Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, II: Lifetimes of LFA-1 Bonds Under Force in Leukocyte Signaling

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant α_Lβ₂ immobilized on microspheres and β₂ integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from β₂ integrin throughout the course of each experiment. In our companion article I, we demonstrate the assay using results from tests of a monovalent ICAM-1 probe against recombinant α_Lβ₂ on microspheres in millimolar solutions of divalent cations (Ca²⁺, Mg²⁺, Mn²⁺). In this article, we examine the impact of inside-out and outside-in signaling in neutrophils on the lifetimes and mechanical strengths of ICAM-1 bonds to β₂ integrin on the cell surface. Even though ICAM-1 bonds to recombinant α_Lβ₂ on microspheres in Mg²⁺ or Mn²⁺ can live for long periods of time under slow pulling, here we show that stimulation of neutrophils in Mg²⁺ plus the chemokine IL-8 (i.e., inside-out signaling) induces several-hundred-fold longer lifetimes for ICAM-1 attachments to LFA-1, creating strong bonds at very slow pulling speeds where none are perceived in Mg²⁺ or Mn²⁺ alone. Similar changes are observed with outside-in signaling, i.e., long lifetimes and increased bond strength also occur when neutrophils are bound with the activating (anti-CD18) monoclonal 240Q. Limiting our investigation to rare events using very dilute ICAM-1 probes, we show that although the prolonged lifetimes of cell surface attachments for both inside-out and outside-in signaling exhibit single-bond-like statistics for dissociation under force, they are consistent with a tightly coupled dimeric ICAM-1 interaction with a pair of LFA-1 heterodimers.     

Dishevelled,a Wnt signalling component,is involved in mitotic progression in cooperation with Plk1

Wnt signalling is known to promote G1/S progression through the stimulation of gene expression, but whether this signalling regulates mitotic progression is not clear. Here, the function of dishevelled 2 (Dvl2), which transmits the Wnt signal, in mitosis was examined. Dvl2 localized to the spindles and spindle poles during mitosis. When cells were treated with nocodazole, Dvl2 was observed at the kinetochores (KTs). Dvl2 bound to and was phosphorylated at Thr206 by a mitotic kinase, Polo‐like kinase 1 (Plk1), and this phosphorylation was required for spindle orientation and stable microtubule (MT)‐KT attachment. Dvl2 was also found to be involved in the activation of a spindle assembly checkpoint (SAC) kinase, Mps1, and the recruitment of other SAC components, Bub1 and BubR1, to the KTs. However, the phosphorylation of Dvl2 by Plk1 was dispensable for SAC. Furthermore, Wnt receptors were involved in spindle orientation, but not in MT‐KT attachment or SAC. These results suggested that Dvl2 is involved in mitotic progression by regulating the dynamics of MT plus‐ends and the SAC in Plk1‐dependent and ‐independent manners.     

Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes

Both insulin and the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. However, regulation of the CIDE family by insulin and the contribution of the CIDE family to insulin actions remain unclear. Here, we investigated whether insulin regulates expression of the CIDE family and which subtypes contribute to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. Insulin decreased CIDEA and increased CIDEC but not CIDEB mRNA expression. Starvation-induced apoptosis in adipocytes was significantly inhibited when insulin decreased the CIDEA mRNA level. Small interfering RNA-mediated depletion of CIDEA inhibited starvation-induced apoptosis similarly to insulin and restored insulin deprivation-reduced adipocyte number, whereas CIDEC depletion did not. Lipid droplet size of adipocytes was increased when insulin increased the CIDEC mRNA level. In contrast, insulin-induced enlargement of lipid droplets was markedly abrogated by depletion of CIDEC but not CIDEA. Furthermore, depletion of CIDEC, but not CIDEA, significantly increased glycerol release from adipocytes. These results suggest that CIDEA and CIDEC are novel genes regulated by insulin in human adipocytes and may play key roles in the effects of insulin, such as anti-apoptosis and lipid droplet formation.     

