Eosinophils and cysteinyl leukotrienes

Eosinophils are the main source of the cysteinyl leukotrienes, LTC(4)/D(4)/E(4), which are lipid mediators that play major roles in the pathogenesis of asthma and other forms of allergic inflammation. Here, we review the mechanisms governing eosinophil LTC(4) synthesis, focusing on the distinct intracellular domains that regulate eicosanoid formation and function within eosinophils. Cysteinyl leukotrienes exert their actions by engaging specific receptors. As recently shown, eosinophils express CysLT1 and CysLT2, the only cloned receptors for cysteinyl leukotrienes. Therefore, here we also present some of the new findings regarding the paracrine/autocrine activation of these CysLT receptors on eosinophils, and discuss some data on novel intracrine effects of LTC(4) triggered by a putative third CysLT receptor expressed intracellularly within eosinophils.     

Twenty-four hour activity budgets and patterns of behavior in captive ocelots (Leopardus pardalis)

Activity budgets of captive ocelots (Leopardus pardalis) were assessed from over 547h of observational data obtained from six ocelots; two females at the Dallas Zoo (Dallas, TX), two females at the Caldwell Zoo (Tyler, TX) and a male and female at the Fossil Rim Wildlife Center (Glen Rose, TX). Data were examined for the percentage of active behaviors exhibited during the day and nighttime hours; temporal patterns of active, pace, exploratory and marking behavior, and for significance in pacing behavior between pre- and post-feeding times. The captive cats had a bimodal pattern of active behavior similar to field studies of wild ocelots, except that the timing of the active peaks were closer to the diurnal hours for the captive cats. The captive ocelots were less active than wild ocelots, and more diurnal. Also, the captive cats exhibited stereotypic pacing. When the percentage of time of active behavior was assessed for each cat, a strong variation between individuals and institution was not seen. Pacing behavior was highest prior to the feeding times for the cats. In assessing patterns of behavior, peaks in marking and exploratory behavior in the cats did not occur at the same time as the peaks in active behavior. However, we did see institutional differences in the pattern of exploratory and marking behavior, which may have been influenced by differing management practices.     

Induction of endothelial cell cytoplasmic lipid bodies during hypoxia

Lipid bodies (LBs), lipid-rich cytoplasmic inclusions found in many cell types, seem to act as nonmembrane sites of eicosanoid formation. Because alterations in eicosanoid products have been demonstrated in endothelial cells (ECs) during hypoxia, we investigated induction of LBs in systemic and pulmonary ECs exposed to acute and/or chronic hypoxia. LBs in ECs were O(2)-concentration dependent, increasing approximately fivefold during acute exposure to 0% O(2) in both cell types. During chronic exposure to 3% O(2), LBs were induced only in systemic ECs. LBs were not induced by other cellular stresses (heat shock or glucose deprivation). Subsequent studies suggested that protein kinase C-dependent and tyrosine kinase-dependent pathways are important in LB induction during hypoxia. PGH synthase was demonstrated in LBs in every case in which they were induced. These are the initial studies to demonstrate induction of LBs in ECs and to demonstrate LB induction during exposure to hypoxia in any cell type. These results imply that in ECs, LBs are structurally distinct inducible sites for synthesis of eicosanoid mediators.     

Light-induced nuclear translocation of endogenous pea phytochrome A visualized by immunocytochemical procedures

Although the physiological functions of phytochrome A (PhyA) are now known, the distribution of endogenous PhyA has not been examined. We have visualized endogenous PhyA apoprotein (PHYA) by immunolabeling cryosections of pea tissue, using PHYA-deficient mutants as negative controls. By this method, we examined the distribution of PHYA in different tissues and changes in its intracellular distribution in response to light. In apical hook cells of etiolated seedlings, PHYA immunolabeling was distributed diffusely in the cytosol. Exposure to continuous far-red (cFR) light caused a redistribution of the immunolabeling to the nucleus, first detectable after 1.5 hr and greatest at 4.5 hr. During this time, the amounts of spectrally active phytochrome and PHYA did not decline substantially. Exposure to continuous red (cR) light or to a brief pulse of red light also resulted in redistribution of immunolabeling to the nucleus, but this occurred much more rapidly and with a different pattern of intranuclear distribution than it did in response to cFR light. Exposures to cR light resulted in loss of immunolabeling, which was associated with PHYA degradation. These results indicate that the light-induced intracellular location of PHYA is wavelength dependent and imply that this is important for PhyA activity.     

