A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Ferritin and superoxide-dependent lipid peroxidation

Ferritin was found to promote the peroxidation of phospholipid liposomes, as evidenced by malondialdehyde formation, when incubated with xanthine oxidase, xanthine, and ADP. Activity was inhibited by superoxide dismutase but markedly stimulated by the addition of catalase. Xanthine oxidase-dependent iron release from ferritin, measured spectrophotometrically using the ferrous iron chelator 2,2'-dipyridyl, was also inhibited by superoxide dismutase, suggesting that superoxide can mediate the reductive release of iron from ferritin. Potassium superoxide in crown ether also promoted superoxide dismutase-inhibitable release of iron from ferritin. Catalase had little effect on the rate of iron release from ferritin; thus hydrogen peroxide appears to inhibit lipid peroxidation by preventing the formation of an initiating species rather than by inhibiting iron release from ferritin. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide was used to observe free radical production in this system. Addition of ferritin to the xanthine oxidase system resulted in loss of the superoxide spin trap adduct suggesting an interaction between superoxide and ferritin. The resultant spectrum was that of a hydroxyl radical spin trap adduct which was abolished by the addition of catalase. These data suggest that ferritin may function in vivo as a source of iron for promotion of superoxide-dependent lipid peroxidation. Stimulation of lipid peroxidation but inhibition of hydroxyl radical formation by catalase suggests that, in this system, initiation is not via an iron-catalyzed Haber-Weiss reaction.     

An electron paramagnetic resonance study of the interactions between the adriamycin semiquinone, hydrogen peroxide, iron-chelators, and radical scavengers.

Anaerobic reduction of hydrogen peroxide in a xanthine/xanthine oxidase system by adriamycin semiquinone in the presence of chelators and radical scavengers was investigated by direct electron paramagnetic resonance and spin trapping techniques. Under these conditions, adriamycin semiquinone appears to react with hydrogen peroxide forming the hydroxyl radical in the presence of chelators such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid. In the absence of chelators, a related, but unknown oxidant is formed. In the presence of desferrioxamine, adriamycin semiquinone does not disappear in the presence of hydrogen peroxide at a detectable rate. The presence of adventitious iron is therefore implicated during adriamycin semiquinone-catalyzed reduction of hydrogen peroxide. Formation of alpha-hydroxyethyl radical and carbon dioxide radical anion from ethanol and formate, respectively, was detected by spin trapping. Both the hydroxyl radical and the related oxidant react with these scavengers, forming the corresponding radical. In the presence of scavengers from which reducing radicals are formed, the rate of consumption of hydrogen peroxide in this system is increased. This result can be explained by a radical-driven Fenton reaction.     

Inhibition by glutamate of phosphate-dependent glutaminase of rat kidney.

A membrane-associated form of phosphate-dependent glutaminase was derived from sonicated mitochondria and purified essentially free of gamma-glutamyl transpeptidase activity. Increasing concentrations of phosphate cause a sigmoidal activation of the membrane-bound glutaminase. Phosphate also causes a similar effect on the rate of glutaminase inactivation by the two affinity labels, L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo-L-norleucine, as observed previously for the solubilized and purified enzyme. Therefore the two forms of glutaminase undergo similar phosphate-induced changes in conformation. A sensitive radioactive assay was developed and used to determine the kinetics of glutamate inhibition of the membrane-associated glutaminase. The Km for glutamine decreases from 36 to 4 mM when the phosphate concentration is increased from 5 to 100 mM. Glutamate is a competitive inhibitor with respect to glutamine at both high and low concentrations of phosphate. However, the Ki for glutamate is increased from 5 to 52 mM with increasing phosphate concentration. Therefore glutamine and glutamate interact with the same site on the glutaminase, but the specificity of the site is determined by the available phosphate concentration.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.     

Dihydropyridine calcium channel blockers inhibit ascorbic acid accumulation in human intestinal Caco-2 cells

We investigated the effect of commonly used medications on the accumulation of ascorbic acid in human intestinal Caco-2 cells. Although ascorbic acid is negatively charged at physiological pH, anionic compounds including drugs and metabolites had little effect on its accumulation. On the other hand, hydrophobic 1,4-dihydropyridine compounds (nifedipine and nicardipine), but not other structurally unrelated calcium channel blockers, were found to be potent inhibitors. They inhibited both Na+-dependent and Na+-independent (K+ substituting Na+) accumulation of ascorbic acid. The inhibition was non-competitive with a Ki of 108 microM and 9 microM for nifedipine and nicardipine, respectively. The efflux of ascorbic acid from cells was not affected. Previously, we reported a similar inhibition of ascorbic acid accumulation by estrogens. When nifedipine and estrogens were included in the buffer together, the combined inhibitory effect was less than additive implying that they may act through the same mechanism. The potential clinical significance of dihydropyridine usage on ascorbic acid status in human needs to be considered.     

Studies on beta-D-Gal(f)-(1-->4)-alpha-L-Rha(p) octyl analogues as substrates for mycobacterial galactosyl transferase activity

The biochemically unique structures of sugar residues in the outer cell wall of Mycobacterium tuberculosis (MTB) make the pathways for their biosynthesis and utilization attractive targets for the development of new and selective anti-tubercular agents. A cell-free assay system for galactosyltransferase activity using UDP[14C]Gal as the glycosyl donor, as well as an in vitro colorimetric broth micro-dilution assay system, were used to determine the activities of three beta-D-gal(f)(1-->4)-alpha-L-rham(p) octyl disaccharides as substrates and antimycobacterial agents respectively. The cell-free enzymatic studies using compounds 8 and 10 suggested that these disaccharides bind to and are effective substrates for a putative mycobacterial galactosyltransferase. The modified acceptor 8 was found to be a slower but prolonged binder as compared to the less substituted analogue 10 as evidenced by their Km and Vmax values. Moderate antimycobacterial activity was observed with compounds 8 and 9 against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC).     

Comparison of synthetic saponin cholesterol absorption inhibitors in rabbits: evidence for a non-stoichiometric, intestinal mechanism of action

The hypocholesterolemic activities of pamaqueside and tiqueside, two structurally similar saponins, were evaluated in cholesterol-fed rabbits. The pharmacological profiles of the saponins were virtually identical: both dose-dependently decreased the intestinal absorption of labeled cholesterol 25-75%, increased fecal neutral sterol excretion up to 2.5-fold, and decreased hepatic cholesterol content 10-55%. High doses of pamaqueside (>5 mg/kg) or tiqueside (>125 mg/kg) completely prevented hypercholesterolemia. Decreases in plasma and hepatic cholesterol levels were strongly correlated with increased neutral sterol excretion. Ratios of neutral sterol excreted to pamaqueside administered were greater than 1:1 at all doses, in opposition to the formation of a stoichiometric complex previously suggested for tiqueside and other saponins. Ratios in tiqueside-treated rabbits were less than unity, a reflection of its lower potency. Pamaqueside-treated rabbits exhibited a more rapid decline in plasma cholesterol concentrations than control animals fed a cholesterol-free diet, indicating that the compound also inhibited the absorption of biliary cholesterol. Intravenous administration of pamaqueside had no effect on plasma cholesterol levels despite plasma levels twice those observed in rabbits given pamaqueside orally.These data indicate that pamaqueside and tiqueside induce hypocholesterolemia by blocking lumenal cholesterol absorption via a mechanism that apparently differs from the stoichiometric complexation of cholesterol hypothesized for other saponins.