Ferrocenylnaphthalene diimide-based electrochemical hybridization assay for a heterozygous deficiency of the lipoprotein lipase gene

An electrochemical hybridization assay has been devised that enables the rapid analysis of a heterozygous deficiency of the human lipoprotein lipase (LPL) gene. PCR products of 350 base pairs (bp) containing the wild-type sequence, a mutated G(818) --> A transition or a G(916) deletion of the LPL gene were subjected to hybridization with a probe DNA of 13 or 15 bases that represented either the wild-type or the mutated sequence immobilized on a gold electrode. The differential pulse voltammetry of the electrode before and after hybridization was determined in the presence of ferrocenylnaphthalene diimide (FND) at 460 mV. The measured change in peak current, Deltai, was defined by (i - i(o))/i(o) x 100%, where i(o) and i represent the current before and after hybridization, respectively. Matched combinations of sample and probe gave Deltai values of 40-90%, whereas mismatched combinations gave values of 20-35%, enabling the discrimination of matched hybrids from mismatched ones across a slim margin. Because the heterozygote contains both the wild-type and mutated sequences, however, it alone gives large Deltai values with both the wild- and mutant-type probes. This system was validated on 10 unknown samples of each of the two types of LPL mutation, which were correctly identified in every case.     

Isolation and culture conditions of a Klebsiella pneumoniae strain that can utilize ammonium and nitrate ions simultaneously with controlled iron and molybdate ion concentrations

A bacterial strain F-5-2, isolated from soil and identified as Klebsiella pneumoniae, removed NH4+ completely in 24 h of aerobic cultivation in a medium containing 1 mg/ml of NH4NO3. However, 70% of the NO3- originally provided remained. When 100 microM Fe2+ was added to the medium, both NH4+ and NO3- were removed simultaneously and completely from the culture within 6 h of incubation. In addition, the amount of MoO4- in the medium markedly affected the bacterial cell growth and utilization of NH4+ and NO3-. The bacterium could remove 4 mg/ml of NH4NO3 completely in 48 h of aerobic cultivation in a medium containing 100 microM Fe2+ and 0.8 pM MoO4(2-). The total nitrogen in the culture containing its cells was decreased to 14% of that in the NH4NO3 originally provided. GC-MS analysis demonstrated that N2 was generated from the nitrogen atoms of both NH4+ and NO3-.     

Loss-of-function of a rice brassinosteroid biosynthetic enzyme,C-6 oxidase,prevents the organized arrangement and polar elongation of cells in the leaves and stem

Molecular genetic and physiological studies on brassinosteroid (BR)-related mutants of dicot plants have revealed that BRs play important roles in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of a rice mutant partially insensitive to BR signaling. To investigate the function of BR in monocots, we identified and characterized BR-deficient mutants of rice, BR-deficient dwarf1 (brd1). The brd1 mutants showed a range of abnormalities in organ development and growth, the most striking of which were defects in the elongation of the stem and leaves. Light microscopic observations revealed that this abnormality was primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. The accumulation profile of BR compounds in the brd1 mutants suggested that these plants may be deficient in the activity of BR C-6 oxidase. Therefore, we cloned a rice gene, OsDWARF, which has a high sequence similarity to the tomato C-6 oxidase gene, DWARF. Introduction of the wild-type OsDWARF gene into brd1 rescued the abnormal phenotype of the mutants. The OsDWARF gene was expressed at a low level in all of the examined tissues, with preferential expression in the leaf sheath, and the expression was negatively regulated by brassinolide treatment. On the basis of these findings, we discuss the biological function of BRs in rice plants.     

Pistillody, homeotic transformation of stamens into pistil-like structures, caused by nuclear-cytoplasm interaction in wheat

Homeotic transformation of stamens into pistil-like structures (pistillody) has been observed in a cytoplasmic substitution (alloplasmic) line of wheat (Triticum aestivum L.) cv. Norin 26, which has the cytoplasm of a wild relative species, Aegilops crassa L. On the other hand, an alloplasmic line of wheat cv. Chinese Spring (CS) with Ae. crassa cytoplasm has normal flowers. This is due to the presence in the CS nucleus of a fertility-restoring gene, Rfd1. Deletion mapping analysis revealed that Rfd1 is located on the middle part of the long arm of chromosome 7B. To investigate the function of the Rfd1 gene by a loss-of-function strategy, we produced alloplasmic lines of CS ditelosomic 7BS [(cr)-CSdt7BS] and CS monotelodisomic 7BS [(cr)-CSmd7BS] with the Ae. crassa cytoplasm, and characterized their phenotypes. The line (cr)-CSdt7BS without Rfd1 exhibited pistillody in all florets, and also female sterility. Scanning electron microscopy of the young spikes revealed that the pistillody was induced at an early stage of stamen development. The pistillate stamens often developed incomplete ovule-like structures with integuments instead of tapetum and pollen grains. It is possible that MADS box genes are associated with the induction of pistillody, because the expression of wheat APETALA3 homologue (WAP3) was reduced in the young spikes of (cr)-CSdt7BS. In addition, a histological study indicated that the female sterility in (cr)-CSdt7BS is due to the abnormality of the ovule, which fails to form an inner epidermis and integuments in the chalaza region. The line (cr)-CSmd7BS, hemizygous for Rfd1, showed partial pistillody (51%) and restored female fertility up to 72%. These results suggest that the induction of both pistillody and ovule deficiency caused by the Ae. crassa cytoplasm is inhibited by the Rfd1 gene in a dose-dependent manner.     

