Evolution of interleukin-18 binding proteins and interleukin-1 receptor, type II proteins

Interleukin-18 (IL-18) is one of the pivotal cytokines controlling the defense mechanism called inflammation. As a first step to develop proteins for controlling the IL-18 level, we initiated a study of IL-18-binding proteins (IL-18BPs). Twenty-four IL-18BP family members, 11 from vertebrates and 13 from chordopoxviruses, were picked from the NCBI database. Eight of these vertebrate IL-18BPs and two of the chordopoxvirus IL18-BPs were identified here and characterized as new members of the IL-18BP family. Their IL-18 binding domains were aligned and the distribution of highly conserved critical amino acid residues was analyzed and used to construct a phylogenetic tree. From this tree it was inferred that at least two independent events created two different ancestral viral IL-18BP genes by retroposition of IL-18BP genes from the vertebrate lineage. These two events are estimated to have occurred after an ancient mammalian IL-18BP gene diverged from birds, and before the mammalian IL-18BP gene diverged into human, ungulate and rodent IL-18BP genes. Moreover, our results suggest that IL-18BP and interleukin-1 receptor, type II (IL-1R2) had a common ancestral gene and diverged from the ancestral gene into IL-18BP and IL-1R2 genes in the fish period.     

Thin-filament-binding domains of cardiac and fast skeletal muscle troponin I isoforms as studied by epitope tagging

We examined the binding domains of cardiac and fast skeletal muscle troponin I (CTnI and FTnI, respectively) to myofibrils (MFs). Deletion mutants containing CTnI amino acid residues 1–79, 43–207 and 80–207 (CTnI-head, CTnI-tail-1 and CTnI-tail-2, respectively) and FTnI amino acid residues 1–54 and 55–182 (FTnI-head and FTnI-tail, respectively) were transiently expressed in cardiac and fast skeletal muscle cells. To monitor the intracellular localization of these exogenously introduced truncated TnIs, epitope tagging was used. CTnI-tail-1 was incorporated into cardiac MFs specifically, but CTnI-tail-2 was not assembled onto any MFs examined. This suggests that there is no potent actin filament-binding site in CTnI-tail-2. Since CTnI-tail-1 has an amino acid extension (CTnI residues 43–79) whose sequence is longer than that of CTnI-head-2; it appears that this sequence extension is important in binding to cardiac MFs. FTnI-tail, containing the inhibitory domain of actomyosin ATPase, showed intensive and specific incorporation into fast MFs. FTnI-tail was a homologous fragment of CTnI-tail-2, but the binding patterns of these two domains differed greatly from each other. It is possible that the absence of potent binding affinity of CTnI-tail-2 corresponding to the inhibitory domain of actomyosin ATPase is advantageous for continuous cardiac muscle contraction, since a potent inhibitory activity is a serious obstacle to cardiac muscle contraction. It can be assumed that distinctive binding ability of functional domains of TnI-tails reflect unique adaptations to muscles with different physiological properties.     

Human T-cell receptor BV6 gene polymorphism in relation to expression level and CD4/CD8 skewness

Using 50 samples of umbilical cord blood lymphocytes from Japanese donors, we analysed two human T-cell receptor beta variable (TCRBV) genes, BV6S4 and BV6S5, for their polymorphism, usage frequencies and CD4/CD8 skewness. They showed contrasting CD4/CD8 skewness, BV6S4 to CD8+ T cells and BV6S5 to CD4+ T cells. Genotyping of the BV6S4 alleles (A1, A2 and A3) revealed two of the six possible BV6S4 genotypes, A1/A2 and A2/A2. Among the two BV6S4 genotypes, no significant difference was detected in usage frequency or CD4/CD8 skewness. On the other hand, genotyping of the BV6S5 alleles (A1 and A2) revealed all three possible BV6S5 genotypes, A1/A1, A1/A2 and A2/A2, and the gene usage frequency was high, in the order A1/A1 > A1/A2 > A2/A2. These results indicate that the amino acid substitutions in BV6S5 (S36R and G70E) are in some way associated with the expression level of this gene. In the analysis of CD4/CD8 skewness, the three BV6S5 genotypes had similar skewness, indicating that A1 alleles are expressed more frequently than A2 alleles in both CD4+ and CD8+ T-cell populations. Although BV6S5 exhibits marked skewness to CD4+ T cells, our results indicate that the higher expression of A1 alleles is not associated with the increased ratio of CD4+ T cells.     

Further characterization of memory T cells existing in a case of CD8 deficiency

CD8 deficiency is a rare primary immunodeficiency caused by a defect of ZAP-70, which plays a pivotal role in T cell activation. We previously reported the existence of memory phenotype-CD4+ T cells in a case of CD8 deficiency, which demonstrates that activation signals through ZAP-70 are not essential to the phenotypic conversion of T cells from "naive" to "memory." In this study, we further characterized CD45RO+ T cells in a CD8 deficient patient. We showed that the patient's CD45RO+ T cell population had a wide variety of T cell receptor Vbeta-chain gene usage, and contained few clonally expanded T cells, while many clonally expanded T cells were present in the memory T cell population of age-matched healthy children. These results suggest that various kinds of antigens were involved in the differentiation of the patient's T cells, and that the differentiation into memory T cells was not accompanied by profound T cell proliferation. Moreover, our findings confirmed that the patient's CD45RO+CD4+ T cells had acquired effector-cytokine producing ability, indicating that there exists an alternative activation pathway which is independent of ZAP-70 for the acquisition of effector-cytokine producing ability.     

