Synthesis of modified siRNA bearing C-5 polyamine-substituted pyrimidine nucleoside in their 3'-overhang regions and its RNAi activity

Short interfering RNA (siRNA) induces specific gene silencing by the RNA interference (RNAi) pathway. Nucleosides in the 3′-overhang regions of siRNAs were replaced with 5-bis(aminoethyl)aminoethylcarbamoylmethyl-2′-deoxyuridine or thymidine. siRNA bearing modified nucleoside was more active in silencing the gene expression of hepatocyte nuclear factor 4α (HNF4α) compared with siRNA bearing thymidine.     

Side-necked turtle (Pleurodira, Chelonia, reptilia) hemoglobin: cDNA-derived primary structures and X-ray crystal structures of Hb A

Red blood cells of yellow-spotted river turtles (Podocnemis unifilis, Pleurodira, Chelonia, REPTILIA) have two hemoglobin (Hb) components, Hb A and Hb D. We purified the hemoglobin component homologous to amniote (reptiles, birds, and mammals) adult Hb A which comprises two identical α(A) -globin polypeptides and two identical β-globin polypeptides. To establish the crystal structure of Podocnemis Hb A, we first determined the globin primary structures using cDNA nucleotide sequencing with the assistance of protein sequencing. The purified Podocnemis Hb A produced a different form of crystal for each of the two different buffer systems used: form A, tetragonal crystals (space group, P4?2?2), produced under neutral pH (pH 7-8) conditions; and form B, hexagonal crystals (space group, P6?22), produced under high alkaline pH (pH 11-13) conditions. Single crystals of the two forms were examined by Raman microscopy with an excitation of 532 nm, indicating their structural differences. The crystal structures of the two forms were constructed by X-ray crystallographic diffraction at a resolution of 2.20 ? for form A and 2.35 ? for form B. The differences of the tertiary and quaternary structures of the two forms were marginal; however, one clear difference was found in helix structure. When comparing Podocnemis Hb A with Hb A from specimens in other taxa, such as Anser indicus (birds) and Homo sapiens (mammals) by SHELXPRO, the root mean square deviation (RMSD) between the corresponding Cα atoms of the two globins does not exceed 2.0 ?. These low values indicate the crystal structures resemble each other. Our data on X-ray crystal structures and Raman spectra not only reveal the first findings on the two crystal forms of Podocnemis unifilis Hb A but also provide the first refined models for reptilian adult Hb A.     

Experimental challenge of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) in rainbow trout and olive flounder

The third-stage larvae of Anisakis simplex sensu lato (s.l.) are found in many marine fishes. To ensure food safety, it is important to determine whether these larvae are present in the body muscle of commercial fish species. However, there is little information regarding the tissue specificity of Anisakis and two of its sibling species, A. simplex sensu stricto (s.s.) and Anisakis pegreffii, that are common in marine fish in Japanese waters. We orally challenged rainbow trout (Oncorhynchus mykiss (Walbaum)), and olive flounder (Paralichthys olivaceus (Temminck and Schlegel)) with L3 larvae of these two sibling species and monitored infection for 5weeks. In rainbow trout, A. simplex s.s., but not A. pegreffii larvae, migrated into the body muscle. A small number of freely moving A. pegreffii larvae were recovered within the body cavity. In olive flounder, A. simplex s.s. larvae were found in both the body cavity and body muscle. A. pegreffii larvae were found only in the body cavity and primarily encapsulated in lumps. Our results indicate that there are differences in the sites of infection and host specificity between the two sibling species of A. simplex s.l.     

Effects of changing illuminance on somatosensory function

Artificial sources of illumination can be easily used, regardless of the time and place, to improve visibility at night and in dark places. Illuminance and color temperature are particularly important factors since they are known to elicit physiological effects. However, the relationship between changes in illuminance and somatosensory function has not been sufficiently clarified. Thus, the purpose of this study was to construct a laboratorial model to examine the effects of lowering or raising illuminance on somatosensory function. Three illuminance levels (200 lx, 50 lx, and 0 lx), which were changed using all combinations, and an artificial sensory stimulus maintained at a constant intensity were presented to the subjects of this study. Objective sensory function in response to the sensory stimulus was investigated by somatosensory evoked potential (SEP), and subjective sensory evaluation in response to the stimulus was investigated using a visual analogue scale (VAS) and by interview. In many cases, the SEP amplitude and VAS value tended to decrease when illuminance was lowered and tended to increase when illuminance was raised. However, in a few cases, SEP amplitude and VAS value tended to increase in spite of the low illuminance. The occurrence of attention responses and unpleasant emotional responses caused by lowering the illuminance seems to be related to this study finding.     

