Casein kinase Iepsilon down-regulates phospho-Akt via PTEN, following genotoxic stress-induced apoptosis in hematopoietic cells

Here, we show a functional role of casein kinase I (CKI) epsilon in hematopoietic cell survival through the modification of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Introduction of wild-type (WT)-CKIepsilon into interleukin-3 (IL-3)-dependent 32D cells increased the sensitivity to genotoxic stresses, such as gamma-irradiation, etoposide, and IL-3 deprivation, whereas kinase-negative (KN)-CKIepsilon suppressed it. Contrary to KN-CKIepsilon, WT-CKIepsilon attenuated the IL-3-induced activation of Akt with the increase of PTEN activity. Similarly, the increase of Akt activation, as well as PTEN inactivation, was accompanied both by a decrease of CKIepsilon expression induced by all-trans retinoic acid and by the addition of a specific inhibitor for CKIepsilon in HL-60 cells. CKIepsilon seems to activate PTEN by physical interaction. These results suggest that the CKIepsilon-induced down-regulation of PI3K/Akt signaling through PTEN lead to amplified sensitivity to apoptosis. Thus, the suppression of CKIepsilon in many human leukemia cell lines may play a role in the cell immortalization.     

Structural requirements for activation in alphaIIb beta3 integrin

Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant αIIbβ3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the β-head/β-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the α-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the α-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.     

Role of xyloglucan in gravitropic bending of azuki bean epicotyl

The mechanism of the gravitropic bending was studied in azuki bean epicotyls. The cell wall extensibility of the lower side became higher than that of the upper side in the epicotyl bending upward. The contents of matrix polysaccharides of the cell wall (pectin and xyloglucan in hemicellulose-II) in the lower side became smaller than those in the upper side. The molecular mass of xyloglucans in the lower side decreased. After an epicotyl was fixed to a metal rod to prevent the bending, gravistimulation was applied. Fundamentally the same results were obtained with respect to rheological and chemical characteristics of the cell wall as those of epicotyls showing gravitropic bending. The present results suggested that the initial gravitropic bending was caused by the increase in extensibility of the lower side and the decrease in extensibility of the upper side via the change of the cell wall matrix, especially xyloglucans.     

MyD88-dependent pathway in T cells directly modulates the expansion of colitogenic CD4+ T cells in chronic colitis

TLRs that mediate the recognition of pathogen-associated molecular patterns are widely expressed on/in cells of the innate immune system. However, recent findings demonstrate that certain TLRs are also expressed in conventional TCRalphabeta(+) T cells that are critically involved in the acquired immune system, suggesting that TLR ligands can directly modulate T cell function in addition to various innate immune cells. In this study, we report that in a murine model of chronic colitis induced in RAG-2(-/-) mice by adoptive transfer of CD4(+)CD45RB(high) T cells, both CD4(+)CD45RB(high) donor cells and the expanding colitogenic lamina propria CD4(+)CD44(high) memory cells expresses a wide variety of TLRs along with MyD88, a key adaptor molecule required for signal transduction through TLRs. Although RAG-2(-/-) mice transferred with MyD88(-/-)CD4(+)CD45RB(high) cells developed colitis, the severity was reduced with the delayed kinetics of clinical course, and the expansion of colitogenic CD4(+) T cells was significantly impaired as compared with control mice transferred with MyD88(+/+)CD4(+)CD45RB(high) cells. When RAG-2(-/-) mice were transferred with the same number of MyD88(+/+) (Ly5.1(+)) and MyD88(-/-) (Ly5.2(+)) CD4(+)CD45RB(high) cells, MyD88(-/-)CD4(+) T cells showed significantly lower proliferative responses assessed by in vivo CFSE division assay, and also lower expression of antiapoptotic Bcl-2/Bcl-x(L) molecules and less production of IFN-gamma and IL-17, compared with the paired MyD88(+/+)CD4(+) T cells. Collectively, the MyD88-dependent pathway that controls TLR signaling in T cells may directly promote the proliferation and survival of colitogenic CD4(+) T cells to sustain chronic colitis.     

Differential requirement for the CD40-CD154 costimulatory pathway during Th cell priming by CD8 alpha+ and CD8 alpha- murine dendritic cell subsets

Dendritic cells (DCs) regulate the development of distinct Th populations and thereby provoke appropriate immune responses to various kinds of Ags. In the present work, we investigated the role CD40-CD154 interactions play during the process of Th cell priming by CD8 alpha(+) and CD8 alpha(-) murine DC subsets, which have been reported to differently regulate the Th response. Adoptive transfer of Ag-pulsed CD8 alpha(+) DCs induced a Th1 response and the production of IgG2a Abs, whereas transfer of CD8 alpha(-) DCs induced Th2 cells and IgE Abs in vivo. Induction of distinct Th populations by each DC subset was also confirmed in vitro. Although interruption of CD80/CD86-CD28 interactions inhibited Th cell priming by both DC subsets, disruption of CD40-CD154 interactions only inhibited the induction of the Th1 response by CD8 alpha(+) DCs in vivo. CD40-CD154 interactions were not required for the proliferation of Ag-specific naive Th cells stimulated by either DC subset, but were indispensable in the production of IL-12 from CD8 alpha(+) DCs and their induction of Th1 cells in vitro. Taken together, in our immunization model of Ag-pulsed DC transfer, CD40-CD154 interactions play an important role in the development of CD8 alpha(+) DC-driven Th1 responses but not CD8 alpha(-) DC-driven Th2 responses to protein Ags.     