Classification of brassinosteroid-regulated genes based on expression profiles in bri1 and in response to a protein kinase inhibitor, staurosporin

To gain insight into the brassinosteroid (BR) signaling pathway, the expression of BR-regulated genes was analysed in the BR-signaling mutant br-insensitive 1 (bri1), and in the presence of a protein kinase inhibitor, staurosporin. BR-regulated genes were classified based on the results. This classification will perhaps prove useful in BR-signalling studies using BR-regulated genes as molecular markers.     

Preparation and biological activity of molecular probes to identify and analyze jasmonic acid-binding proteins

Several types of jasomonic acid (JA) derivatives, including JA--amino acid conjugates, a JA--biotin conjugate, a JA--dexamethasone heterodimer, and a JA-fluoresceine conjugate, were prepared as candidates for molecular probes to identify JA--binding proteins. These JA derivatives, excepting the JA--fluoresceine conjugate, exhibited significant biological activities in a rice seedling assay, a rice phytoalexin-inducing assay, and/or a soybean phenylalanine ammonia-lyase-inducing assay. These JA derivatives could therefore be useful probes for identifying JA--binding proteins. The activity spectra of the prepared compounds were different from each other, suggesting that different types of JA receptors were involved in the perception of JA derivatives in the respective bioassays.     

Effects of light intensity distribution on growth of Rhodobacter capsulatus

For cultivation of photosynthetic cells under defined light intensity distributions, the repeated batch culture, in which a part of culture broth containing grown cells was repeatedly replaced at predetermined time intervals with a fresh medium to keep the cell concentration constant at an initial value, was employed. By use of this method the effects of the light intensity distribution on the growth characteristics of Rhodobacter capsulatus were studied. Unexpected decreases in the specific growth rate were observed in culture of R. capsulatus at high cell concentrations and a long light path length. Big differences in the light intensities of lightly and darkly illuminated portions in photobioreactors, which reflects the light intensity distribution, seemed to cause this phenomenon, which must be taken into consideration for stable growth of photosynthetic cells.     

Development of a dielectric equivalent gel for better impedance matching for human skin

It would be useful to develop a tissue equivalent gel to improve the uniformity of the electromagnetic field in the human body, and for making a tissue equivalent dielectric human phantom. In this study, solid type, water based gelatin-honey gels were developed which have the electrical characteristics of skin tissue. It was demonstrated that a stable and homogeneous gel, with a relative dielectric constant epsilon ' chosen from desired ranges found in skin, can be made for 200-400 MHz.     

Production of xanthones with free radical scavenging properties, emodin and sclerotiorin by the cultured lichen mycobionts of Pyrenula japonica

From the cultures of the spore-derived mycobionts of the lichen Pyrenula japonica, two new xanthones, 1,8-dihydroxy-3-hydroxymethyl-5-methoxyxanthone and 1,2,8-trihydroxy-5-methoxy-3-methylxanthone, were isolated along with 1,7-dihydroxy-3-methylxanthone, 1,5,8-trihydroxy-3-methylxanthone, 1,8-dihydroxy-5-methoxy-3-methylxanthone, emodin and sclerotiorin. Their structures were determined by spectroscopic methods. Sclerotiorin was isolated for the first time from lichen mycobionts. Radical scavenging activities of the isolated xanthones were also studied.     

Differential somatic CAG repeat instability in variable brain cell lineage in dentatorubral pallidoluysian atrophy (DRPLA): a laser-captured microdissection (LCM)-based analysis

Employing a laser-captured microdissection (LCM), we have investigated the somatic instability of CAG repeats in the variable brain cell lineage in three patients with dentatorubral pallidoluysian atrophy (DRPLA). LCM enables the isolation of single lineage brain cells for subsequent molecular analysis. We have found that CAG repeat size and the range of CAG repeats in the cerebellar granular cells is smaller than those in cerebellar glial cells. Similarly, those in the cerebral neuronal cells are significantly shorter than those in cerebral glial cells. These data directly indicate that the CAG repeat is relatively more stable in neuronal cells than in glial cells. Furthermore, cerebellar granular cells show significantly smaller main CAG repeat size and CAG repeat range than either Purkinje cells or cerebral neuronal cells, suggesting that somatic instability in the CAG repeat is markedly variable even among the different types of neuronal populations. The cell-specific CAG repeat instability may thus be more complex than has previously been considered. LCM is a powerful tool for elucidating the mechanism of the triplet repeat instability of each cell type.