Biochemical characterization of the Drosophila wingless signaling pathway based on RNA interference

Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase Ialpha (CKIalpha) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKIalpha and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKIalpha- or Zw3-mediated Arm phosphorylation in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKIalpha and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKIalpha-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIalpha-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIalpha-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphorylation is due to CKIalpha and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.     

Menin missense mutants associated with multiple endocrine neoplasia type 1 are rapidly degraded via the ubiquitin-proteasome pathway

MEN1 is a tumor suppressor gene that is responsible for multiple endocrine neoplasia type 1 (MEN1) and that encodes a 610-amino-acid protein, called menin. While the majority of germ line mutations identified in MEN1 patients are frameshift and nonsense mutations resulting in truncation of the menin protein, various missense mutations have been identified whose effects on menin activity are unclear. For this study, we analyzed a series of menin proteins with single amino acid alterations and found that all of the MEN1-causing missense mutations tested led to greatly diminished levels of the affected proteins in comparison with wild-type and benign polymorphic menin protein levels. We demonstrate here that the reduced levels of the mutant proteins are due to rapid degradation via the ubiquitin-proteasome pathway. Furthermore, the mutants, but not wild-type menin, interact both with the molecular chaperone Hsp70 and with the Hsp70-associated ubiquitin ligase CHIP, and the overexpression of CHIP promotes the ubiquitination of the menin mutants in vivo. These findings reveal that MEN1-causing missense mutations lead to a loss of function of menin due to enhanced proteolytic degradation, which may be a common mechanism for inactivating tumor suppressor gene products in familial cancer.     

Sexual dimorphism of acoustic signals in the oriental white stork: non-invasive identification of sex in birds

Identification of the sex of birds is important for captive breeding of endangered species. In the oriental white stork (Ciconia boyciana), an endangered species, both sexes produce an acoustic signal called "clatter" by rattling their mandibles together to generate sounds. We examined the structure of male and female clatter to determine whether clatter is sexually dimorphic. The acoustic structure of the clatter of the two sexes proved to be dimorphic with respect to the fundamental frequency; female clatter had higher fundamental frequencies. The fundamental frequency correlated significantly and positively with bill length, suggesting that bill morphology contributes to the sexual dimorphism of clatter. Sexing can be done by acoustic signals without capturing birds, and thus is useful as a non-invasive sexing method for ecological and conservation studies of birds.     

A novel method for the measurement of in vitro fatty acid 2-hydroxylase activity by gas chromatography-mass spectrometry

Fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is an enzyme responsible for the de novo synthesis of sphingolipids containing 2-hydroxy fatty acids. 2-Hydroxy sphingolipids are highly abundant in the brain, as major myelin galactolipids (galactosylceramide and sulfatide) contain a uniquely high proportion ( approximately 50%) of 2-hydroxy fatty acids. Other tissues, such as epidermis, epithelia of the digestive tract, and certain cancers, also contain 2-hydroxy sphingolipids. The physiological significance of the 2-hydroxylation on N-acyl chains of subsets of sphingolipids is poorly understood. To study the roles of FA2H and 2-hydroxy sphingolipids in various tissues, we developed a highly sensitive in vitro FA2H assay. FA2H-dependent fatty acid 2-hydroxylation requires an electron transfer system, which was reconstituted in vitro with an NADPH regeneration system and purified NADPH:cytochrome P-450 reductase. A substrate [3,3,5,5-D(4)]tetracosanoic acid was solubilized in alpha-cyclodextrin solution, and the 2-hydroxylated product was quantified by gas chromatography-mass spectrometry after conversion to a trimethylsilyl ether derivative. When the microsomes of FA2H-transfected COS7 cells were incubated with the electron transfer system and deuterated tetracosanoic acid, deuterated 2-hydroxy tetracosanoic acid was formed in a time- and protein-dependent manner. With this method, FA2H activities were reproducibly measured in murine brains and tissue culture cell lines.     

Patterns of cytokine production in human immunodeficiency virus type 1 (HIV-1)-specific human CD8+ T cells after stimulation with HIV-1-infected CD4+ T cells

Although human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells can produce various cytokines that suppress HIV-1 replication or modulate anti-HIV-1 immunity, the extent to which HIV-1-specific CD8+ T cells produce cytokines when they recognize HIV-1-infected CD4+ T cells in vivo still remains unclear. We first analyzed the abilities of 10 cytotoxic T-lymphocyte (CTL) clones specific for three HIV-1 epitopes to produce gamma interferon, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after stimulation with epitope peptide-pulsed cells. These CTL clones produced these cytokines in various combinations within the same specificity and among the different specificities, suggesting a functional heterogeneity of HIV-1-specific effector CD8+ T cells in cytokine production. In contrast, the HIV-1-specific CTL clones for the most part produced a single cytokine, without heterogeneity of cytokine production among the clones, after stimulation with HIV-1-infected CD4+ T cells. The loss of heterogeneity in cytokine production may be explained by low surface expression of HLA class I-epitope peptide complexes. Freshly isolated HIV-1-specific CD8+ T cells with an effector/memory or memory phenotype produced much more of the cytokines than the same epitope-specific CTL clones when stimulated with HIV-1-infected CD4+ T cells. Cytokine production from HIV-1-specific memory/effector and memory CD8+ T cells might be a critical event in the eradication of HIV-1 in HIV-1-infected individuals.     

