A novel function of N-cadherin and Connexin43: marked enhancement of alkaline phosphatase activity in rat calvarial osteoblast exposed to sulfated hyaluronan

In this study, we examined the interaction of the osteoblast which forms bone and sulfated hyaluronan (SHya). For the purpose of the creation of a new functional polysaccharide, we introduced a sulfate group in hyaluronan (Hya) of high molecular weight, and SHya of high molecular weight could be obtained for the first time. When rat calvarial osteoblast (rOB) cells were cultured with a high concentration of SHya, they formed aggregated spheroids after 4h and the spheroids grew to about 200microm after 24h. We examined the expression of cell adhesion molecules in order to clarify the mechanism of aggregate formation. The N-cadherin (N-cad) and Connexin43 (Cx43) expression level of rOB cells cultured with SHya remarkably increased after 2h. A difference in the expression of Integrin beta1 (Intbeta1) could not be observed between the SHya addition and control group. The alkaline phosphatase (ALPase) activity of rOB cells cultured with SHya after 8h was significantly enhanced in comparison with control. Therefore, the sulfate group of SHya seems to enhance expression of cell adhesion protein such as N-cad and Cx43, resulting in aggregate formation and further remarkable induction of the ALPase activity of rOB cells.     

Syntheses and structural characterizations of novel mono- and dinuclear iridium hydrido complexes with polydentate nitrogen donor ligands

New five mono- and dinuclear Ir hydrido complexes with polydentate nitrogen ligands, [Ir(H)₂(PPh₃)₂(tptz)]PF₆ (1), [Ir₂(H)₄(PPh₃)₄(tptz)](PF₆)₂ · 2H₂O (2 · 2H₂O), [Ir(H)₂(PPh₃)₂(tppz)]BF₄ (3), [Ir₂(H)₄(PPh₃)₄(tppz)](BF₄)₂ (4) and [Ir₂(H)₄(PPh₃)₄(bted)](BF₄)₂ · 6CHCl₃ (5 · 6CHCl₃), were systematically prepared by the reactions of the precursor Ir hydrido complex [Ir(H)₂(PPh₃)₂(Me₂CO)₂]X (X=PF₆ and BF₄) with 2,4,6-tris(2-pyridyl)-1,3,5-triazine (tptz), 2,3,5,6-tetrakis(2-pyridyl)pyrazine (tppz) and 1,4-bis(2,2^′:6^′,2″-terpyridine-4^′-yl)benzene (bted), and their structures and properties were characterized in the solid state and in solution. Each of the Ir hydrido complexes with polydentate nitrogen ligands crystallographically described a unique coordination mode. Their ¹H NMR spectra demonstrated unusual ¹H NMR chemical shifts of pyridyl rings that are likely induced by the ring current effect of neighboring ligands.     

In vitro infection of host roots by differentiated calli of the parasitic plant Orobanche

Root parasites of the genus Orobanche are serious weeds in agriculture. An aseptic infection system of host roots using calli of three Orobanche species was developed for the study of host-parasite interaction. The response of calli to various hormonal combinations was studied, because a requirement for infection is the differentiation of root-like protrusions, which are capable of producing haustorial connections to the host. Infectious root-like protrusions develop under the influence of 0.5-1.0 mg l(-1) IAA, and under the combination of 0.2 mg l(-1) NAA with 5.0 mg l(-1) kinetin. These protocols produced root protrusions with pad-like structures that resembled attachment organs of Orobanche seedlings, and proved effective in parasitizing host roots. Direct contact with the medium inhibited haustorium development and prevented infection. To overcome this problem, certain root portions were isolated from the medium by inserting thin glass plates underneath. Calli were then placed on the raised root portions and successfully infected the roots and developed young Orobanche tubercles with vascular system that directly connected to the host.     

