IL-6 enhances IgE-dependent histamine release from human peripheral blood-derived cultured mast cells

We examined whether interleukin (IL)-6 exerts the stimulatory effects on the secretion of histamine from human mast cells triggered by crosslinking of the high affinity IgE receptor (FcepsilonRI) with IgE and anti-IgE. As target cells, we used peripheral blood-derived cultured mast cells grown with SCF, because they were superior in FcepsilonRIalpha expression to cord blood-derived mast cells. Incubation with SCF+IL-6 for 1 week increased the IgE-dependent release as well as intracellular content of histamine in the cultured mast cells, as compared with the values obtained by incubation with SCF alone. The magnitude of these increases was higher than that for priming with SCF+IL-4. A striking difference was also found in the expression of FcepsilonRIalpha between the two-factor combinations. The addition of IL-6 during FcepsilonRI crosslinking with IgE/anti-IgE in the presence of SCF did not influence histamine secretion. When SCF, IL-6 and IL-4 were used together, a further increase was observed in the anti-IgE-dependent liberation of histamine from the cultured mast cells, compared with the two-factor combinations. These results suggest that IL-6 functions as a secretagogue for the inflammatory mediator of human mast cells in the presence of SCF.     

Identification of a new member of the GLWamide peptide family: physiological activity and cellular localization in cnidarian polyps

KPNAYKGKLPIGLWamide, a novel member of the GLWamide peptide family, was isolated from Hydra magnipapillata. The purification was monitored with a bioassay: contraction of the retractor muscle of a sea anemone, Anthopleura fuscoviridis. The new peptide, termed Hym-370, is longer than the other GLWamides previously isolated from H. magnipapillata and another sea anemone, A. elegantissima. The amino acid sequence of Hym-370 is six residues longer at its N-terminal than a putative sequence previously deduced from the cDNA encoding the precursor protein. The new longer isoform, like the shorter GLWamides, evoked concentration-dependent muscle contractions in both H. magnipapillata and A. fuscoviridis. In contrast, Hym-248, one of the shorter GLWamide peptides, specifically induced contraction of the endodermal muscles in H. magnipapillata. This is the first case in which a member of the hydra GLWamide family (Hym-GLWamides) has exhibited an activity not shared by the others. Polyclonal antibodies were raised to the common C-terminal tripeptide GLWamide and were used in immunohistochemistry to localize the GLWamides in the tissue of two species of hydra, H. magnipapillata and H. oligactis, and one species of sea anemone, A. fuscoviridis. In each case, nerve cells were specifically labeled. These results suggest that the GLWamides are ubiquitous among cnidarians and are involved in regulating the excitability of specific muscles.     

Differences between maize and rice in N-use efficiency for photosynthesis and protein allocation

The N-use efficiency for photosynthesis was higher in a C(4) plant, maize, than in a C(3) plant, rice, including rbcS antisense rice with optimal ribulose-1,5-bisphosphate carboxylase (Rubisco) content for CO(2)-saturated photosynthesis, even when photosynthesis was measured under saturating CO(2) conditions. The N cost for the C(4) cycle enzymes in maize was not large, and the lower amount of Rubisco allowed a greater N investment in the thylakoid components. This greater content of the thylakoid components as well as the CO(2) concentrating mechanism may support higher N-use efficiency for photosynthesis in maize.     

Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms

We report an allele-specific sequencing method using allele-specific long-range polymerase chain reaction (PCR) to determine if multiple (specifically, more than three) single nucleotide polymorphisms (SNPs) are located on the same allele. We sequenced the glucocorticoid receptor (GR) gene as a model and detected four nucleotide changes, including two novel variations, in intron 4 and exons 6, 8, and 9 alpha in four of the investigated cell lines. The terminal SNPs (intron 4 and exon 9 alpha) were separated by 19 kb. Following SNP identification, the first round PCR allele-specific primers are designed at the both distal SNP sites (intron 4 and exon 9 alpha), placing the SNP positions at the primer 3'-end. Using these first round PCR products as template, the second round PCR was performed to separately amplify exons 6 and 8. These second round PCR products were subsequently sequenced. The sequencing results showed that the four SNPs were located on the same allele, i.e., forming a haplotype. This allele-specific long-range PCR/sequencing (ALP/S) method is rapid and applicable to the allelic assignment for more than three SNPs.     

Acetylcholine increases intracellular Ca2+ in the rat pituitary folliculostellate cells in primary culture

Pituitary folliculostellate cells (FSCs) are thought to partially inhibit pituitary hormone secretion through a paracrine mechanism. In this process, one of the important questions is what factors regulate the function of FSCs. Because ACh is synthesized in and possibly released from the corticotrophs and lactotrophs, we examined whether FSCs respond to ACh by the method of Ca2+ imaging in primary cultured FSCs from male Wistar rats. ACh (30 nM-3 microM) increased intracellular calcium concentration ([Ca2+](i)) of FSCs in a concentration-dependent manner, with an initial rapid rise followed by a relatively sustained increase. The complete block of the response by atropine and pirenzepine suggests involvement of muscarinic receptors. Depletion of the stored Ca2+ by thapsigargin blocked the response completely. Blockers of phospholipase C, U-73122 and neomycin, suppressed significantly the rise of [Ca2+](i). These results suggest that ACh increases [Ca2+](i) in FSCs by activating phospholipase C, presumably through activation of M(1) receptors. The rise in [Ca2+](i) could trigger a variety of Ca2+-dependent cellular processes, including the synthesis and release of bioactive substances, which in turn act on endocrine cells.     

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gialpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 approximately 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 microM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [3H]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gialpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gialpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gialpha-2 was found by immunoblot analysis with anti-Gialpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gialpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of Gbetagamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.