The keratan sulfate disaccharide Gal(6S03) beta1,4-GlcNAc(6S03) modulates interleukin 12 production by macrophages in murine Thy-1 type autoimmune disease

It has been reported that disaccharides of the glycosaminoglycans (GAGs), heparin, or heparan sulfate suppress the production of cytokines. Therefore, we examined the effects of GAGs (keratan sulfate, hyaluronan, chondroitin, chondroitin sulfate, and heparin sulfate) disaccharides on production of interleukin (IL)-12, a pivotal cytokine in the Th-1 type immune system. Among the GAG disaccharides, only a keratan sulfate disaccharide, Gal(6-SO(3))-GlcNAc(6-SO(3)) (L4), suppressed IL-12 production in macrophages stimulated with lipopolysaccharides and interferon-gamma. Neither keratan sulfate chains nor keratan sulfate tetrasaccharides elicited any change in the IL-12 production. N-Acetyl-lactosamine, Gal-GlcNAc (LacNAc), also did not change IL-12 production. These results indicated that a certain size, i.e. disaccharide and sulfate, are essential to suppress IL-12 production. L4 was then applied to MRL-lpr/lpr mice, a Th-1 type autoimmune disease model. The treatment of MRL-lpr/lpr mice with L4 1) decreased in serum IL-12, 2) induced apoptosis in T cells in lymph nodes thereby suppressing lymphoaccumulation, and 3) suppressed hypergammaglobulinemia and glomerulonephritis. We showed previously that IL-12 suppresses cell death of T cells, thereby enhancing the lymphoaccumulation in MRL-lpr/lpr mice. Moreover, it has been reported that IL-12 deficiency in MRL-lpr/lpr mice diminishes lymphoaccumulation and delays glomerulonephritis. The treatment with L4 suppressed phosphoprotein kinase C and phosphoinositide 3-kinase expression in macrophages, suggesting that L4 suppresses IL-12 production by inhibiting phosphoprotein kinase C and phosphoinositide 3-kinase pathways.     

ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles

Rho-associated kinase (ROCK) I mediates signaling from Rho to the actin cytoskeleton. To investigate the in vivo functions of ROCK-I, we generated ROCK-I-deficient mice. Loss of ROCK-I resulted in failure of eyelid closure and closure of the ventral body wall, which gave rise to the eyes open at birth and omphalocele phenotypes in neonates. Most ROCK-I(-/-) mice died soon after birth as a result of cannibalization of the omphalocele by the mother. Actin cables that encircle the eye in the epithelial cells of the eyelid were disorganized and accumulation of filamentous actin at the umbilical ring was impaired, with loss of phosphorylation of the myosin regulatory light chain (MLC) at both sites, in ROCK-I(-/-) embryos. Stress fiber formation and MLC phosphorylation induced by EGF were also attenuated in primary keratinocytes from ROCK-I(-/-) mice. These results suggest that ROCK-I regulates closure of the eyelids and ventral body wall through organization of actomyosin bundles.     

CXCL10 DNA vaccination prevents spontaneous diabetes through enhanced beta cell proliferation in NOD mice

CXCL10, a chemokine for Th1 cells, is involved in the pathogenesis of various Th1-dominant autoimmune diseases. Type 1 diabetes is considered to be a Th1-dominant autoimmune disease, and a suppressive effect of CXCL10 neutralization on diabetes development has been reported in a cyclophosphamide-induced accelerated diabetes model through induction of beta cell proliferation. However, intervention in a diabetes model might bring about opposite effects, depending on the timing, amount, or method of treatment. In the present study, we examined the effect of CXCL10 neutralization in a "spontaneous diabetes" model of NOD mice, using CXCL10 DNA vaccination (pCAGGS-CXCL10). pCAGGS-CXCL10 treatment in young NOD mice induced the production of anti-CXCL10 Ab in vivo and suppressed the incidence of spontaneous diabetes, although this treatment did not inhibit insulitis or alter the immunological response. pCAGGS-CXCL10 treatment enhanced the proliferation of pancreatic beta cells, resulting in an increase of beta cell mass in this spontaneous diabetes model as well. Therefore, CXCL10 neutralization is suggested to be useful for maintaining beta cell mass at any stage of autoimmune diabetes.