CXC chemokine ligand 10 neutralization suppresses the occurrence of diabetes in nonobese diabetic mice through enhanced beta cell proliferation without affecting insulitis

We have shown that neutralization of IFN-inducible protein 10/CXCL10, a chemokine for Th1 cells, breaks Th1 retention in the draining lymph nodes, resulting in exacerbation in Th1-dominant autoimmune disease models induced by immunization with external Ags. However, there have been no studies on the role of CXCL10 neutralization in Th1-dominant disease models induced by constitutive intrinsic self Ags. So, we have examined the effect of CXCL10 neutralization using a type 1 diabetes model initiated by developmentally regulated presentation of beta cell Ags. CXCL10 neutralization suppressed the occurrence of diabetes after administration with cyclophosphamide in NOD mice, although CXCL10 neutralization did not significantly inhibit insulitis and gave no influence on the trafficking of effector T cells into the islets. Because both CXCL10 and CXCR3 were, unexpectedly, coexpressed on insulin-producing cells, CXCL10 was considered to affect mature and premature beta cells in an autocrine and/or paracrine fashion. In fact, CXCL10 neutralization enhanced proliferative response of beta cells and resultantly increased beta cell mass without inhibiting insulitis. Thus, CXCL10 neutralization can be a new therapeutic target for beta cell survival, not only during the early stage of type 1 diabetes, but also after islet transplantation.     

The stem cell niche: theme and variations

Stem cells in animal tissues are often located and controlled by special tissue microenvironments known as niches. Studies of stem cell niches in model systems such as Drosophila have revealed adhesive interactions, cell cycle modifications and intercellular signals that operate to control stem cell behavior. Candidate niches and regulatory molecules have also been identified in many mammalian tissues, including bone marrow, skin, gut and brain. While niches are an ancient evolutionary device with conserved features across diverse organisms, we suggest that certain niches display important differences in their organization and function.     

Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively.     

Endogenous levels of polyamines in the organs of cucumber plant (Cucumis sativus) and factors affecting leaf polyamine contents

Polyamine compositions of various organs from hydroponically cultivated cucumber plants ( Cucumis sativus L. cv. Sharp-1) and factors affecting the leaf polyamine content were examined. Diamine putrescine was found most abundantly in the root, while a relatively large amount of spermine was detected in the reproductive organs such as the immature fruit and the calyx (+stamen). Spermidine was present at the highest level in rapidly growing tissues such as newly expanded leaf and fruit at an early developing stage, implying the possible involvement of spermidine in the growth and development of these young tissues. Polyamine content of cucumber leaves changed during the day. Especially, the putrescine content of upper leaves showed a striking decrease from the morning to the night. Alterations of leaf Ca or Mg content did not significantly affect leaf polyamine composition. On the other hand, abnormal cucumber leaves showed altered polyamine composition. Yellowing of the leaf intervein resulted in a striking decrease in spermidine content without a significant change in putrescine and spermine content. By contrast, the leaves infected with the phytopathogen, powdery mildew, showed decreased putrescine and increased spermine content in response to the degree of fungi infection. The possible usefulness of polyamines as a diagnostic marker of plant development and physiological disorder is discussed.     

Analyses of virus-induced homomeric and heteromeric protein associations between IRF-3 and coactivator CBP/p300

Cellular genes including the type I interferon genes are activated in response to viral infection. We previously reported that IRF-3 (interferon regulatory factor 3) is specifically phosphorylated on serine residues and directly transmits a virus-induced signal from the cytoplasm to the nucleus, and then participates in the primary phase of gene induction. In this study, we analyzed the molecular mechanism of IRF-3 activation further. The formation of a stable homomeric complex of IRF-3 between the specifically phosphorylated IRF-3 molecules occurred. While virus-induced IRF-7 did not bind to p300, the phosphorylated IRF-3 complex formed a stable multimeric complex with p300 (active holocomplex). Competition using a synthetic phosphopeptide corresponding to the activated IRF-3 demonstrated that p300 directly recognizes the structure in the vicinity of the phosphorylated residues of IRF-3. These results indicated that the phosphorylation of serine residues at positions 385 and 386 is critical for the formation of the holocomplex, presumably through a conformational switch facilitating homodimer formation and the generation of the interaction interface with CBP/p300.