Novel method of the synthesis and hybridization properties of an oligonucleotide containing non-ionic diisopropylsilyl internucleotide linkage

Efficient synthesis of a dithymidine dinucleotide analog bearing a diisopropylsilyl linkage instead of a phosphodiester linkage is described with respect to its incorporation into oligonucleotides. The diisopropylsilyl linkage was introduced into the oligonucleotide by preparation of the phosphoramidite derivative of a dithymidine dimer unit. The diisopropylsilyl-modified oligonucleotide exhibited hybridization behavior with both single strand and duplex DNA. The thermal stability of both the duplex and triplex showed a relative instability compared to the corresponding natural phosphodiester DNA, because of the steric hindrance of the isopropyl group on the silicon atom.     

Role of the Sec14-like domain of Dbl family exchange factors in the regulation of Rho family GTPases in different subcellular sites

Mechanisms underlying subcellular region-specific regulation of Rho family GTPases through Dbl family guanine nucleotide exchange factors (GEFs) remain totally unknown. Here we show that the Sec14-like domain, which lies in the N-terminus of the Dbl family GEFs Dbl and Ost, directs the subcellular localization of these GEFs and also their substrate Cdc42. When coexpressed with Cdc42 in human adenocarcinoma HeLa cells, Dbl-I and Ost-I, which lack the Sec14-like domain, translocated Cdc42 to the plasma membrane, where Dbl-I or Ost-I was colocalized. In marked contrast, Dbl-II and Ost-II, which contain the Sec14-like domain, were colocalized with Cdc42 in endomembrane compartments. Furthermore, ruffle membrane formation upon epidermal growth factor treatment was mediated by Dbl-I or Ost-I, but neither Dbl-II nor Ost-II, supporting a notion that GEFs with or without the Sec14-like domain are linked to different upstream signals. By employing a novel method to detect the active GTP-bound form of Cdc42 in situ, we demonstrate that Dbl-I and Ost-I, but neither Dbl-II nor Ost-II, indeed activate colocalized Cdc42.     

Nitrate reductase-formate dehydrogenase couple involved in the fungal denitrification by Fusarium oxysporum

Dissimilatory nitrate reductase (Nar) was solubilized and partially purified from the large particle (mitochondrial) fraction of the denitrifying fungus Fusarium oxysporum and characterized. Many lines of evidence showed that the membrane-bound Nar is distinct from the soluble, assimilatory nitrate reductase. Further, the spectral and other properties of the fungal Nar were similar to those of dissimilatory Nars of Escherichia coli and denitrifying bacteria, which are comprised of a molybdoprotein, a cytochrome b, and an iron-sulfur protein. Formate-nitrate oxidoreductase activity was also detected in the mitochondrial fraction, which was shown to arise from the coupling of formate dehydrogenase (Fdh), Nar, and a ubiquinone/ubiquinol pool. This is the first report of the occurrence in a eukaryote of Fdh that is associated with the respiratory chain. The coupling with Fdh showed that the fungal Nar system is more similar to that involved in the nitrate respiration by Escherichia coli than that in the bacterial denitrifying system. Analyses of the mutant species of F. oxysporum that were defective in Nar and/or assimilatory nitrate reductase conclusively showed that Nar is essential for the fungal denitrification.     

The light/dark cycle operation with an hour-scale period enhances caffeine production by Coffea arabica cells

The intermittent light irradiation with an hour-scale period is used for producing caffeine by Coffea arabica cells. Three factors concerning the light/dark cycle operation such as light intensity, the length of the cycle (period), and the ratio of the illumination time to the dark time (light/dark ratio) were investigated to optimize the caffeine production efficiency regarding light consumption. The light/dark ratio of 1/1 enhanced caffeine production, reaching the same level as continuous light; thus, the intermittent light irradiation improved the production efficiency twofold. The production was not influenced by the period, but was determined by light intensity regardless of intermittent or continuous light irradiation.     

Functional expression and mutational analysis of flavonol synthase from Citrus unshiu.

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII/2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m/z 37888 +/- 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N-terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5-6 with dihydroquercetin as substrate and a temperature optimum at about 37 degrees C. Km values of 272, 11 and 36 micro m were determined for dihydroquercetin, FeII and 2-oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (Km 45 micro m). Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30-60% similarity were apparent with other dioxygenases. Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for FeII-coordination, an arginine (Arg287) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality. Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity. Alternatively, the substitution of Pro207 by glycine hardly affected the activity. The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.     

Effects of sodium chloride,guanidine hydrochloride,and sucrose on the viscoelastic properties of sodium hyaluronate solutions

Effects of sodium chloride (NaCl), guanidine hydrochloride (GuHCl), or sucrose on the viscoelasticity of sodium hyaluronate (NaHA) solutions were studied. NaCl and GuHCl decreased both storage and loss moduli, while sucrose increased both moduli. The critical concentration C* was determined as an inflection point in the plot of zero shear specific viscosity vs concentration for NaHA solutions with and without NaCl, GuHCl, or sucrose. It is suggested that sodium ions or guanidinium ions shield the electrostatic repulsion of NaHA molecules, hence reduce the coil dimension, and C* shifted to higher concentrations. However, sucrose enhances the entanglement coupling between NaHA molecules and retards the disentanglement of molecular chains or promotes to create hydrogen bonds, and then C* for NaHA solutions with sucrose shifts to lower concentrations. This is in agreement with the results of light scattering measurements in the presence of 0.2M NaCl. Both the radius of gyration and hydrodynamic radius of NaHA were reduced in dilute solutions by the addition of sucrose, and added sucrose enhances the interaction between NaHA monomer units. In the case of concentrated NaHA solution, such interactions result to increase the storage and loss moduli because of the enhancement of temporary network formation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 23–34, 1999     

Emergence of the circadian sleep-wake rhythm might depend on conception not on birth timing

Sleep and Biological Rhythms - Developmental changes in the sleep-wake rhythm of preterm infants were compared with those of full-term infants, to clarify the timing of the developmental change of...     

Common-intersection hypothesis of development rate lines of ectotherms within a taxon revisited

The relationship between development rate of an ectotherm and temperature in experiments where insects or mites are reared under several constant temperatures, while non-linear over the entire range, can be approximately described by a line in the mid-temperature range. It was hypothesized that the development rate lines of ectotherms within a taxon such as a family would have a common intersection. Thus for a given temperature, the development time should be the same for all the species within any specified taxon. In the current study, the data on temperature-dependent development rates of species of the Aphididae and Tetranychidae families were used to test the validity of the common-intersection hypothesis. The hypothesis was supported with either dataset used. In addition, the current study tested the common-intersection hypothesis through mathematical analysis on the basis of two linear relationships (i.e., the linear relationship between temperature and development rate and that between the lower developmental threshold and the sum of effective temperatures). An attempt was made to combine the rate isomorphy hypothesis that all the lower developmental thresholds of different stages were assumed to be equal for a single species with the common-intersection hypothesis for related species within a taxon and hypothesize that if these two hypotheses hold, then for any developmental stages a common temperature of all stages should exist.     

Synthesis and Duplex-Forming Property of Oligonucleotides Bearing a Novel Polyamine-Modified Intercalator at the Terminal or the Internal Position

Novel oligonucleotides bearing a polyamine-intercalator conjugate modified at the terminal or the internal position were reported. These modified oligonucleotides showed duplex-stabilization effect, and the thermodynamic analysis and the salt concentration dependency of the duplex stability revealed that the polyamine moiety also acted as the duplex stabilizer by neutralization of the phosphate negative charge.