a select group of pathogenic isolates of Candida albicans was capable of switching heritably, reversibly and at a high frequency (10^–2 to 10^–3) between two phenotypes (white or opaque) readily distinguishable by the size, shape, and color of colonies formed on agar at 25°C. This paper describes experiments designed to determine the ability of these two phenotypes to attach to buccal epithelial cells (BECs) and plastic, and to compare the cell surface hydrophobicities of white and opaque phenotypes from three clinical isolates. White cells were found to be significantly more adhesive to BECs, and a strong correlation was also found between phenotype adhesiveness and the percentage of BECs to which C. albicans had attached. The percentage of BECs with one or more attached C. albicans was approximately 90% for the white phenotype and approximately 50% for the opaque phenotype. Opaque cells, in contrast, were twice as hydrophobic as white cells, and the percentage of opaque cells bound to BECs by coadhesion was also double that of white cells. The differences in adhesion to plastic between the two phenotypes were not statistically significant and there was no distinct trend to suggest which phenotype might be more adhesive to plastic. These results indicate that several factors are involved in the adhesion of C. albicans to plastic, and confirm the hypothesis that cell surface hydrophobicity is of minor importance in direct adhesion to epithelial cells but that it may contribute to indirect attachment to epithelial cells by promoting yeast coadhesion. Moreover, the data presented in this paper also revealed that under identical growth conditions, adhesion of C. albicans was significantly altered depending on the phenotypic state of the organism tested. Therefore, because C. albicans can switch at a high frequency to various phenotypes in vitro, it may be that in future adhesion studies involving Candida the phenotypic state of the organism at the time of testing will have to be determined. Otherwise, the results, even within the same laboratory, may be difficult to interpret.     

Osmotic effectors in kidneys of xeric and mesic rodents: corticomedullary distributions and changes with water availability

Summary Urea, sodium, the methylamines glycine betaine and glycerophosphorylcholine (GPC), and the polyols sorbitol and myo-inositol are reported to be the major osmolytes in kidneys of laboratory mammals. These were measured (millimoles per kilogram wet weight) in kidney regions and urines of three species of wild rodents with different dehydration tolerances: the pocket mousePerognathus parvus (xeric), voleMicrotus montanus (mesic), and deer mousePeromyscus m. gambeli (intermediate). In animals kept without water for 4–6 days, sodium, urea, betaine and GPC+choline were found in gradients increasing from cortex to outer to inner medulla in all species, withPerognathus having the highest levels. Sorbitol was high in the inner medulla but low in the cortex and outer medulla; inositol was highest in the outer medulla. Totals of methylamines and methylamines plus polyols in the medulla showed high linear correlations (positive) with urea and with sodium values.Whole medullae were analyzed at several time points inMicrotus andPeromyscus subject to water diuresis followed by antidiuresis. In 102 h diuresis inMicrotus, all osmolytes decreased except inositol; however, only urea, sodium and sorbitol reached new steady states within 24 h. Urea returned to initial values in 18 h antidiuresis, while other osmolytes required up to 90 h. InPeromyscus, all osmolytes except the polyols declined in diuresis (max. 78 h test period). During antidiuresis, urea and GPC+choline rose to initial values in 18 h, with sodium and betaine requiring more time. In plots of both species combined, total methylamines+polyols correlated linearly (positive) with sodium, and GPC+choline with urea.Estimates of tissue concentrations suggest that total methylamines+polyols can account for intracellular osmotic balance in all species in antidiuresis and that sufficient concentrations of methylamines may be present to counteract perturbing effects of urea on proteins.Abbrevations GPC Glycero-3-phosphorylcholine - TCA trichloroacetic acid - M+P methylamines plus polyols     

The DNA unwinding reaction catalyzed by Rep protein is facilitated by an RHSP-DNA interaction.

The unwinding reaction catalyzed by the Escherichia coli Rep protein is stimulated by a small 15 kDa protein called Rep helicase stimulatory protein (RHSP)(1). The RHSP-stimulated unwinding reaction catalyzed by Rep protein proceeded at a rapid rate after a time lag of 1-2 min at 37 degrees C. This time lag was eliminated by preincubating RHSP with the DNA substrate, indicating that stimulation resulted from an interaction between RHSP and DNA. RHSP was shown to increase the rate as well as the extent of the unwinding reaction catalyzed by Rep protein. RHSP bound both single- and double-stranded DNA with apparent equal affinity, forming an unusually stable complex. Electron microscopy illustrated that the RHSP-DNA complex consisted of large protein aggregates bound to DNA forming a highly condensed, aggregated DNA-protein complex. The protein aggregates were not observed in the absence of DNA and appeared to form cooperatively in the presence of DNA. NH2-terminal amino acid sequence analysis suggested that RHSP was identical to E. coli ribosomal-protein L14. Binding assays showed that the interaction between RHSP and rRNA was similar to the RHSP-DNA interaction. Several models are put forth to explain the stimulation of the unwinding reaction catalyzed by Rep protein. In addition, the potential physiological significance of the RHSP-stimulated Rep protein unwinding reaction is discussed.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Trimethylamine oxide, betaine and other osmolytes in deep-sea animals: depth trends and effects on enzymes under hydrostatic pressure.

Most shallow teleosts have low organic osmolyte contents, e.g. 70 mmol/kg or less of trimethylamine oxide (TMAO). Our previous work showed that TMAO contents increase with depth in muscles of several Pacific families of teleost fishes, to about 180 mmol/kg wet wt at 2.9 km depth in grenadiers. We now report that abyssal grenadiers (Coryphaenoides armatus, Macrouridae) from the Atlantic at 4.8 km depth contain 261 mmol/kg wet wt in muscle tissue. This precisely fits a linear trend extrapolated from the earlier data. We also found that anemones show a trend of increasing contents of methylamines (TMAO, betaine) and scyllo-inositol with increasing depth. Previously we found that TMAO counteracts the inhibitory effects of hydrostatic pressure on a variety of proteins. We now report that TMAO and, to a lesser extent, betaine, are generally better stabilizers than other common osmolytes (myo-inositol, taurine and glycine), in terms of counteracting the effects of pressure on NADH Km of grenadier lactate dehydrogenase and ADP Km of anemone and rabbit pyruvate kinase.     

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.