The genetic profile and molecular diagnosis of thiophanate-methyl resistant strains of Fusarium asiaticum in Japan

Fusarium asiaticum strains resistant to thiophanate-methyl were detected in four prefectures in Japan though their proportion in the total population was low in all instances. The F167Y or F200Y mutation in the β2-tubulin gene (FGSG06611.3) was detected in thiophanate-methyl resistant (TMR) strains. A PCR-based diagnostic method based on these mutations was developed and applied for all 17 TMR strains that have been detected so far in Japan. Three and 14 TMR strains were the F167Y and F200Y mutation types, respectively. Analysis by 11 variable number of tandem repeat markers showed that TMR strains from the same site had an identical genotype, while TMR strains from different sites were dissimilar. This result indicates that TMR strains did not spread clonally to the different sites.     

Conformation and structure of 2-amino-8-(β-d-ribofuranosyl)imidazo [1,2-a]-s-triazin-4-one (5-aza-7-deaza guanosine), a potent antiviral nucleoside

The crystal and molecular structure of one imidazo[1,2-a]-s-triazine nucleoside and its antiviral activity are described. The crystal structure of 2-amino-8-(β-d-ribofuranosyl)imidazo-[1,2-a]-s-triazin-4-one monohydrate (C₁₀H₁₃N₅O₅·H₂O) was solved by X-ray counter data. The compound crystallizes in the monoclinic space group P2₁ with cell dimensions a = 7.353 (1), b = 6.465 (1), c = 13.701 (1) Å, β = 104.64 (1)°. The structure was solved by direct methods and refined by full matrix least-squares technique to a final value of the conventional R-factor of 0.049 using 1998 observed intensities. The orientation of the base relative to the sugar ring defined in terms of rotation about the C(1′)-N(8) glycosyl bond is anti (47.8°). The ribose moiety exhibits C(2′)-endo, ²E conformation. The conformation around C(4′)-C(5′) is gauche^?. Molecular packing is dominated by hydrogen bonds. Base stacking occurs long the b axis. 5-Aza-7-deazaguanosine has shown a marked antiviral activity in vitro against herpes simplex virus despite the fact that N(3) is effective as the hydrogen acceptor only.     

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997     

Identification of new amine acceptor protein substrate candidates of transglutaminase in rat liver extract: use of 5-(biotinamido) pentylamine as a probe

Transglutaminases (TGs) are a family of enzymes that catalyze Ca(2+)-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.     

Participation of prostaglandin E receptor EP4 subtype in duodenal bicarbonate secretion in rats

We examined, by using a specific PGE receptor subtype EP4 agonist and antagonist, the involvement of EP4 receptors in duodenal HCO(3)(-) secretion induced by PGE(2) and mucosal acidification in rats. Mucosal acidification was achieved by exposing a duodenal loop to 10 mM HCl for 10 min, and various EP agonists were given intravenously 10 min before the acidification. Secretion of HCO(3)(-) was dose-dependently stimulated by AE1-329 (EP4 agonist), the maximal response being equivalent to that induced by sulprostone (EP1/EP3 agonist) or PGE(2). The stimulatory action of AE1-329 and PGE(2) but not sulprostone was attenuated by AE3-208, a specific EP4 antagonist. This antagonist also significantly mitigated the acid-induced HCO(3)(-) secretion. Coadministration of sulprostone and AE1-329 caused a greater secretory response than either agent alone. IBMX potentiated the stimulatory action of both sulprostone and AE1-329, whereas verapamil mitigated the effect of sulprostone but not AE1-329. Chemical ablation of capsaicin-sensitive afferent neurons did not affect the response to any of the EP agonists used. We conclude that EP4 receptors are involved in the duodenal HCO(3)(-) response induced by PGE(2) or acidification in addition to EP3 receptors. The process by which HCO(3)(-) is secreted through these receptors differs regarding second-messenger coupling. Stimulation through EP4 receptors is mediated by cAMP, whereas that through EP3 receptors is regulated by both cAMP and Ca(2+); yet there is cooperation between the actions mediated by these two receptors. The neuronal reflex pathway is not involved in stimulatory actions of these prostanoids.     

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.