Hepatoprotective effect of sesquiterpenes in turmeric

Hepatoprotective effect of turmeric together with its sesquiterpenes and curcuminoids fractions were examined on D-galactosamine induced liver injury in rats. All the diets individually contained the turmeric extract, the curcuminoids fraction, and the sesquiterpenes fraction suppressed the increase of LDH, ALT, and AST levels caused by D-GalN treatment. Since few anti-oxidative activities are expected in the sesquiterpenes fraction, it is presumed that hepatoprotective mechanism of sesquiterpenes in turmeric is different from that of curuminoids.     

Membrane-anchored CD40 is processed by the tumor necrosis factor-alpha-converting enzyme. Implications for CD40 signaling

The soluble form of CD40 (sCD40), which co-exists with the membrane-anchored form (mCD40), is a natural antagonist of mCD40/CD154 interaction. However, the mechanism leading to the production of sCD40 has never been investigated. Here, we show that the engagement of mCD40 on the surface of B lymphocytes by anti-CD40 antibody led to enhanced sCD40 release associated with decreased amounts of mCD40. This sCD40 production was not affected by vesicular traffic inhibitors but was completely blocked by a broad-spectrum synthetic metalloproteinase (MP) inhibitor (GM6001) or a membrane-anchored MP-specific inhibitor (dec-RVKR-cmk). Recombinant MP disintegrin tumor necrosis factor-alpha converting enzyme (TACE) cleaved the purified CD40 ectodomain/Fc chimeric protein in vitro, giving rise to an sCD40 form similar to that shed from B cell cultures. Moreover, spontaneous production of sCD40 by mCD40-transfected human embryonic kidney cells (constitutively expressing TACE) was enhanced by the overexpression of TACE and abrogated by co-transfection with a dominant-negative TACE mutant. These results provide strong evidence that sCD40 production is an active process regulated by the engagement of mCD40 and its proteolytic cleavage by TACE or a related MP disintegrin. Given the antagonistic activity of sCD40 on the CD40/CD154 interaction, this shedding mechanism might represent an important negative feedback control of CD40 functions.     

Circulating interleukin-18 and osteopontin are useful to evaluate disease activity in patients with tuberculosis

BACKGROUND: T helper type 1 (Th1) responses have been implicated in the protective immunity, pathophysiology and development of tuberculosis. However, it is still unclear which molecule(s) reflect disease activity in patients with tuberculosis. METHODS: By specific enzyme immunoassays, circulating interferon-gamma. (IFN-gamma), interleukin-12 (IL-12), IL-18 and osteopontin (OPN) were measured in 47 patients with pulmonary tuberculosis and 7 patients with miliary tuberculosis before anti-tuberculosis therapy, and also measured in 19 patients with tuberculosis before and after anti-tuberculosis therapy. RESULTS: Circulating IFN-gamma, IL-18 and OPN levels were significantly higher in patients with pulmonary tuberculosis than in healthy controls, while there was no significant difference in levels of circulating IL-12 between tuberculosis patients and controls. Circulating IFN-gamma, IL-12, IL-18 and OPN paralleled the extent of lung lesions, and circulating IFN-gamma, IL-18 and OPN paralleled the magnitude of fever in patients with pulmonary tuberculosis. Patients with miliary tuberculosis had extremely high levels of circulating OPN, IFN-gamma and IL-18. Circulating IL-18 and OPN were significantly decreased with anti-tuberculosis therapy, whereas circulating IL-12 and IFN-gamma were not. CONCLUSIONS: Among Th1 response associated molecules, circulating levels of IL-18 and OPN, but not IFN-gamma or IL-12, reflect disease activity in patients with tuberculosis.     

Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum

The molecular machinery governing glutamatergic-GABAergic neuronal subtype specification is unclear. Here we describe a cerebellar mutant, cerebelless, which lacks the entire cerebellar cortex in adults. The primary defect of the mutant brains was a specific inhibition of GABAergic neuron production from the cerebellar ventricular zone (VZ), resulting in secondary and complete loss of external germinal layer, pontine, and olivary nuclei during development. We identified the responsible gene, Ptf1a, whose expression was lost in the cerebellar VZ but was maintained in the pancreas in cerebelless. Lineage tracing revealed that two types of neural precursors exist in the cerebellar VZ: Ptf1a-expressing and -nonexpressing precursors, which generate GABAergic and glutamatergic neurons, respectively. Introduction of Ptf1a into glutamatergic neuron precursors in the dorsal telencephalon generated GABAergic neurons with representative morphological and migratory features. Our results suggest that Ptf1a is involved in driving neural precursors to differentiate into GABAergic neurons in the cerebellum.