The hedgehog-PKA pathway regulates two distinct steps of the differentiation of retinal ganglion cells: the cell-cycle exit of retinoblasts and their neuronal maturation

In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. Here, we show that cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye.     

The Wnt antagonist Frzb-1 regulates chondrocyte maturation and long bone development during limb skeletogenesis

The Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, we determined Frzb-1 gene expression patterns during chick long bone development and carried out gain- and loss-of-function studies by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression was quite strong in mesenchymal prechondrogenic condensations and then characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression caused shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintained the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promoted chondrocyte maturation, hypertrophy, and calcification. Immunostaining revealed that the distribution of endogenous Wnt mediator beta-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevented beta-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action.     

Hansenula polymorpha Pex14p phosphorylated in vivo

Hansenula polymorpha Pex14p is a novel peroxisomal membrane protein essential for peroxisome biogenesis. In vivo labeling experiment of wild-type cells with ³²P-orthophosphate and alkaline phosphatase treatment of labeled Pex14p indicate that Pex14p is phosphorylated in vivo. Analysis of the phosphoamino acid in the phosphorylated Pex14p suggested that the major phosphoamino acid was acid labile. Using expression system of several truncated Pex14ps in a PEX14-deletion strain it is suggested that the phosphorylation site of Pex14p resides in the C-terminal 58 residues.     

Comparative study of three phospholipase A2s from the venom of Vipera aspis

1. Three phospholipase A2s, PLA2-I, PLA2-II and PLA2-III, were isolated from Vipera aspis venom by gel filtration and ion exchange chromatography. 2. Purified PLA2-I, -II and -III have mol. wts of 30,200, 16,000 and 13,500, and isoelectric points of 9.45, 7.65 and less than 4.1, respectively. 3. PLA2-I consists of an acidic subunit (mol. wt 13,700, pI: less than 3.5) and a basic subunit (mol. wt 16,500, pI: 10.6), which can be separated under highly acidic conditions. 4. PLA2-I possessed lethal activity and LD50 for this preparation was estimated to be 0.288 (0.209-0.397) micrograms/g, while lethality was not observed when PLA2-II, -III or each subunit of PLA2-I were administered. 5. Capillary permeability-increasing activity was found in the samples which possessed basic isoelectric points. Additionally, PLA2-I and its basic subunit drastically prolonged activated partial thromboplastin time of platelet rich plasma. 6. Intramuscular injections of PLA2-I, -II and -III increased serum creatine phosphokinase activity in mice, indicating that damage in muscle was caused by these enzymes. 7. NH2-terminal sequences of the three PLA2s were compared with other phospholipase A2s from snake venoms. Furthermore, antigenicities were tested using antiserum prepared against each sample.     

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

Cytochrome c₅₅₅ from hyperthermophilic bacteria Aquifex aeolicus (AA cyt c₅₅₅) is a hyperstable protein belonging to the cyt c protein family, which possesses a unique long 3₁₀‐α‐3₁₀ helix containing the heme‐ligating Met61. Herein, we show that AA cyt c₅₅₅ forms dimers by swapping the region containing the extra 3₁₀‐α‐3₁₀ helix and C‐terminal α‐helix. The asymmetric unit of the crystal of dimeric AA cyt c₅₅₅ contained two dimer structures, where the structure of the hinge region (Val53–Lys57) was different among all four protomers. Dimeric AA cyt c₅₅₅ dissociated to monomers at 92 ± 1°C according to DSC measurements, showing that the dimer was thermostable. According to CD measurements, the secondary structures of dimeric AA cyt c₅₅₅ were maintained at pH 2.2–11.0. CN^‐ and CO bound to dimeric AA cyt c₅₅₅ in the ferric and ferrous states, respectively, owing to the flexibility of the hinge region close to Met61 in the dimer, whereas these ligands did not bind to the monomer under the same conditions. In addition, CN^‐ and CO bound to the oxidized and reduced dimer at neutral pH and a wide range of pH (pH 2.2–11.0), respectively, in a wide range of temperature (25–85°C), owing to the thermostability and pH tolerance of the dimer. These results show that the ligand binding character of hyperstable AA cyt c₅₅₅ changes upon dimerization by domain swapping.     