Overwintering leaves of a forest-floor fern, Dryopteris crassirhizoma (Dryopteridaceae): a small contribution to the resource storage and photosynthetic carbon gain

BACKGROUND AND AIMS: Dryopteris crassirhizoma is a semi-evergreen fern growing on the floor of deciduous forests. The present study aimed to clarify the photosynthetic and storage functions of overwintering leaves in this species. METHODS: A 2-year experiment with defoliation and shading of overwintering leaves was conducted. Photosynthetic light response was measured in early spring (for overwintering leaves) and summer (for current-year leaves). KEY RESULTS: No nitrogen limitation of growth was detected in plants subjected to defoliation. The number of leaves, their size, reproductive activity (production of sori) and total leaf mass were not affected by the treatment. The defoliation of overwintering leaves significantly reduced the bulk density of rhizomes and the root weight. The carbohydrates consumed by the rhizomes were assumed to be translocated for leaf production. Photosynthetic products of overwintering leaves were estimated to be small. CONCLUSION: Overwintering leaves served very little as nutrient-storage and photosynthetic organs. They partly functioned as a carbon-storage organ but by contrast to previous studies, their physiological contribution to growth was found to be modest, probably because this species has a large rhizome system. The small contribution of overwintering leaves during the short-term period of this study may be explained by the significant storage ability of rhizomes in this long-living species. Other ecological functions of overwintering leaves, such as suppression of neighbouring plants in spring, are suggested.     

Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria

In amino acid production by coryneform bacteria, study on relationship between change in enzyme activities and production of a target amino acid is important. In glutamate production, Kawahara et al. discovered that the effect of decrease in 2-oxoglutamate dehydrogenase complex (ODHC) on glutamate production is essential (Kawahara et al., Biosci. Biotechnol. Biochem. 61(7) (1997) 1109). Significant reduction of the ODHC activity was observed in the cells under the several glutamate-productive conditions in Corynebacterium glutamicum. Recent progress in metabolic engineering enables us to quantitatively compare the flux redistribution of the different strains after change in enzyme activity precisely. In this paper, relationship between flux redistribution and change in enzyme activities after biotin deletion and addition of detergent (Tween 40) was studied in two coryneform bacteria, C. glutamicum and a newly isolated strain, Corynebacterium efficiens (Fudou et al., Int. J. Syst. Evol. Microbiol. 52(Part 4) 1127), based on metabolic flux analysis (MFA). It was observed that in both species the specific activities of isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) did not significantly change throughout the fermentation, while that of the ODHC significantly decreased after biotin depletion and Tween 40 addition. Flux redistribution clearly occurred after the decrease in ODHC specific activity. The difference in glutamate production between C. glutamicum and C. efficiens was caused by the difference in the degree of decrease in ODHC specific activity. The difference in Michaelis-Menten constants or K(m) value between ICDH, GDH, and ODHC explained the mechanism of flux redistribution at the branch point of 2-oxoglutarate. It was found that the K(m) values of ICDH and ODHC were much lower than that of GDH for both strains. It was quantitatively proved that the ODHC plays the most important role in controlling flux distribution at the key branch point of 2-oxoglutarate in both coryneform bacteria. Flux redistribution mechanism was well simulated by a Michaelis-Menten-based model with kinetic parameters. The knowledge of the mechanism of flux redistribution will contribute to improvement of glutamate production in coryneform bacteria.     

Interstrand photocrosslinking of DNA via p-carbamoylvinyl phenol nucleoside

We report a novel interstrand photocrosslinking of oligodeoxynucleotides (ODNs). In this system, a modified ODN containing p-carbamoylvinyl phenol nucleoside reacts by photoirradiation at 366 nm with adenine residue of a complementary template ODN to yield a crosslinked ODN in 97% yield.     

