Constitutive Turnover of Phosphorylation at Thr-412 of Human p57/Coronin-1 Regulates the Interaction with Actin

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in hematopoietic cells and plays crucial roles in the immune response through reorganization of the actin cytoskeleton. We previously reported that p57/coronin-1 is phosphorylated by protein kinase C, and the phosphorylation down-regulates the association of this protein with actin. In this study we analyzed the phosphorylation sites of p57/coronin-1 derived from HL60 human leukemic cells by MALDI-TOF-MS, two-dimensional gel electrophoresis, and Phos-tag® acrylamide gel electrophoresis in combination with site-directed mutagenesis and identified Ser-2 and Thr-412 as major phosphorylation sites. A major part of p57/coronin-1 was found as an unphosphorylated form in HL60 cells, but phosphorylation at Thr-412 of p57/coronin-1 was detected after the cells were treated with calyculin A, a Ser/Thr phosphatase inhibitor, suggesting that p57/coronin-1 undergoes constitutive turnover of phosphorylation/dephosphorylation at Thr-412. A diphosphorylated form of p57/coronin-1 was detected after the cells were treated with phorbol 12-myristate 13-acetate plus calyculin A. We then assessed the effects of phosphorylation at Thr-412 on the association of p57/coronin-1 with actin. A co-immunoprecipitation experiment with anti-p57/coronin-1 antibodies and HL60 cell lysates revealed that β-actin was co-precipitated with the unphosphorylated form but not with the phosphorylated form at Thr-412 of p57/coronin-1. Furthermore, the phosphorylation mimic (T412D) of p57/coronin-1 expressed in HEK293T cells exhibited lower affinity for actin than the wild-type or the unphosphorylation mimic (T412A) did. These results indicate that the constitutive turnover of phosphorylation at Thr-412 of p57/coronin-1 regulates its interaction with actin.     

An inducible lactone hydrolase yielding 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid from pulvinic acid

The metabolism of vulpinic acid by an unclassified soil micro-organism was studied. A new compound, 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid (DHOHA) was isolated from the reaction mixture of a cell-free preparation and pulvinic acid. The existence of a hydrolase which catalyses the conversion of vulpinic acid to pulvinic acid was detected in cell-free preparation, and an inducible lactone hydrolase capable of converting pulvinic acid to DHOHA was purified 130-fold and characterized. This enzyme had a MW of ca 34 000, a K_m for pulvinic acid at pH optimum (pH 7.0) less than 10 ^{? 6} M, pI = 5.0, and was inhibited by p-chloromercuriphenylsulfonate and diethylpyrocarbonate. The enzyme was highly specific for pulvinic acid. The initial degradative steps proposed for this organism are vulpinic acid → pulvinic acid → DHOHA.     

Effects of vanillin and o-vanillin on induction of DNA-repair networks: modulation of mutagenesis in Escherichia coli

Vanillin and its isomer o-vanillin have an effect on the adaptive and SOS responses, as well as mutagenesis, induced in Escherichia coli by N-methyl-N-nitrosourea (MNU) and UV irradiation, potentiating in some cases and suppressing in others. o-Vanillin markedly inhibited the MNU-induced adaptive response, while both vanillins potentiated the UV-induced SOS response. These phenomena appear to be responsible for the comutagenic or antimutagenic role of these chemicals in MNU and UV mutagenesis.     

Chromosome numbers of some North American Salix

Chromosome numbers are reported for 19 species of North AmericanSalix, one natural hybrid, and one introduced species. The following 17 species are here examined cytologically for the first time:Salix amygdaloides Anderss.,S. arbusculoides Anderss.,S. brachycarpa Nutt.,S. Candida Willd.,S. discolor Muhl.,S. exigua Nutt.,S. kumilis Marsh.,S. interior Rowlee,S. lutea Nutt.,S. maccalliana Rowlee,S. monticola Rydb.,S. myrtillifolia Anderss.,S. pellita Anderss.,S. petiolaris J. E. Sm,S. scouleriana Barr.,S. silicicola Raup, andS. subcoerulea Piper. Of the 19 native North American species 11 are diploid, four are tetraploid, one is triploid, one hexaploid, one dodecaploid, and one exhibits more than one ploidal level.     

