Cloning of cDNAs encoding cell-wall hydrolases from pear (Pyrus communis) fruit and their involvement in fruit softening and development of melting texture

'La France' pear ( Pyrus communis L.) fruit stored at 1°C for 1 month (short-term storage) before transfer to 20°C softened and developed a melting texture during ripening, whereas fruit stored for 5 months (long-term storage) before transfer to 20°C softened but did not develop a melting texture. To clarify the mechanisms involved in fruit softening and textural changes, the cDNAs encoding cell-wall hydrolases were isolated by RT-PCR, and their expression and localization were investigated in 'La France' pears. Genes encoding three polygalacturonases (PG; EC 3.2.1.15), four pectin methylesterases (PME; EC 3.1.1.11), one α -arabinofuranosidase (ARF; EC 3.2.1.55), three β -galactosidases (GAL; EC 3.2.1.23), and two endo-1,4- β - d -glucanases (Cel; EC 3.2.1.4) were isolated. Among these 13 isolated genes, PcPG1 was the only gene for which the mRNA expression levels increased in both the short- and long-term stored fruits. This suggested that PcPG1 is involved in fruit softening rather than in the development of the melting texture. In contrast, the expression levels of PcPG3 , PcPME1 , PcPME2 , PcPME3 , PcGAL1 , PcGAL2 , and PcCel2 increased during ripening only in the short-term stored fruit. These genes might thus be involved in the development of the melting texture.     

Light activates H2 15O flow in rice: Detailed monitoring using a positron-emitting tracer imaging system (PETIS)

Water (H₂ ¹⁵O) translocation from the roots to the top of rice plants ( Oryza saliva L. cv. Nipponbare) was visualized over time by a positron-emitting tracer imaging system (PETIS). H₂ ¹⁵O flow was activated 8 min after plants were exposed to bright light (1 500 μmol m⁻² s⁻¹). When the light was subsequently removed, the flow gradually slowed and completely stopped after 12 min. In plants exposed to low light (500 μmol m⁻² s⁻¹), H₂ ¹⁵O flow was activated more slowly, and a higher translocation rate of H₂ ¹⁵O was observed in the same low light at the end of the next dark period. NaCl (80 m M ) and methylmercury (1 m M ) directly suppressed absorption of H₂ ¹⁵O by the roots, while methionine sulfoximine (1 m M ), abscisic acid (10 μ M ) and carbonyl cyanide m -chlorophenylhydrazone (10 m M ) were transported to the leaves and enhanced stomatal closure, reducing H₂ ¹⁵O translocation.     

RBMX is a novel hepatic transcriptional regulator of SREBP-1c gene response to high-fructose diet

In rodents a high-fructose diet induces metabolic derangements similar to those in metabolic syndrome. Previously we suggested that in mouse liver an unidentified nuclear protein binding to the sterol regulatory element (SRE)-binding protein-1c (SREBP-1c) promoter region plays a key role for the response to high-fructose diet. Here, using MALDI-TOF MASS technique, we identified an X-chromosome-linked RNA binding motif protein (RBMX) as a new candidate molecule. In electrophoretic mobility shift assay, anti-RBMX antibody displaced the bands induced by fructose-feeding. Overexpression or suppression of RBMX on rat hepatoma cells regulated the SREBP-1c promoter activity. RBMX may control SREBP-1c expression in mouse liver in response to high-fructose diet.     

Pre-activation strategy for oligodeoxyribonucleotide synthesis using triaryloxydichlorophosphoranes in the phosphotriester method.

Triaryloxydichlorophosphoranes were tested as condensing agents for oligodeoxyribonucleotide synthesis in the phosphotriester method. Tris(2,4,6-tribromophenoxy)dichlorophosphorane (BDCP) was found to be a relatively stable crystalline material which could be used as a chemical reagent. A notable feature of the BDCP-promoted condensation reaction was studied by 31P-NMR. A small amount of BDCP compared to the conventional condensing agent was effective for the generation of active nucleotide intermediates and BDCP itself was quantitatively converted into an inert material, tris(2,4,6-tribromophenyl)phosphate (2). Thus, BDCP enabled us to separate the activation step from the condensation process in the phosphotriester method. This preactivation method was applied to the solid-phase synthesis.     

