Construction of Libraries for Methylation Sites by In-gel Competitive Reassociation (IGCR)

The in-gel competitive reassociation (IGCR) procedure was successfullyapplied to construct a comprehensive library enriched in DNAfragments containing C^5mCGG sequences from mouse liver and braingenomic DNA. For IGCR, methylation-insensitive restriction enzyme(Msp I) digests were used as target DNA and methylation-sensitiverestriction enzyme (Hpa II) digests as competitor DNA. Southernblot analysis indicated that 60 to 70% of the clones in thelibrary were derived from the methylated sites and overall enrichmentwas 200- to 1000-fold. IGCR was further applied to constructa library for the sites differentially methylated between brainand liver DNA. In the library, approximately 20% of the HpaII sites exhibited different degrees of methylation betweenthese tissues.     

Molecular phylogeny in the Lardizabalaceae

Eleven species belonging to seven genera in the Lardizabalaceae were analyzed in terms of restriction fragment length polymorphism (RFLP) of chloroplast DNA and the sequence of the chloroplast gene,rbcL, of Lardizabalaceae and its related families. Phylogenetic trees inferred from parsimony, neighbor joining and maximum likelihood methods based on RFLP data showed that two South American genera,Boquila andLardizabala, and three East Asian genera,Akebia, Holboellia andStauntonia are closely related to each other, respectively. On the other hand, the parsimony, neighbor joining and maximum likelihood trees constructed using sequence data of therbcL gene showed thatAkebia, Stauntonia, Boquila andLardizabala clustered as(((Akebia, Stauntonia), Boquila), Lardizabala). This difference may be attributable to fewer informative sites inrbcL genes than in RFLP in this family.Decaisnea diverges at the very base of the Lardizabalaceae.     

Purification and characterization of phosphoenolpyruvate phosphomutase from Pseudomonas gladioli B-1.

Phosphoenolpyruvate phosphomutase (PEPPM) catalyzes C-P bond formation by intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvate (PnPy). We purified PEPPM from a gram-negative bacterium, Pseudomonas gladioli B-1 isolated as a C-P compound producer. The equilibrium of this reaction favors the formation of the phosphate ester by cleaving the C-P bond of PnPy, but the C-P bond-forming reaction is physiologically significant. The C-P bond-forming activity of PEPPM was confirmed with a purified protein. The molecular mass of the native enzyme was estimated to be 263 and 220 kDa by gel filtration and polyacrylamide gel electrophoresis, respectively. A subunit molecular mass of 61 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the native protein was a tetramer. The optimum pH and temperature were 7.5 to 8.0 and 40 degrees C, respectively. The Km value for PnPy was 19 +/- 3.5 microM, and the maximum initial velocity of the conversion of PnPy to phosphoenolpyruvate was 200 microM/s/mg. PEPPM was activated by the presence of the divalent metal ion, and the Km values were 3.5 +/- 1.4 microM for Mg2+, 16 +/- 5 nM for Mn2+, 3.0 +/- 1.5 microM for Zn2+, and 1.2 +/- 0.2 microM for Co2+.     

Characteristics of stable isotope signature of diet in tissues of captive Japanese macaques as revealed by controlled feeding

We determined the magnitude of isotopic fractionation of carbon and nitrogen stable isotope ratios (as enrichment factors, Δδ¹³C and Δδ¹⁵N, respectively) between the tissues and diets of captive Japanese macaques (Macaca fuscata) using a controlled feeding experiment, to provide basic data for reconstructing their feeding habits. The Δδ¹³C and Δδ¹⁵N values, respectively, were 0.9 ± 0.2 ‰ (mean ± standard deviation, SD) and 3.0 ± 0.3 ‰ for whole blood, 1.3 ± 0.2 ‰ and 4.3 ± 0.3 ‰ for plasma, and 0.8 ± 0.2 ‰ and 3.0 ± 0.2 ‰ for red blood cells. However, the Δδ¹³C and Δδ¹⁵N values for hair were 2.8 ± 0.3 ‰ and 3.4 ± 0.2 ‰, respectively. No difference was detected in the δ¹³C and δ¹⁵N values of hair sampled from different parts of the body. We investigated the effects of diet on δ¹³C in growing hair by alternating the diet of the macaques each month between two diets that differed markedly in δ¹³C. Hair regrown after shaving repeatedly recorded the δ¹³C of the diet consumed during the time of hair growth. On the other hand, hair naturally grown during the diet-change experiment did not show a clear pattern. One possible reason is that the hair had grown abnormally under unnatural indoor conditions and showed complicated isotope signatures. To reconstruct the long-term feeding history of Japanese macaques, we need to further clarify the relationships between the stable isotope signature of diet and various body tissues.     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.     

