Expression of mouse Coiled-coil-DIX1 (Ccd1), a positive regulator of Wnt signaling, during embryonic development

Wnt signaling plays an important role in cell growth, differentiation, polarity formation, and neural development. We have recently identified the Coiled-coil-DIX1 (Ccd1) gene encoding a third type of a DIX domain-containing protein. Ccd1 forms homomeric and heteromeric complexes with Dishevelled and Axin, and positively regulates the Wnt/beta-catenin pathway. Here, we examined the spatiotemporal expression pattern of Ccd1 mRNA in mouse embryos from embryonic day 6.5 (E6.5) to E17.5 by in situ hybridization. Ccd1 expression was detected in the node region in gastrula embryos, in the cephalic mesenchyme and tail bud at E8.5, and in the branchial arch and forelimb bud at E9.5. In the central nervous system, Ccd1 expression began and persisted in the regions where the neurons differentiated, so that it was observed throughout the brain and spinal cord at E17.5. Ccd1 expression was also strong in the peripheral nervous system, including sensory cranial ganglia (trigeminal, facial, and vestibulocochlear ganglia), dorsal root ganglia, and autonomic ganglia (sympathetic ganglia, celiac ganglion, and hypogastric plexus). Ccd1 was detected in the sensory organs, such as the inner nuclear layer of the neural retina, saccule and cochlea of the inner ear, and nasal epithelium. Outside the nervous system, Ccd1 mRNA was observed in the cartilage, tongue, lung bud, stomach, and gonad at E12.5-E14.5, and in the tooth bud, bronchial epithelium, and kidney at E17.5. Taken together, these findings demonstrate that Ccd1 expression is observed in all the neurons in the nervous system, closely associated with neural crest-derived tissues, and largely overlapping with the regions where several Wnt genes are reported to play a role.     

ER-resident Gi2 protein controls sar1 translocation onto the ER during budding of transport vesicles

In our previous study, fluoride ([AlF(4) ](-) ) disturbed ER-to-Golgi transport through the activation of ER-resident heterotrimeric G protein (ER-G protein). Therefore, ER-G protein may be implicated in ER-to-Golgi transport at the early stage prior to coat protein assembly. Sar1 translocation onto the endoplasmic reticulum (ER) membrane is suppressed by non-selective protein kinase inhibitor H89, suggesting the participation of H89-sensitive kinase in this process. To investigate the involvement of ER-G protein in ER-to-Golgi transport, the effect of G(i) protein activator (mastoparan 7) was examined on Sar1 translocation onto the ER in a cell-free system consisting of microsome membrane and cytosol. Sar1 translocation onto the microsome membrane was induced by addition of GTPγS in the cell-free system. Translocation of Sar1 by GTPγS was suppressed significantly by both H89 and mastoparan 7. Mastoparan 7 suppressed the translocation of Sar1 onto the microsome membrane with dosage dependency, but mastoparan 17, the inactive analog of mastoparan 7, had no effect on Sar1 translocation. The suppressive effect of mastoparan 7 was recovered by treatment with pertussis toxin (IAP). Moreover, G(i2) protein was detected on the microsome membrane by western blotting for heterotrimeric G(i) proteins. These results indicate that ER-G(i2) protein modulated Sar1 translocation onto the ER, suggesting that ER-resident G(i2) protein is an important negative regulator of vesicular transport at the early stage of vesicle formation before coat protein assembly on the ER.     

Synthesis of 2′,3′-Dideoxypurinenucleosides via the Palladium Catalyzed Reduction of 9-(2,5-Di-O-acetyl-3-bromo-3-deoxy-β-d-xylofuranosyl)purine Derivatives

Abstract

Practical method to produce 2′,3′-dideoxypurinenucleosides from 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)purines (1) was developed. High ratio of 2′,3′-dideoxynucleoside to 3′-deoxyribonucleoside was obtained by selecting the reaction conditions (solvent, pH and/or base), or changing 2′-acyloxy leaving group. The reaction mechanism was studied by deuteration experiments of 1a and 1-(3,5-di-O-acety1-2-bromo-2-deoxy-β-D-ribofuranosyl)thymine (12).

     

Involvement of proton-sensing receptor TDAG8 in the anti-inflammatory actions of dexamethasone in peritoneal macrophages

Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophages with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-α, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-α production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable.     