Functional differentiation in the leucine-rich repeat domains of closely related plant virus-resistance proteins that recognize common avr proteins

The N' gene of Nicotiana sylvestris and L genes of Capsicum plants confer the resistance response accompanying the hypersensitive response (HR) elicited by tobamovirus coat proteins (CP) but with different viral specificities. Here, we report the identification of the N' gene. We amplified and cloned an N' candidate using polymerase chain reaction primers designed from L gene sequences. The N' candidate gene was a single 4143 base pairs fragment encoding a coiled-coil nucleotide-binding leucine-rich repeat (LRR)-type resistance protein of 1,380 amino acids. The candidate gene induced the HR in response to the coexpression of tobamovirus CP with the identical specificity as reported for N'. Analysis of N'-containing and tobamovirus-susceptible N. tabacum accessions supported the hypothesis that the candidate is the N' gene itself. Chimera analysis between N' and L(3) revealed that their LRR domains determine the spectrum of their tobamovirus CP recognition. Deletion and mutation analyses of N' and L(3) revealed that the conserved sequences in their C-terminal regions have important roles but contribute differentially to the recognition of common avirulence proteins. The results collectively suggest that Nicotiana N' and Capsicum L genes, which most likely evolved from a common ancestor, differentiated in their recognition specificity through changes in the structural requirements for LRR function.     

Blocking O-linked GlcNAc cycling in Drosophila insulin-producing cells perturbs glucose-insulin homeostasis

A dynamic cycle of O-linked GlcNAc (O-GlcNAc) addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively, in a process that serves as the final step in a nutrient-driven "hexosamine-signaling pathway." Evidence points to a role for O-GlcNAc cycling in diabetes and insulin resistance. We have used Drosophila melanogaster to determine whether O-GlcNAc metabolism plays a role in modulating Drosophila insulin-like peptide (dilp) production and insulin signaling. We employed transgenesis to either overexpress or knock down Drosophila Ogt(sxc) and Oga in insulin-producing cells (IPCs) or fat bodies using the GAL4-UAS system. Knockdown of Ogt decreased Dilp2, Dilp3, and Dilp5 production, with reduced body size and decreased phosphorylation of Akt in vivo. In contrast, knockdown of Oga increased Dilp2, Dilp3, and Dilp5 production, increased body size, and enhanced phosphorylation of Akt in vivo. However, knockdown of either Ogt(sxc) or Oga in the IPCs increased the hemolymph carbohydrate concentration. Furthermore, phosphorylation of Akt stimulated by extraneous insulin in an ex vivo cultured fat body of third instar larvae was diminished in strains subjected to IPC knockdown of Ogt or Oga. Knockdown of O-GlcNAc cycling enzymes in the fat body dramatically reduced neutral lipid stores. These results demonstrate that altered O-GlcNAc cycling in Drosophila IPCs modulates insulin production and influences the insulin responsiveness of peripheral tissues. The observed phenotypes in O-GlcNAc cycling mimic pancreatic β-cell dysfunction and glucose toxicity related to sustained hyperglycemia in mammals.     

Hybridization-dependent fluorescence of oligodeoxynucleotides incorporating new pyrene-modified adenosine residues

We report the synthesis and properties of oligonucleotides incorporating N(6)-[N-(pyren-1-ylmethyl)carbamoyl]-deoxyadenosine (dA(pymcm)). We designed the ODN which incorporated two consecutive dA(pymcm) residues. It was revealed that on hybridization with the target DNA and RNA oligomers, the fluorescence spectra of ODNs having two consecutive dA(pymcm) molecules near the 5'-terminal position can change from the pyrene monomer emission to the excimer, depending on the chain length of the target DNA and RNA. These results indicated that dA(pymcm)-modified ODNs can be used as interesting hybridization sensors that are sensitive to the size of the target strand.     

Myocardial tissue engineering: toward a bioartificial pump

Regenerative therapies, including cell injection and bioengineered tissue transplantation, have the potential to treat severe heart failure. Direct implantation of isolated skeletal myoblasts and bone-marrow-derived cells has already been clinically performed and research on fabricating three-dimensional (3-D) cardiac grafts using tissue engineering technologies has also now been initiated. In contrast to conventional scaffold-based methods, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cell sheets to construct 3-D cell-dense tissues. Upon layering, individual cardiac cell sheets integrate to form a single, continuous, cell-dense tissue that resembles native cardiac tissue. The transplantation of layered cardiac cell sheets is able to repair damaged hearts. As the next step, we have attempted to promote neovascularization within bioengineered myocardial tissues to overcome the longstanding limitations of engineered tissue thickness. Finally, as a possible advanced therapy, we are now trying to fabricate functional myocardial tubes that may have a potential for circulatory support. Cell sheet-based tissue engineering technologies therefore show an enormous promise as a novel approach in the field of myocardial tissue engineering.