N-cyanomethyl-2-chloroisonicotinamide induces systemic acquired resistance in arabidopsis without salicylic acid accumulation

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid-mediated pathway. N-cyanomethyl-2-chloroisonicotinamide (NCI) is able to induce a broad range of disease resistance in tobacco and rice and induces SAR marker gene expression without SA accumulation in tobacco. To clarify the detailed mode of action of NCI, we analyzed its ability to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with NCI exhibited increased expression of several pathogenesis-related genes and enhanced resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. NCI induced disease resistance and PR gene expression in NahG transgenic plants, but not in the npr1 mutant. NCI could induce PR gene expression in the etr1-1, ein2-1 and jar1-1 mutants. Thus, NCI activates SAR, independently from ethylene and jasmonic acid, by stimulating the site between SA and NPR1.     

Minimal motif peptide structure of metzincin clan zinc peptidases in micelles

It is well known that the functions of metalloproteins generally originate from their metal‐binding motifs. However, the intrinsic nature of individual motifs remains unknown, particularly the details about metal‐binding effects on the folding of motifs; the converse is also unknown, although there is no doubt that the motif is the core of the reactivity for each metalloprotein. In this study, we focused our attention on the zinc‐binding motif of the metzincin clan family, HEXXHXXGXXH; this family contains the general zinc‐binding sequence His–Glu–Xaa–Xaa–His (HEXXH) and the extended GXXH region. We adopted the motif sequence of stromelysin‐1 and investigated the folding properties of the Trp‐labeled peptides WAHEIAHSLGLFHA (STR‐W1), AWHEIAHSLGLFHA (STR‐W2), AHEIAHSLGWFHA (STR‐W11), and AHEIAHSLGLFHWA (STR‐W14) in the presence and absence of zinc ions in hydrophobic micellar environments by circular dichroism (CD) measurements. We accessed successful incorporation of these zinc peptides into micelles using quenching of Trp fluorescence. Results of CD studies indicated that two of the Trp‐incorporated peptides, STR‐W1 and STR‐W14, exhibited helical folding in the hydrophobic region of cetyltrimethylammonium chloride micelle. The NMR structural analysis of the apo STR‐W14 revealed that the conformation in the C‐terminus GXXH region significantly differred between the apo state in the micelle and the reported Zn‐bound state of stromelysin‐1 in crystal structures. The structural analyses of the qualitative Zn‐binding properties of this motif peptide provide an interesting Zn‐binding mechanism: the minimum consensus motif in the metzincin clan, a basic zinc‐binding motif with an extended GXXH region, has the potential to serve as a preorganized Zn binding scaffold in a hydrophobic environment. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Thalidomide and its analogues as cyclooxygenase inhibitors

Thalidomide showed cyclooxygenase (COX)-1/2 inhibitory activity with a potency comparable to that of aspirin. Structural development studies of thalidomide resulted in potent COX-1/2 inhibitors, and COX-1-selective and COX-2-selective inhibitors.     

Design and synthesis of dysidiolide analogs from vitamin D3: novel class of Cdc25A inhibitors

Potent dysidiolide analogs were synthesized by structural hybridization of dysidiolide and vitamin D(3). These analogs exhibited strong inhibitory activity toward dual-specificity phosphatase Cdc25A (IC(50)=0.44-0.89 microM).     

Atrial chamber-specific expression of sarcolipin is regulated during development and hypertrophic remodeling

Intracellular Ca2+ regulation is critical in the normal cardiac function and development of pathologic hearts. Phospholamban, an endogenous inhibitor of sarcoplasmic reticulum Ca2+ ATPase in the sarcoplasmic reticulum, plays an important role in Ca2+ cycling in heart. Recently, sarcolipin has been identified as having a similar function as phospholamban in skeletal muscle. Because phospholamban is differentially expressed in atrial and ventricular myocardia and its expression is often altered in diseased hearts, we investigated the cardiac chamber specificity of sarcolipin expression and its regulation during development and hypertrophic remodeling. Northern blot analysis revealed that the expression of mouse sarcolipin mRNA was most abundant in the atria and was undetectable in the ventricles, indicating an atrial chamber-specific expression pattern. Atrial chamber-specific expression of sarcolipin mRNA was increased during development. These findings were confirmed by in situ hybridization studies. In addition, sarcolipin expression was down-regulated in the atria of hypertrophic heart when induced by ventricular specific overexpression of the activated H-ras gene. In humans, sarcolipin mRNA was also expressed in the atria but not detected in the ventricles, although sarcolipin expression was most abundant in skeletal muscle. Taken together, sarcolipin is likely to be an atrial chamber-specific regulator of Ca2+ cycling in heart.