H89 sensitive kinase regulates the translocation of Sar1 onto the ER membrane through phosphorylation of ER-coupled β-tubulin

ER-to-Golgi protein transport is carried out by transport vesicles which are formed at the ER-exit sites with recruitment of cytoplasmic coat proteins. Vesicle formation is initiated by assembly of the small G protein (Sar1) onto the ER membrane. Sar1 assembly onto the ER membrane is suppressed by protein kinase inhibitor H89, suggesting participation of H89-sensitive kinase in this process. The present study identified an effector of H89-sensitive kinase by LC-MS PMF analysis combined with 1D- and 2D-PAGE autoradiography, and examined the changes on the effector and Sar1 translocation induced by H89. H89 significantly suppressed the phosphorylation of 55 kDa protein with dosage dependency, and phosphorylation of 55 kDa, pI 5.5 protein spot in 2-D-autoradiography was drastically diminished by H89. LC-MS PMF analysis showed that the protein spot was β-tubulin. H89 significantly suppressed Sar1 translocation onto the ER. These findings indicate that β-tubulin is one of downstream effectors of H89-sensitive kinase, and that suppression of ER-coupled β-tubulin phosphorylation decreases Sar1 translocation onto the ER, suggesting that phosphorylation of β-tubulin regulates Sar1 translocation.     

Lack of Oncostatin M Receptor β Leads to Adipose Tissue Inflammation and Insulin Resistance by Switching Macrophage Phenotype

Oncostatin M (OSM), a member of the IL-6 family of cytokines, plays important roles in a variety of biological functions, including inflammatory responses. However, the roles of OSM in metabolic diseases are unknown. We herein analyzed the metabolic parameters of OSM receptor β subunit-deficient (OSMRβ^−/−) mice under normal diet conditions. At 32 weeks of age, OSMRβ^−/− mice exhibited mature-onset obesity, severer hepatic steatosis, and insulin resistance. Surprisingly, insulin resistance without obesity was observed in OSMRβ^−/− mice at 16 weeks of age, suggesting that insulin resistance precedes obesity in OSMRβ^−/− mice. Both OSM and OSMRβ were expressed strongly in the adipose tissue and little in some other metabolic organs, including the liver and skeletal muscle. In addition, OSMRβ is mainly expressed in the adipose tissue macrophages (ATMs) but not in adipocytes. In OSMRβ^−/− mice, the ATMs were polarized to M1 phenotypes with the augmentation of adipose tissue inflammation. Treatment of OSMRβ^−/− mice with an anti-inflammatory agent, sodium salicylate, improved insulin resistance. In addition, the stimulation of a macrophage cell line, RAW264.7, and peritoneal exudate macrophages with OSM resulted in the increased expression of M2 markers, IL-10, arginase-1, and CD206. Furthermore, treatment of C57BL/6J mice with OSM increased insulin sensitivity and polarized the phenotypes of ATMs to M2. Thus, OSM suppresses the development of insulin resistance at least in part through the polarization of the macrophage phenotypes to M2, and OSMRβ^−/− mice provide a unique mouse model of metabolic diseases.     

Microbial Production of Abscisic Acid by Botrytis cinerea

The effect of oryzalexin D, which has been isolated as a group of novel phytoalexins of rice plant, on DNA, RNA, protein, lipid and chitin biosyntheses, respiration and cell membrane permeability was investigated in Pyricularia oryzae. The concentration for 50% inhibition (ED₅₀) by oryzalexin D of the mycelial growth of P. oryzae was 230 ppm. At this concentration, oryzalexin D inhibited equally the incorporation of [2–¹⁴C]thymidine, [2–¹⁴C]uridine, l-[U-¹⁴C]amino acid mixture, l-[methyl-¹⁴C]methionine and d-[l-¹⁴C]glucosamine into DNA, RNA, protein, lipid and chitin in intact cells, but did not inhibit these systems in a homogenate of the mycelia of P. oryzae. Oryzalexin D scarcely inhibited the respiration of the homogenate and mitochondria at ED₅₀. On the other hand, oryzalexin D at ED₅₀ caused leakage of potassium and inhibited the uptake of glutamate by mycelial cells of P. oryzae. These results suggest that interference with the cell membrane function is responsible for the primary mode of action.of oryzalexin D against P. oryzae.