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.     

Leukotriene B3, leukotriene B4 and leukotriene B5; binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [3H]-leukotriene B4 have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B4. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [3H] leukotriene B4 binding to these preparations. Displacement of [3H]-leukotriene B4 by leukotriene B4 was compared with displacement by leukotriene B3 and leukotriene B5 which differ from leukotriene B4 only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B3 was shown to be equipotent to leukotriene B4 in ability to displace [3H]-leukotriene B4 from both rat and human leukocyte membranes while leukotriene B5 was 20-50 fold less potent. The relative potencies for the displacement of [3H]-leukotriene B4 by leukotrienes B3, B4 and B5 on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Biological activities of isomers of 8, 15-dihydroxyeicosatetraenoic acid

Chemically synthesized 8(S)-dihydroxy-5, 11-cis-9, 13-transeicosatetraenoic acid and 8(R), 15(S)-dihydroxy-5, 11-cis-9, 13-transeicosatetraenoic acid were inactive, in comparison to leukotriene B₄, in a human polymorphonuclear leukocyte chemokinetic assay and a rat polymorphonuclear leukocyte aggregation assay.     

A comparison of EMG feedback and alternative anxiety treatment programs

Four cohorts of 40 subjects each were randomly assigned to 1 of 10 treatment conditions utilizing EMG feedback, cognitive monitoring training, systematic desensitization, high expectancy discussion group, or waiting list controls either in isolation or in various combinations. A three-way ANOVA for repeated measures indicated that significant anxiety reductions were experienced in all noncontrol treatment conditions. Treatment groups employing EMG feedback demonstrated significantly greater anxiety decrements on Cattell's IPAT Self-Analysis Form, and baseline frontalis EMG. Adding desensitization or cognitive monitoring to EMG feedback did not produce a more powerful effect than using EMG feedback alone. Sex and age differences were also observed. Some implications are discussed.This research was supported in part by a grant from the Medical Services Research Foundation of Alberta.     

Actions of synthetic leukotrienes on platelets and blood vessels in the anesthetised pig: the release of a platelet derived vasodilator

The actions of leukotrienes (LT's) C4, D4, E4 and F4 have been investigated in the perfused hind-limb of the anesthetized pig. In the blood perfused hind limb LTC4, D4 and E4 increased the perfusion pressure in a dose-dependent fashion whereas LTF4 decreased perfusion pressure. In the Tyrode perfused hind limb all LT's increased perfusion pressure (rank order potency LTC4 = LTD4 much greater than LTF4). The actions of LTF4 were not affected by a wide variety of pharmacological treatments, including indomethacin, methysergide and FPL-55712. The LT's aggregated porcine platelets (rank order potency LTC4 greater than LTF4 greater than LTD4) and induced the release of a platelet-derived vasodilatory mediator. The results provide pharmacological evidence of specific leukotriene receptors in vivo and that leukotrienes can independently modulate blood flow. These data suggest that important interactions may occur between platelets, the arachidonate lipoxygenase products and platelet-derived substances in response to inflammatory stimuli in the cardiovascular system.     

A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium

Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can be directly examined by measuring a metabolic endpoint rather than an intermediate (such as a metabolite in the tricarboxylic acid cycle). For instance, oxidation of specific substrates can be measured in vitro by incubation of live cultures with substrates containing radiolabeled carbon and measuring radiolabeled carbon dioxide. To increase throughput, we previously developed a miniaturized platform to measure substrate oxidation of both adherent and suspension cells using multiwell plates rather than flasks. This enabled multiple conditions to be examined simultaneously, ideal for drug screens and mechanistic studies. However, like many metabolic assays, this was not compatible with bicarbonate-buffered media, which is susceptible to alkalinization upon exposure to gas containing little carbon dioxide such as air. While other buffers such as HEPES can overcome this problem, bicarbonate has additional biological roles as a metabolic substrate and in modulating hormone signaling. Here, we create a bicarbonate-buffered well-plate platform to measure substrate oxidation. This was achieved by introducing a sealed environment within each well that was equilibrated with carbon dioxide, enabling bicarbonate buffering. As proof of principle, we assessed metabolic flux in cultured adipocytes, demonstrating that bicarbonate-buffered medium increased lipogenesis, glucose oxidation, and sensitivity to insulin in comparison to HEPES-buffered medium. This convenient and high-throughput method facilitates the study and screening of metabolic activity under more physiological conditions to aid biomedical research.     

Detection of sequence polymorphisms in red junglefowl and White Leghorn ESTs

Over 16,000 high quality expressed sequence tags (ESTs) from red junglefowl (RJ) and White Leghorn (WL) brain and testis cDNA libraries were generated. Here, we have used this resource for detection of single nucleotide polymorphisms (SNPs), and also completed full-length sequencing of 46 pairs of clones, representing the same gene from both the RJ and WL libraries. From the main set of ESTs, which were assembled using Phrap, 746 putative SNPs were identified, of which 76% were transitions and 24% were transversions. A subset of SNPs was evaluated by sequence analysis of five RJ and five WL birds. Nine of 12 SNPs were verified in this limited sample, suggesting that a majority of the putative polymorphisms documented in this study represent real SNPs. During full-length sequencing of the 46 RJ/WL clones 100 SNPs were identified, which translated to a frequency of 1.90 SNPs/1000 bp. The number of transitions and transversions were 77% and 23%, respectively, and the proportion of non-synonymous vs. synonymous SNPs was 20% and 80%, respectively. Four large insertions/deletions were identified between the RJ and WL full-length sequences, and they appear to represent different splice variants.