Structural transition of a 15 amino acid residue peptide induced by GM1

The ganglioside GM1-binding peptide, p3, with a sequence of VWRLLAPPFSNRLLP, displayed a clear structural alteration depending on the presence or absence of GM1 micelles. The three-dimensional structures of the p3 peptide in the free and GM1 bound states were analyzed using two-dimensional NMR spectroscopic experiments with distance-restrained simulated annealing calculations. The NMR experiments for the p3 peptide alone indicated that the peptide has two conformers derived from the exchange of cis and trans forms at Pro(7)-Pro(8). Further study with theoretical modeling revealed that the p3 peptide has a curb conformation without regular secondary structure. On the other hand, the NMR studies for the p3 peptide with the GM1 micelles elucidated a trans conformer and gave a structure stabilized by hydrophobic interactions of beta- and helical turns. Based on these structural investigations, tryptophan, a core residue of the hydrophobic cluster, might be an essential residue for the recognition of the GM1 saccharides. The dynamic transition of the p3 peptide may play an important role in the function of GM1 as a multiple receptor as in the traditional pathway of the infection by cholera toxin.     

Direct neurite-osteoblastic cell communication, as demonstrated by use of an in vitro co-culture system

Using an in vitro co-culture approach comprising cultured murine superior cervical ganglia and MC3T3-E1 osteoblast-like cells, we found that the addition of scorpion venom (SV) elicited neurite activation via intracellular Ca2+ mobilization and, after a lag period, osteoblastic Ca2+ mobilization. SV did not have any direct effect on the osteoblastic cells in the absence of neurites. The addition of an alpha1-adrenergic receptor (AR) antagonist, prazosin, dose-dependently prevented the osteoblastic activation that resulted as a consequence of neural activation by SV. These results demonstrate that osteoblastic activation occurred as a direct response to neuronal activation, which activation was mediated by alpha1-ARs in the osteoblastic cells.     

Properties of blocking and non-blocking monoclonal antibodies specific for human macrophage galactose-type C-type lectin (MGL/ClecSF10A/CD301)

Monoclonal antibodies (mAbs) specific for the human macrophage galactose-type calcium-type lectin (MGL) were established. The recombinant extracellular domain of MGL was used to immunize a mouse, and 10 hybridoma clones were obtained. Binding of recombinant MGL to asialo-bovine submaxillary mucin was shown to be blocked by mAbs MLD-1, 4 and 6. Immunoprecipitation of MGL from lysates of COS-1 cells transfected with MGL cDNA (form 6A) was achieved with mAbs MLD-1, 4, 7, 8 and 16. Chimeric recombinant proteins between human MGL and mouse MGL1 were used to determine the location of the epitopes for these mAbs. mAbs MLD-8, 13, 15 and 16 interacted with the amino terminal side of the conserved WVDGTD sequence immediately upstream of QPD, whereas mAbs MLD-7, 12 and 17 interacted with the other side. mAbs MLD-1, 4, and 6 apparently required both sides of this boundary. mAbs MLD-15 and 16 were shown to recognize the protein products of alternatively spliced mRNA 6A/8A and 6C/8A, having deletions at the boundary of exons 7 and 8, in addition to full length and other spliced forms of MGL (6A, 6B and 6C), whereas the other mAbs bound only full length and forms 6A, 6B and 6C.     

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.     

Nuclear shuttling of mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase (ERK) 2) was dynamically controlled by MAP/ERK kinase after antigen stimulation in RBL-2H3 cells

The mitogen-activated protein kinase (MAPK) cascade consists of the MAPK (extracellular signal-regulated kinase 2; ERK2) and its activator, MAPK kinase (MAP/ERK kinase; MEK). However, the mechanisms for activation of ERK2 have not been defined yet in cells. Here, we used fluorescent protein-tagged ERK2 and MEK to examine the localization of ERK2 and MEK in living rat basophilic leukemia (RBL-2H3) cells. ERK2 was mainly in the cytoplasm in resting cells but translocated into the nucleus after the ligation of IgE receptors. The import of ERK2 reached the maximum at 6--7 min, and then the imported ERK2 was exported from the nucleus. MEK mainly resided in the cytoplasm, and no significant MEK translocation was detected statically after ligation of IgE receptors. However, analysis of the dynamics of ERK2 and MEK suggested that both of them rapidly shuttle between the cytoplasm and the nucleus and that MEK regulates the nuclear shuttling of ERK2, whereas MEK remains mainly in the cytoplasm. In addition, the data suggested that the sustained calcium increase was required for the optimal translocation of ERK2 into the nucleus in RBL-2H3 cells. These results gave a new insight of the dynamics of ERK2 and MEK in the nuclear shuttling of RBL-2H3 cells after the ligation of IgE receptors.     

