Interleukin 4 (IL‐4) exerts a decisive role in the coordination of protective immune responses against parasites, particularly helminths. A disregulation of IL‐4 function is possibly involved in the genesis of allergic disease states. The search for important amino acid residues in human IL‐4 by mutational analysis of charged invariant amino acid positions identified two distinct functional sites in the 4‐helix‐bundle protein. Site 1 was marked by amino acid substitutions of the glutamic acid at position 9 in helix A and arginine at position 88 in helix C. Exchanges at both positions led to IL‐4 variants deficient in binding to the extracellular domain of the IL‐4 receptor (IL‐4R(ex)). In parallel, up to 1000‐fold increased concentrations of this type of variant were required to induce T‐cell proliferation and B‐cell CD23 expression. Site 2 was marked by amino acid exchanges in helix D at positions 121, 124 and 125 (arginine, tyrosine and serine respectively in the wild‐type). IL‐4 variants affected at site 2 exhibited partial agonist activity during T‐cell proliferation; however, they still bound with high affinity to IL‐4R(ex). [The generation of an IL‐4 antagonist by replacing tyrosine 124 with aspartic acid has been described before by Kruse et al. (1992) (EMBO J., 11, 3237‐3244)]. These findings indicate that IL‐4 functions by binding IL‐4R(ex) via site 1 which is constituted by residues on helices A and C.(ABSTRACT TRUNCATED AT 250 WORDS)
For many years we have investigated the earliest crystal formations of different developing hard tissues (matrix vesicle, bone, dentine, enamel, etc.) by different electron microscopic measurements. It was observed that primarily Ca-phosphate (apatite) "chains," composed of nanometer sized particles (dots, islands), exist, which coalesce rapidly to needles. For the mineralization of collagen (e.g., bone, dentine) the center to center distances between the dots in the mineral chains represent the distances between nucleating sites, so-called "active sites" of collagen which bind primarily Ca for a subsequent nucleation. For the mineralization of noncollagen macromolecules (e.g., enamel) the same principle of mineral nucleation at such "active sites" exists being represented indirectly by corresponding center to center distances between the dots in the mineral chains.
SummaryThe primary crystallites of the different developing hard tissues have an apatite structure. However, they have crystal lattice distortions representing an intermediate state between amorphous and fully crystalline. We have applied energy-filtering transmission electron microscopy in the selected area electron diffraction mode to analyse different stages of crystal formation in dentine, bone, enamel and inorganic apatite mineral. We have obtained quantitative information on the degree of crystal lattice distortion using the paracrystal theory of Hosemann and Bagchi.We have found that the early formed crystallites of the hard tissues being analysed have a paracrystalline character comparable to biopolymers. However, with maturation, the lattice fluctuations of the crystallites of the hard tissues bone, enamel and dentine decrease to form a typical (para)crystalline character. Also the decrease of the organic proportion in the matrix corresponds to the decrease of the lattice fluctuation of the crystallites in the different hard tissues during maturation.
Interleukin-4 (IL-4) triggers cellular responses by interaction with the bipartite interleukin-4 receptor (IL-4R). IL-4-responsive cells specifically endocytose IL-4. We studied the ligand internalization properties of the human IL-4R and analyzed the specific functions of its two subunits IL-4Ra and gc in this process. IL-4 mutant RY, which binds to IL-4Ra but does not recruit gc into the receptor complex was used as a tool to show that IL-4Ra can promote independent ligand uptake in human T cells. Internalization was limited, however, by rapid IL-4 dissociation, suggesting that one important function of gc in IL-4 endocytosis is to retain the ligand sufficiently long within the ternary receptor complex. We then measured IL-4 internalization by murine Ba/F3 cells that were stably transfected with various human IL-4R constructs. Efficient IL-4 uptake required the cytoplasmic section of the receptor. The intracellular domains of IL-4Ra and gc were responsible for independent endocytosis processes with distinct kinetics. IL-4Ra-mediated internalization resulted in long-term intracellular maintainance of IL-4, whereas gc directed the associated radioligand to intracellular breakdown and rapid release in the form of degraded protein. Mutants of either IL-4R subunit deficient in Janus kinase activation were not impaired in internalization, indicating that IL-4 endocytosis is not functionally connected to signal transduction.Keywords: cytokine receptor; endocytosis; internalization; interleukin-4; signal transduction.Interleukin-4 (IL-4) is a key regulator of the immune system and is a central mediator of type I allergy [1]. It exerts its function on various target cells by activating the bipartite interleukin-4 receptor (IL-4R), which consists of the IL-4Ra subunit and the common g receptor chain (gc). The latter is also a constituent of the receptor systems for IL-2, IL-7, IL-9 and IL-15 [2]. The interaction of IL-4 with the IL-4R in the course of heterodimerization is a crucial event for the onset of IL-4 driven cellular reactions and has therefore been extensively studied, also with the aim of potential pharmacological intervention. Antagonistic inhibitors of IL-4 have been developed based on their properties to tightly bind IL-4Ra, but to fail contacting gc [3].Ligand-induced dimerization of IL-4Ra and gc triggers signal cascades that involve reversible tyrosine phosphorylation of cytoplasmic proteins, e.g. the JAK/STAT pathway [4]. Whereas the initiation of these signal transduction events has been quite well characterized in recent years, the limitations and termination of IL-4-induced signaling is still poorly understood. One important general mechanism of signal down-regulation and control of cell responsiveness is the internalization of ligand±receptor complexes. Endocytosis of IL-4 by IL-4R-expressing cells has been demonstrated [5], but has not yet been investigated in detail.The most thoroughly analyzed endocytosis pathway proceeds via clathrin-coated pits as examplified by the transferrin receptor [6]. T...
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