Aldehyde oxidase (AO) in the root nodules of Lupinus albus and Medicago truncatula: identification of AO in meristematic and infection zones

Phytohormones are involved in the organogenesis of legume root nodules. The source of the auxin indole-3-acetic acid (IAA) in nodules has not been clearly determined. We studied the enzyme aldehyde oxidase (AO; EC 1.2.3.1), that catalyzes the last step of IAA biosynthesis in plants, in the nodules of Lupinus albus and Medicago truncatula. Primordia and young lupin nodules and mature M. truncatula nodules showed AO activity bands after native polyacrylamide gel electrophoresis. Gel activity analyses using indole-3-aldehyde as substrate indicated that the nodules of white lupin and M. truncatula have the capability to synthesize IAA via the indole-3-pyruvic acid pathway. Immunolocalization and in situ hybridization experiments revealed that AO is preferentially expressed in the meristematic and the invasion zones in Medicago nodules and in the lateral meristematic zone of Lupinus nodules. High IAA immunolabeling was also detected in the meristematic and invasion zones. Low expression levels and no AO activity were detected in lupin Fix- nodules that displayed restricted growth and early senescence. We propose that local synthesis of IAA in the root nodule meristem and modulation of AO expression and activity are involved in regulation of nodule development.     

Proinsulin lacking the A7-B7 disulfide bond, Ins2Akita, tends to aggregate due to the exposed hydrophobic surface

A single mutation (C96Y) in the Ins2 gene, which disrupts the A7-B7 disulfide bond, causes the diabetic phenotype in Akita mice. We biochemically analyzed the conformation of wild-type and Akita mutant recombinant proinsulins. Gel filtration chromatography and dynamic light scattering revealed that the apparent size of the mutant proinsulin molecules was significantly larger than that of wild-type proinsulin, even in the absence of intermolecular disulfide bonds. Titration with a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate, demonstrated that the mutant proinsulin was more hydrophobic than the wild type. In addition, circular dichroism studies revealed that the conformation of the mutant proinsulin was less stable than the wild type, which is consistent with the observation that hydrophobic residues are exposed on the surface of the proinsulin molecules. Studies with antiserum against the C-peptide of proinsulin indicated that the mutant proinsulin had an immunoreactivity that was at least one-tenth weaker than wild-type proinsulin, suggesting that the C-peptide of mutant proinsulin is buried inside the aggregate of the proinsulin molecule. These findings indicate that increased hydrophobicity of mutant proinsulin facilitates aggregate formation, providing a clue to the dominant negative effect in the Akita mouse.     

Genetic diversity of JC virus in the Saami and the Finns: implications for their population history

The JC virus (JCV) genotyping method was used to gain insights into the population history of the Saami and the Finns, both speaking Finno-Ugric languages and living in close geographic proximity. Urine samples from Saami and Finns, collected in northern and southern Finland, respectively, were used to amplify a 610-bp JCV-DNA region containing abundant type-specific mutations. Based on restriction site polymorphisms in the amplified fragments, we classified JCV isolates into one of the three superclusters of JCV, type A, B, or C. All 15 Saami isolates analyzed and 41 of 43 Finnish isolates analyzed were classified as type A, the European type, and two samples from Finns were classified as type B, the African/Asian type. We then amplified and sequenced a 583-bp JCV-DNA region from the type A isolates of Saami and Finns. According to type-determining nucleotides within the region, we classified type A isolates into EU-a1, -a2, or -b. Most type A isolates from Saami were classified as EU-a1, while type A isolates from Finns were distributed among EU-a1, EU-a2, and EU-b. This trend in the JCV-genotype distribution was statistically significant. On a phylogenetic tree based on complete sequences, most of the type A isolates from Saami were clustered in a single clade within EU-a1, while those from Finns were distributed throughout EU-a1, EU-a2, and EU-b. These findings are discussed in the context of the population history of the Saami and the Finns. This study provides new complete JCV DNA sequences derived from populations of anthropological interest.