A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

Mitochondrial oxidative stress is considered as a key accelerator of fibrosis in various organs including the liver. However, the production of oxidative stress and progression of liver fibrosis may merely represent the independent consequences of hepatocellular injury caused by the primary disease. Because of a lack of appropriate experimental models to evaluate the sole effects of oxidative stress, it is virtually unknown whether this stress is causatively linked to the progression of liver fibrosis. Here, we examined the direct effects of mitochondrial reactive oxygen species (ROS) on the progression of high fat/calorie diet-induced steatohepatitis using Tet-mev-1 mice, in which a mutated succinate dehydrogenase transgene impairs the mitochondrial electron transport and generates an excess amount of ROS in response to doxycycline administration. Wild type and Tet-mev-1 mice that had been continuously given doxycycline-containing water were subsequently fed either normal chow or a cholesterol-free high-fat/high-sucrose diet for 4 months at approximately 1 or 2 years of age. Histopathological examinations indicated that neither the mitochondrial ROS induced in Tet-mev-1 mice nor the feeding of wild type animals with high-fat/high-sucrose diet alone caused significant liver fibrosis. Only when the Tet-mev-1 mice were fed a high-fat/high-sucrose diet, it induced lipid peroxidation in hepatocytes and enhanced hepatic CC chemokine expression. These events were accompanied by increased infiltration of CCR5-positive cells and activation of myofibroblasts, resulting in extensive liver fibrosis. Interestingly, this combinatorial effect of mitochondrial ROS and excess fat/calorie intake on liver fibrosis was observed only in 2-year-old Tet-mev-1 mice, not in the 1-year-old animals. Collectively, these results indicate that mitochondrial ROS in combination with excess fat/calorie intake accelerates liver fibrosis by enhancing CC chemokine production in aged animals. We have provided a good experimental model to explore how high fat/calorie intake increases the susceptibility to nonalcoholic steatohepatitis in aged individuals who have impaired mitochondrial adaptation.     

Follistatin-derived peptide expression in muscle decreases adipose tissue mass and prevents hepatic steatosis

Myostatin, a member of the transforming growth factor (TGF)-β superfamily, plays a potent inhibitory role in regulating skeletal muscle mass. Inhibition of myostatin by gene disruption, transgenic (Tg) expression of myostatin propeptide, or injection of propeptide or myostatin antibodies causes a widespread increase in skeletal muscle mass. Several peptides, in addition to myostatin propeptide and myostatin antibodies, can bind directly to and neutralize the activity of myostatin. These include follistatin and follistatin-related gene. Overexpression of follistatin or follistatin-related gene in mice increased the muscle mass as in myostatin knockout mice. Follistatin binds to myostatin but also binds to and inhibits other members of the TGF-β superfamily, notably activins. Therefore, follistatin regulates both myostatin and activins in vivo. We previously reported the development and characterization of several follistatin-derived peptides, including FS I-I (Nakatani M, Takehara Y, Sugino H, Matsumoto M, Hashimoto O, Hasegawa Y, Murakami T, Uezumi A, Takeda S, Noji S, Sunada Y, Tsuchida K. FASEB J 22: 477-487, 2008). FS I-I retained myostatin-inhibitory activity without affecting the bioactivity of activins. Here, we found that inhibition of myostatin increases skeletal muscle mass and decreases fat accumulation in FS I-I Tg mice. FS I-I Tg mice also showed decreased fat accumulation even on a control diet. Interestingly, the adipocytes in FS I-I Tg mice were much smaller than those of wild-type mice. Furthermore, FS I-I Tg mice were resistant to high-fat diet-induced obesity and hepatic steatosis and had lower hepatic fatty acid levels and altered fatty acid composition compared with control mice. FS I-I Tg mice have improved glucose tolerance when placed on a high-fat diet. These data indicate that inhibiting myostatin with a follistatin-derived peptide provides a novel therapeutic option to decrease adipocyte size, prevent obesity and hepatic steatosis, and improve glucose tolerance.     

The role of sphingosine 1-phosphate in migration of osteoclast precursors; an application of intravital two-photon microscopy

Sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. Through intravital multiphoton imaging of bone tissues, we reveal that the bidirectional function of S1P temporospatially regulates the migration of osteoclast precursors within intact bone tissues. Imaging technologies have enabled in situ visualization of the behaviors of several players in intact tissues. In addition, intravital microscopy has the potential to be more widely applied to functional analysis and intervention.     

Tyrosine phosphorylation-dependent activation of TRPC6 regulated by PLC-γ1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis

Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6-PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type-specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity.     

Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques―photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis—are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.