Expression of Arabidopsis plastidial phosphoglucomutase in tobacco stimulates photosynthetic carbon flow into starch synthesis

Phosphoglucomutase (PGM, EC 2.7.5.1) is one of the enzymes constituting the carbohydrate synthesis pathway in higher plants. It catalyzes the reversible conversion of glucose 6-phosphate (Glc6P) to glucose 1-phosphate (Glc1P). Previously, metabolic turnover analysis using (13)CO(2) in tobacco leaves demonstrated that conversion of Glc6P to Glc1P may limit carbon flow into carbohydrate synthesis. In order to assess the effects of PGM, Arabidopsis thaliana cytosolic or plastidial PGM was expressed under the control of cauliflower mosaic virus 35S promoter in tobacco plants (Nicotiana tabacum cv. Xanthi) and phenotypic analysis was performed. The transgenic plants expressing Arabidopsis plastidial PGM showed 3.5-8.2-fold higher PGM activity than that of wild-type, and leaf starch and sucrose contents increased 2.3-3.2-fold and 1.3-1.4-fold, respectively over wild-type levels. In vivo(13)C-labeling experiments indicated that photosynthetically fixed carbon in the transgenic plants could be converted faster to Glc1P and adenosine 5'-diphosphate glucose than in wild-type, suggesting that elevation of plastidial PGM activity should accelerate conversion of Glc6P to Glc1P in chloroplasts and increase carbon flow into starch. On the other hand, transgenic plants expressing Arabidopsis cytosolic PGM showed a 2.1-3.4-fold increase in PGM activity over wild-type and a decrease of leaf starch content, but no change in sucrose content. These results suggest that plastidial PGM limits photosynthetic carbon flow into starch.     

Brown Adipose Tissue in Cetacean Blubber

Brown adipose tissue (BAT) plays an important role in thermoregulation in species living in cold environments, given heat can be generated from its chemical energy reserves. Here we investigate the existence of BAT in blubber in four species of delphinoid cetacean, the Pacific white-sided and bottlenose dolphins, Lagenorhynchus obliquidens and Tursiops truncates, and Dall’s and harbour porpoises, Phocoenoides dalli and Phocoena phocoena. Histology revealed adipocytes with small unilocular fat droplets and a large eosinophilic cytoplasm intermingled with connective tissue in the innermost layers of blubber. Chemistry revealed a brown adipocyte-specific mitochondrial protein, uncoupling protein 1 (UCP1), within these same adipocytes, but not those distributed elsewhere throughout the blubber. Western blot analysis of extracts from the inner blubber layer confirmed that the immunohistochemical positive reaction was specific to UCP1 and that this adipose tissue was BAT. To better understand the distribution of BAT throughout the entire cetacean body, cadavers were subjected to computed tomography (CT) scanning. Resulting imagery, coupled with histological corroboration of fine tissue structure, revealed adipocytes intermingled with connective tissue in the lowest layer of blubber were distributed within a thin, highly dense layer that extended the length of the body, with the exception of the rostrum, fin and fluke regions. As such, we describe BAT effectively enveloping the cetacean body. Our results suggest that delphinoid blubber could serve a role additional to those frequently attributed to it: simple insulation blanket, energy storage, hydrodynamic streamlining or contributor to positive buoyancy. We believe delphinoid BAT might also function like an electric blanket, enabling animals to frequent waters cooler than blubber as an insulator alone might otherwise allow an animal to withstand, or allow animals to maintain body temperature in cool waters during sustained periods of physical inactivity.     

Characterization of Oseltamivir-Resistant 2009 H1N1 Pandemic Influenza A Viruses

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.     

Chloroplast division in cultured cells of the hornwortAnthoceros punctatus

Using cultured cells of the hornwortAnthoceros punctatus, the change in the relative chloroplast DNA content in each stage of chloroplast division was investigated to clarify the relationship between the division cycle of a chloroplast and a cell nucleus. Samples of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then observed with an epifluorescence microscope and a chromosome image analyzing system (CHIAS). A chloropiast in cultured cells duplicated DNA with an increase in size. When a chloroplast began to divide, it was constricted in the middle, taking a dumbbell shape, and then divided into two daughter chloroplasts. In cultured cells of this species, the pattern of quantitative change of chloroplast DNA, that is, the DNA replication pattern of chloroplasts, corresponded to that of cell nuclear DNA in mitosis.     

The initiation of fetal hemoglobin biosynthesis.

Biosynthesis of the alpha and beta chains of rabbit and human adult hemoglobin is initiated with a methionyl residue, which is removed during elongation of the peptide chain. To study the initiation of biosynthesis of the delta chain of human fetal hemoglobin, fresh placental blood was used for labeling experiments with radioactive amino acids. Labeled nascent peptide chains were purified from the polysomal fraction of placental blood reticulocytes. The number of amino acid residues in nascent gamma chain at the time of removal of its N-terminal methionine was estimated to be 40--60 from the relative yields of labeled tryptic peptides.