Increased numbers of B-1 cells and enhanced responses against TI-2 antigen in mice lacking APS, an adaptor molecule containing PH and SH2 domains

APS (adaptor molecule containing PH and SH2 domains) is an intracellular adaptor protein that forms an adaptor family along with Lnk and SH2-B. While experiments using cultured cell lines have demonstrated that APS is phosphorylated in response to various stimuli, its in vivo functions remain unclear. We attempted to determine the physiological roles of APS by generating APS-deficient (APS(-/-)) mice. APS(-/-) mice were viable and fertile and showed no abnormalities or growth retardation. Immunologically, APS(-/-) mice showed normal development and distribution of lymphocytes and myeloid cells, except for increased numbers of B-1 cells in the peritoneal cavity. APS(-/-) mice exhibited an enhanced humoral immune response against trinitrophenol-Ficoll, a thymus-independent type 2 antigen, while APS(-/-) B-2 cells exhibited normal proliferative responses and tyrosine phosphorylation of intracellular proteins upon B-cell receptor (BCR) cross-linking. APS colocalized with filamentous actin (F-actin) accumulated during the capping of BCRs in APS-transgenic B cells. After BCR stimulation, F-actin contents were lower in APS(-/-) B-1 cells than in wild-type B-1 cells. Our results indicate that APS might have a novel regulatory role in actin reorganization and control of B-1 cell compartment size.     

Developmental origin of wiring specificity in the olfactory system of Drosophila

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.     

The Induction of Intercellular Adhesion Molecule-1 on Human Umbilical Vein Endothelial Cells by a Heat-Stable Component of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Live Porphyromonas gingivalis enhanced the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of human umbilical vein endothelial cells (HUVECs) in a bacterial dose-dependent manner. Inactivation of P. gingivalis by ultraviolet (UV), heat (56°C, 30 min), or sonication did not alter its stimulatory activity. ICAM-1 expression began to increase at 4 h after stimulation, reached a maximum at 12 h, and remained at the maximum for at least the next 8 h. This time course was similar to that of expression by Escherichia coli LPS. Furthermore, the effect of UV-inactivated P. gingivalis was not inhibited by boiling or polymyxin B treatment. In addition, the effect of P. gingivalis strain W83 on ICAM-1 expression was stronger than that of strain ATCC 33277. Our results suggested that some unidentified, heat-stable proteins, polysaccharides, or lipids may be the stimulatory factor(s), although the participation of LPS could not be completely ruled out. The ability of P. gingivalis to stimulate ICAM-1 expression on endothelial cells may play an important role in the pathogenesis of periodontal disease.     

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Arterial anatomical features of the upper palpebra

The arterial anatomical features of the upper palpebra were examined in both sides of seven fresh cadavers that had been systemically injected with a lead oxide/gelatin mixture. All specimens were stereoscopically radiographed for analysis of the three-dimensional structure of the arteries and were macroscopically dissected for observation of the relationships between the arteries and the other tissues. Cross-sections were prepared from one specimen and examined histologically. In all cases, there were four arterial arcades in the upper palpebra, namely, the marginal, peripheral, superficial orbital, and deep orbital arcades. Each arcade provided small vertical branches. The vertical branches coursed on both sides of the orbicularis oculi muscle and on both sides of the tarsal plate. From these small vertical branches, fine vessels branched off to the skin, muscle, and tarsal plate. These findings are important for avoiding complications such as bleeding and are useful for designing local flaps, such as switch flaps, for reconstructive surgical procedures.