Zinc tetraruthenated porphyrin binding and photoinduced oxidation of calf-thymus DNA

The photooxidation of calf-thymus DNA has been investigated in the presence of a supramolecular tetraruthenated zincporphyrin (ZnTRP) sensitizer. A strong interaction of ZnTRP with DNA has been observed, exhibiting a gradual transition from a non-specific electrostatic binding mode to a more specific one at high DNA concentrations. Formation of O2(1delta(g)) has been detected from its near-infrared emission, after the excitation of ZnTRP in dioxygen-containing solutions. In the presence of DNA and dioxygen, ZnTRP promotes efficient photocatalytic oxidation of the 2'-deoxyguanosine sites, via their direct reaction with O2(1delta(g)), as in a previous work on the ZnTRP-photoinduced oxidation of the free nucleosides.     

Correlations of calcium accumulations in arteries, veins, cartilages, ligaments, and bones in single humans

To show the relationships of calcium accumulation in the thoracic aorta to the other tissues, calcium contents were determined with a microwave-induced plasma-atomic emission spectrometer on arteries, veins, cartilages, ligaments, and bones. These tissues were resected from 18 individuals, consisting of 11 men and 7 women who died in the age range 59–91 yr. As thoracic and abdominal aortas are routinely used for radiographic examination of arterial calcification, they appear to be standard tissues of the calcium accumulation. The calcium accumulations were determined in the femoral artery, the superior and inferior venae cavae, the internal jugular vein, cartilages of the articular disk of the temporomandibular joint and the intervertebral disk, both the ligaments of the anterior cruciate ligament and the ligamentum capitis femoris, and the calcaneus, in contrast with the thoracic aorta. As calcium increased in the thoracic aorta, it increased in the femoral artery, the articular disk of the temporomandibular joint, the intervertebral disk, both ligaments of the anterior cruciate ligament, and the ligamentum capitis femoris, but it did not increase in veins, such as the superior and inferior venae cavae and the internal jugular vein. In contrast, it decreased in the calcaneus.     

Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in the silver mutation of the Japanese quail

The neural retina and retinal pigment epithelium (RPE) diverge from the optic vesicle during early embryonic development. They originate from different portions of the optic vesicle, the more distal part developing as the neural retina and the proximal part as RPE. As the distal part appears to make contact with the epidermis and the proximal part faces mesenchymal tissues, these two portions would encounter different environmental signals. In the present study, an attempt has been made to investigate the significance of interactions between the RPE and mesenchymal tissues that derive from neural crest cells, using a unique quail mutant silver (B/B) as the experimental model. The silver mutation is considered to affect neural crest-derived tissues, including the epidermal melanocytes. The homozygotes of the silver mutation have abnormal eyes, with double neural retinal layers, as a result of aberrant differentation of RPE to form a new neural retina. Retinal pigment epithelium was removed from early embryonic eyes (before the process began) and cultured to see whether it expressed any phenotype characteristic of neural retinal cells. When RPE of the B/B mutant was cultured with surrounding mesenchymal tissue, neural retinal cells were differentiated that expressed markers of amacrine, cone or rod cells. When isolated RPE of the B/B mutant was cultured alone, it acquired pigmentation and did not show any property characteristic of neural retinal cells. The RPE of wild type quail always differentiated to pigment epithelial cells. In the presence of either acidic fibroblast growth factor (aFGF) or basic FGF (bFGF), the RPE of the B/B mutant differentiated to neural retinal cells in the absence of mesenchymal tissue, but the RPE of wild type embryos only did so in the presence of 10–40 times as much aFGF or bFGF. These observations indicate that genes responsible for the B/B mutation are expressed in the RPE as well as in those cells that have a role in the differentiation of neural crest cells. They further suggest that development of the neural retina and RPE is regulated by some soluble factor(s) that is derived from or localized in the surrounding embryonic mesenchyme and other ocular tissues, and that FGF may be among possible candidates.     

Production of ubiquinone-10 using bacteria

Among the bacterial strains known to contain ubiquinone-10, three strains, Agrobacterium tumefaciens KY-3085 (ATCC4452), Paracoccus denitrificans KY-3940 (ATCC19367) and Rhodobacter sphaeroides KY-4113 (FERM-P4675), were selected as excellent producers of this ubiquinone. The ubiquinone-10 production by the Agrobacterium and Rhodobacter strains was affected by aeration. An ethionine-resistant mutant (M-37) derived from A. tumefaciens KY-3085 promoted increased production of ubiquinone-10 (20% higher than the parent). Another Agrobacterium mutant (AU-55), which was induced by the successive addition of four genetic markers, showed a tolerance to the suppression of ubiquinone-10 production caused by aeration, and the fermentation time for production was remarkably shortened. The amount of ubiquinone-10 produced by this Agrobacterium mutant reached 180 mg/l in a 58 h culture. A green mutant (carotenoid-deficient mutant, Co-22-11) derived from R. sphaeroides KY-4113 produced 350 mg/l of ubiquinone-10 under culturing conditions with a limited supply of air, the ubiquinone-10 content being 8.7 mg/g-dry cell. In this case, the amount and content corresponded to 2.8 and 3.6 times larger than those given by the wild-type strain, respectively. A multiple-layer structure of cell membrane was observed in the highly ubiquinone-10 accumulating cell of the green mutant by electron microscopy. The amount of ubiquinone-10 produced by P. denitrificans was much lower than those of the other two strains.     