Freeze-mount microautoradiographic study in the mouse hippocampus after intravenous injection of tritiated 2-deoxyglucose and glucose

Differences in the uptake of tracers from radioactive 2-deoxyglucose ([1,2-3H] and [2,6-3H]), and glucose ([1-3H], [3-3H]) into hippocampal regions were investigated by freeze-mount microautoradiography after 45 min for 2-deoxyglucose, and after 15 and 45 min for glucose. Silver grains were assessed quantitatively by an image analyser. (1) The radioactivity (silver grains/mm2) in the stratum lacunosum-moleculare of Ammon's horn from 2-deoxyglucose autoradiograms was significantly higher than that in other hippocampal regions (P less than 0.01), while lowest in the hilus fascia dentata (P less than 0.01). (2) Autoradiograms of [1-3H]glucose and 15 min of [3-3H]glucose showed the radioactivity in the dentate molecular layer to be significantly higher than that in other regions, excepting the stratum lacunosummoleculare (P less than 0.05). (3) The 2-deoxyglucose and 45-min glucose autoradiograms showed intensely labeled perikarya of pyramidal cells in the CA3a sector. (4) Radioactivity in the dentate granular layer from the 45-min autoradiogram of [3-3H]glucose was significantly higher than that in the molecular layer (P less than 0.05). The results imply that the metabolic fate of glucose, i.e. whether it is mainly used for energy production or amino acid synthesis, depends on each structure of the hippocampus.     

Activation of Human Monocytes for Enhanced Production of Interleukin 8 During Transendothelial Migration in Vitro

Interleukin-8 (IL-8) is a chemokine for polymorphonuclear leukocytes (PMNs) and lymphocytes, which promotes the extravasation of these inflammatory cells. In this study, we investigated IL-8 synthesis induced by the adhesive interaction between monocytes and endothelial cells during transmigration and the capacity of transmigrated monocytes to produce IL-8. Cocultured human monocytes and human umbilical vein endothelial cell (HUVEC) monolayers induced the synefgistic production of IL-8, compared with cultures of either monocytes or HUVEC monolayers alone. Coculture-induced IL-8 production almost doubled after HUVECs were stimulated with IL-1. The induced IL-8 mRNA expression was consistent with the protein data, indicating the de novo synthesis of IL-8 by the coculture. Monoclonal antibodies (mAbs) against IL-8 inhibited the transendothelial chemotactic activity of the supernatants for PMNs by 55%. Immunohistochemistry revealed that both adherent and transmigrated monocytes and unstimulated HUVECs expressed IL-8 protein, whereas nonadherent monocytes did little. Transmigrated monocytes spontaneously secreted a 3.8-fold greater amount of IL-8 than the initial monocytes. Coculture-induced IL-8 production was inhibited about 30% by polyclonal Abs against IL-, IL-1, or tumor necrosis factor , while it was not affected by mAbs against intercellular adhesion molecule 1 or vascular cell adhesion molecule 1. The results suggested that adhesive interaction during the transmigration of monocytes through HUVEC monolayers activates both cell types to produce IL-8 and that transmigrated monocytes are capable of producing ample IL-8.     

Disconnected optic axons persist in the visual pathway during regeneration of the retino-tectal projection in the frog

Summary In this study, we crushed one optic nerve in the frog Litoria (Hyla) moorei and at intervals thereafter anterogradely labelled optic axons with horseradish peroxidase (HRP). For one series, HRP was applied between the eye and the crush site and in a second series between the crush site and the chiasm. A tectal projection of regenerating axons was seen in both series but, in addition, up to 12 weeks post-crush, the second series displayed an additional projection. Its appearance matched that of the disconnected, but persisting, optic axon terminals which are found after enucleation or optic nerve ligation. We conclude that, in the frog, many disconnected optic axons persist throughout the period of optic nerve regeneration and of restoration of an orderly retino-tectal map.Abbreviation HRP horseradish peroxidase     

Association and crystallization of poly(ethylene oxide)

The discontinuous change of the lamellar thickness with crystallization temperature was studied for low molecular weight fractions of OH-terminated poly(ethylene oxide) (PEO). IR analyses demonstrated that almost all of the molecular chain ends were associated in the molten state, whereas a large part of their ends were free in dilute solution. Discontinuous changes were observed for low molecular weight PEO fractions crystallized from the melt, whereas continuous changes were found both for PEO's crystallized from dilute solution and those with phenylated end groups crystallized from the melt. Accordingly, it was pointed out that the association of the end groups could play an important role in the crystallization mechanism and the conformation of the resultant PEO crystals.