Elevated A beta and apolipoprotein E in A betaPP transgenic mice and its relationship to amyloid accumulation in Alzheimer's disease

BACKGROUND: Amyloid-beta (A beta) accumulates in plaques and as cerebral amyloid angiopathy (CAA) in the brains of both Alzheimer's disease (AD) patients and transgenic A betaPPswe/tg2576 (tg2576) mice. Increasingly, evidence in humans and mice shows this process to be modulated by apolipoprotein E (apoE). MATERIALS AND METHODS: To explore this relationship, we measured apoE and A beta levels in brains of tg2576 mice and controls at intervals between 2 and 20 months. In addition, A beta concentrations in plasma and muscle of these animals were also quantified. RESULTS: Quite strikingly, we found that the amount of tg2576 mice brain apoE was elevated by an average of 45%, relative to the control mice from 2 months on. The level of brain apoE soared after 14 months to almost 60% greater than the level found in control mice. A beta concentrations in brains before 9 months were less than 2 ng/mg of protein, but by 14 months concentrations rose to 8.7 ng/mg, and by 20 months to 47 ng/mg. In plasma, we noted that the levels of A beta in tg2576 mice declined from above 30 ng/ml prior to 12 months to 14 ng/ml by 14 months. Histology showed that A beta plaques and CAA began to be discernible in the tg2576 mice at about 9 and 20 months of age, respectively. CONCLUSIONS: ApoE was immunocytochemically detected in neuritic plaques that were positive for thioflavine-S. We suggest that the elevation of brain apoE in tg2576 mice participates in an age-related dysregulation of A beta clearance and signals the start of A beta sequestration during the time of cognitive dysfunction.     

16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides

Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically important members of many microbial communities. A suite of five 16S rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats. For this we initially performed a literature survey-for the sources and sites of isolation of hitherto described members of the CFB-phylum. Probe CFB286 is mostly complementary to the 16S rRNA of species originally isolated from freshwater habitats, however, detects some marine and soil isolates and is the only probe which includes some food isolates. Probe CFB563 detects marine as well as animal-associated isolates. Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates. All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the data set investigated (October 1998), made up 46% of all 16S rRNA sequences from the CFB-phylum. We conclude that it is difficult to find habitat-specific probes for members of the CFB-phylum and that the design of probes for monophyletic groups should remain the standard approach. Applicability of the probes for fluorescence in situ hybridization and specificity for single cell detection were evaluated for the four mentioned probes whereas the fifth, probe CFB1082, which almost exclusively targets human-associated species, was not further characterized. The new probes are of limited determinative value and should be used together with the already established probes for the CFB-phylum. It is the hybridization pattern observed for a given cell or culture with the enlarged probe set that is suggestive for its affiliation with a defined group within the CFB-phylum.     

Transforming growth factors beta(1) (TGF-beta(1)) and TGF-beta(2) promote glioma cell migration via Up-regulation of alpha(V)beta(3) integrin expression

The migratory behaviour of malignant gliomas relies on the interaction of integrins with extracellular matrix (ECM) components. Transforming growth factor-beta(1) (TGF-beta(1)) potently stimulates glioma cell motility whereas TGF-beta(2) is known for its immunosuppressive properties. Here, we show that both TGF-beta(1) and TGF-beta(2) promote migration of glioma cells. In parallel, TGF-beta(1) and TGF-beta(2) induce alpha(V) and beta(3) intergrin mRNA expression and enhance cell surface expression of alpha(V)beta(3) integrin. TGF-beta-mediated promotion of migration is abrogated by echistatin, a Arg-Gly-Asp (RGD) peptide antagonist of alpha(V)beta(3) integrin, and by a neutralizing anti-alpha(V)beta(3) integrin antibody. Taken together, we report a novel mechanism by which TGF-beta modulates cell ECM interactions and promotes glioma cell motility.     

Role of Calcineurin in Ca2+-Induced Release of Catecholamines and Neuropeptides

Abstract: Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca²⁺-regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munc18, MARCKS, dynamin I, and B-50/GAP-43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin-neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca²⁺-induced release of [³H]noradrenaline and neuropeptide cholecystokinin-8 in a specific and concentration-dependent manner. Our data show that the Ca²⁺/calmodulin-dependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin-8, transmitters stored in large dense-cored vesicles.     

Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia

In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.