The flowering-time geneFT and regulation of flowering inArabidopsis

Transition from vegetative to reproductive development (flowering) is one of the most important decisions during the post-embryonic development of flowering plants. More than twenty loci are known to regulate this process inArabidopsis. Some of these flowering-time genes may act at the shoot apical meristem to regulate its competence to respond to floral inductive signals and floral evocation. Genetic and phenotypic analyses of mutants suggest that the late-flowering geneFT may be a good candidate for such genes. To test this, we have cloned theFT gene using aFT-deficiency line associated with a T-DNA insertion. Cloned genes and loss-of-function mutants in hand, it is now possible to analyse the role ofFT and other genes in flowering at the biochemical and cellular levels as well as at the genetic level. The deduced FT protein has homology with TFL1 and CEN proteins believed to be involved in regulation of inflorescence meristem identity. Phylogenetic analysis suggests that theFT group and theTFL1/CEN group of genes diverged before the diversification of major angiosperm clades. This raises the interesting question of the evolutionary relationship between the regulation of vegetative/reproductive switching in the shoot apical meristem and the regulation of inflorescence architecture in angiosperms. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Fronitier of Plant Biology”     

The human poly(ADP-ribose) glycohydrolase maps to chromosome 10q11.23-21.1 by fluorescence in situ hybridization.

Poly(ADP-ribose) glycohydrolase (PARG) digests poly(ADP-ribose), which is synthesized by poly(ADP-ribose) polymerase (PARP) after DNA damage. We mapped the human poly(ADP-ribose) glycohydrolase gene to chromosome 10q11.23-21.1 by fluorescence in situ hybridization analysis. Since chromosomal rearrangements in thyroid papillary carcinoma and loss of heterozygosity in glioblastoma are frequently observed in this region, genetic alteration of PARG could be implicated in these diseases.     

Sld2, Which Interacts with Dpb11 in Saccharomyces cerevisiae, Is Required for Chromosomal DNA Replication

The DPB11 gene, which genetically interacts with DNA polymerase II (), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint in Saccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal with dpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11 suppressed the temperature-sensitive growth defect caused by sld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25°C but not at 34°C when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25°C. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1 cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.     

Generation of long-lived radical ions through enhanced photoinduced electron transfer processes between [60]fullerene and phenothiazine derivatives.

Photoinduced electron transfer processes between fullerenes (C60) and four phenothiazine derivatives (PTZs) in the absence and presence of hexylviologen dication (HV2+) have been studied by the transient absorption method in the visible and near-IR regions. Electron-transfer takes place from PTZs to the triplet states of fullerenes (3C60*) giving the radical anion of fullerenes (C60.-) and the radical cations of PTZs (PTZ.+). The rate constants and efficiencies of electron transfer are quite high, because of the high electron-donor abilities of PTZs as elucidated by their low oxidation potentials. On addition of HV2+ to the C60 and PTZ systems, the electron-mediating process occurs from C60.- to HV2+, yielding the viologen radical cation (HV.+). In the presence of a sacrificial donor, HV.+ persisted for a long time.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.     

Phase reset affects auditory-visual simultaneity judgment

We continuously receive the external information from multiple sensors simultaneously. The brain must judge a source event of these sensory informations and integrate them. It is thought that judging the simultaneity of such multisensory stimuli is an important cue when we discriminate whether the stimuli are derived from one event or not. Although previous studies have investigated the correspondence between an auditory-visual (AV) simultaneity perceptions and the neural responses, there are still few studies of this. Electrophysiological studies have reported that ongoing oscillations in human cortex affect perception. Especially, the phase resetting of ongoing oscillations has been examined as it plays an important role in multisensory integration. The aim of this study was to investigate the relationship of phase resetting for the judgment of AV simultaneity judgement tasks. The subjects were successively presented with auditory and visual stimuli with intervals that were controlled as SOA_50% and they were asked to report whether they perceived them simultaneously or not. We investigated the effects of the phase of ongoing oscillations on simultaneity judgments with AV stimuli with SOAs in which the detection rate of asynchrony was 50 %. It was found that phase resetting at the beta frequency band in the brain area that related to the modality of the following stimulus occurred after preceding stimulus onset only when the subjects perceived AV stimuli as simultaneous. This result suggested that beta phase resetting occurred in areas that are related to the subsequent stimulus, supporting perception multisensory stimuli as simultaneous.