Involvement of stress kinase mitogen-activated protein kinase kinase 7 in regulation of mammalian circadian clock

The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes, such as cell proliferation, apoptosis, differentiation, and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of an MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1ε. Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.     

Negative regulation of wnt11 expression by Jnk signaling during zebrafish gastrulation

Stress‐induced Sapk/Jnk signaling is involved in cell survival and apoptosis. Recent studies have increased our understanding of the physiological roles of Jnk signaling in embryonic development. However, still unclear is the precise function of Jnk signaling during gastrulation, a critical step in the establishment of the vertebrate body plan. Here we use morpholino‐mediated knockdown of the zebrafish orthologs of the Jnk activators Mkk4 and Mkk7 to examine the effect of Jnk signaling abrogation on early vertebrate embryogenesis. Depletion of zebrafish Mkk4b led to abnormal convergent extension (CE) during gastrulation, whereas Mkk7 morphants exhibited defective somitogenesis. Surprisingly, Mkk4b morphants displayed marked upregulation of wnt11, which is the triggering ligand of CE and stimulates Jnk activation via the non‐canonical Wnt pathway. Conversely, ectopic activation of Jnk signaling by overexpression of an active form of Mkk4b led to wnt11 downregulation. Mosaic lineage tracing studies revealed that Mkk4b‐Jnk signaling suppressed wnt11 expression in a non‐cell‐autonomous manner. These findings provide the first evidence that wnt11 itself is a downstream target of the Jnk cascade in the non‐canonical Wnt pathway. Our work demonstrates that Jnk activation is indispensable for multiple steps during vertebrate body plan formation. Furthermore, non‐canonical Wnt signaling may coordinate vertebrate CE movements by triggering Jnk activation that represses the expression of the CE‐triggering ligand wnt11. J. Cell. Biochem. 110: 1022–1037, 2010. © 2010 Wiley‐Liss, Inc.     

Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes

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Circular dichroism studies on flavoproteins containing covalently bound coenzymes

Absorption and circular dichroism spectra of cholesterol oxidase from Schizophyllum commune and choline oxidase from Alcaligenes sp. were measured and compared. The prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD (1, 2), while that of choline oxidase is 8 alpha-[N(3)-histidyl]-FAD (3). In the CD spectra of the two enzymes in either the oxidized or reduced state, the corresponding bands in the visible region are of approximately the same intensity and shape but of opposite sign. A notable feature in the CD spectra of the two enzymes after light irradiation is the appearance of a CD band in the longer wavelength region (550-650 nm) and the opposite signs of the CD band in this region in the two enzymes. The similarity of the shape and intensity of the CD spectra of the two enzymes suggests that the environments surrounding the flavin moieties are very similar, and the sign reversal of the CD bands suggests that the mutual orientations between the transition moment of flavin and that of its environment differ in the two enzymes.     

A fluorescence study of egg white riboflavin-binding protein

1. Denaturation of riboflavin-binding protein (RBP) by guanidine hydrochloride (Gu-HCl) was investigated by measruing the fluorescence of the protein. The denaturation-renaturation processes of RBP by Gu-HCl were fully reversible. The apo-RBP fluorescence had an emission maximum at 343 nm in the absence of Gu-HCl, and at 350 nm in the presence of 4M Gu-HCl, which completely denatured the protein. The relative fluorescence yield of apo-RBP in the presence of 4 M Gu-HCl was about 170% of that in the absence of Gu-HCl. The affinity of native apo-RBP for riboflavin was very strong, while riboflavin was not bound to the denatured form. The equilibrium system of apo-RBP and riboflavin in solutions containing Gu-HCl at various concentrations was analyzed by measuring riboflavin fluorescence. 2. The quenching of apo-RBP fluorescence, probably the fluorescence of tryptophanyl residues, by iodide anions and cesium cations was measured. The fluorescence of apo-RBP in the presence of 4 M Gu-HCl was quenched considerably by iodide and cesium, and Stern-Volmer plots were linear. However, the fluorescence of native apo-RBP was scarcely quenched by iodide or cesium. This suggested that tryptophanyl residues buried inside apo-RBP were responsible for most of the tryptophanyl fluorescence of native apo-RBP.     

Regenerating islet-derived protein (Reg)3β plays a crucial role in attenuation of ileitis and colitis in mice

Regenerating islet-derived protein (Reg)3β belongs to a member of the Reg family of proteins and has pleiotropic functions, including antimicrobial activity and tissue repair. However, whether Reg3β plays a protective role in the development of colitis and ileitis has not been fully investigated. We generated transgenic mice expressing a short form of cellular FLICE-inhibitory protein (cFLIPs) that promotes necroptosis, a regulated form of cell death. cFLIPs transgenic (CFLARs Tg) mice develop severe ileitis in utero. Although Reg3β is undetectable in the small intestine of wild-type embryos, its expression is aberrantly elevated in the small intestine of CFLARs Tg embryos. To test whether elevated Reg3β attenuates or exacerbates ileitis in CFLARs Tg mice, we generated a Reg3b^?/? strain. Reg3b^?/? mice grew to adulthood without apparent abnormalities. Deletion of Reg3b in CFLARs Tg mice exacerbated the embryonic lethality of CFLARs Tg mice. Dextran sulfate sodium-induced colitis, characterized by body weight loss and infiltration of neutrophils, was exacerbated in Reg3b^?/? compared to wild-type mice. Moreover, the expression of Interleukin 6, an inflammatory cytokine and Chitinase-like 3, a marker for tissue repair macrophages was elevated in the colon of Reg3b^?/? mice compared to wild-type mice after DSS treatment. Together, these results suggest that attenuation of colitis and ileitis is a result of Reg3β′s real function.     

1H- and 13C-NMR spectroscopic study of glucose metabolism in eggs of Angiostrongylus cantonensis during their development

1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy was used to study aerobic glucose metabolism in eggs of Angiostrongylus cantonensis in an NCTC-109 medium supplemented with fetal calf serum. Without any pretreatment of the spent medium, we were able to identify and quantitate, by NMR, the end-products of glucose metabolism in eggs after cultivation for 2, 4, and 8 days. We demonstrated that A. cantonensis eggs took up glucose rapidly; among the major end products were found lactic acid, acetic acid and alanine. The eggs are parasitic in a sense that the energy metabolism in them is dependent mainly upon the energy source present in outer medium.