Conformational behavior of giant DNA through binding with Ag+ and metallization

The conformational behavior of a long single-chain double-stranded DNA in solutions of free silver ions and silver nanoparticles generated via the reduction of AgNO3 by NaBH4 was monitored by fluorescence and electron microscopies and UV spectroscopy. The interaction of monovalent silver ions with DNA induces shrinking of a DNA-coiled polymer chain as a result of a decrease in the DNA persistence length through the complexation of Ag+ with DNA bases. In contrast, the reduction of silver ions by NaBH4 in DNA solutions triggers DNA compaction: a DNA transition from elongated coil state into a compact state. This transition is continuous, unlike the all-or-none discrete DNA compaction that is commonly seen with multications. It is suggested that the collapse of DNA is accompanied by growth aggregation of silver nanoparticles generated on the DNA template.     

Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange

Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector DDeltaNsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.     

Synthesis and absolute configuration of hormone alpha1

An important biological event in phytopathogens of the genus Phytophthora is sexual reproduction, which is conducted by two mating types, A1 and A2. A factor known as hormone alpha1 is secreted by the A1 mating type and induces the formation of sexual spores (oospores) in the A2 mating type. Here we describe the asymmetric synthesis and assignment of the absolute configuration of hormone alpha1 by oospore-inducing assays of the synthesized isomers.     

Identification and genomic structure of chemosensory proteins (CSP) and odorant binding proteins (OBP) genes expressed in foreleg tarsi of the swallowtail butterfly Papilio xuthus

Chemoreception is a key feature for selection of host plants by phytophagous insects. Female swallowtail butterflies recognize their host plants using chemosensilla present on foreleg tarsi. We constructed a cDNA library of female tarsi and a genome library of Papilio xuthus. We identified 11 chemosensory protein (CSP) genes and three odorant binding proteins (OBP) genes from the cDNA library and eight additional CSP genes from the genome library using the ESTs as probes. A sequence similarity tree of insect CSPs showed that lepidopteran CSPs constructed big branches of the order. Small numbers of CSPs have been identified from the whole genomes of several insect orders which belong to branches separated from those of Lepidoptera. The CSP gene family of Lepidoptera may have diverged in at least two steps, the first on a small scale and the second on a large scale before and after the diversification of insect orders, respectively. Seventeen of 19 CSP genes of P. xuthus clustered in a specific region of the genome, suggesting that they were diversified by gene duplication from a common ancestral gene.     

Diversity of staphylocoagulase and identification of novel variants of staphylocoagulase gene in Staphylococcus aureus

Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Serotype of SC (coagulase type) is used as an epidemiological marker and 10 types (I-X) have been discriminated so far. To clarify genetic diversity of SC within a single and among different serotype(s), we determined approximately 1500 bp-nucleotide sequences of SC gene encoding D1, D2, and central regions (N-terminal half and central regions of SC; SC(NC)) for a total of 33 S. aureus strains comprising two to three strains from individual coagulase types (I-VIII, X) and 10 strains which were not determined as previously known SC serotypes (ND-strains). Amino acid sequence identities of SC(NC) among strains with a single coagulase type of II, III, IV, V, VI and X were extremely high (more than 99%), whereas lower identity (56-87%) was observed among different types. In contrast, within a single coagulase type of I, VII, or VIII, sequence divergence was found (lowest identity; 82%). SC(NC) sequences from the ND-strains were discriminated into two genetic groups with an identity of 71% to each other (tentatively assigned to genotypes [XI] and [XII]), and exhibited less than 86% sequence identities to those of most known coagulase types. All the types [XI] and [XII] strains were methicillin susceptible and belonged to different sequence types from those of coagulase types I-X strains reported so far by multilocus sequence typing. These findings indicated genetic heterogeneity of SC in coagulase types I, VII, and VIII strains, and the presence of two novel SC genotypes related to antigenicity of SC serotypes.     

Real-time PCR method using capturing oligo-immobilized PCR tubes to determine the specific gene for soybean and genetically modified soybean in food matrices

A new real-time PCR method using capturing oligo-immobilized PCR tubes is described. This method was used to detect specific genes for soybean and genetically modified (GM) soybean in food matrices. In a standard reaction using soybean genomic DNA and a capturing oligo for the lectin gene (Le1) immobilized on the tube, we examined the effects of such hybridization conditions as the location, length, and amount of the capturing oligo, and the incubation time and temperature. Under optimized conditions, the copy number of Le1 was determined in a concentration-dependent manner from soybean genomic DNA and soybean lysate (DNA 10-1000 ng, r=0.99; lysate 1-100%, r=0.99). The copy number of a Roundup Ready soybean (RRS) gene was also successfully detected in a concentration-dependent manner (1-100%, r=0.99) from GM soybean lysate, using PCR tubes with an immobilized capturing oligo for the transgene. Our data indicate that this is a rapid and simple method to determine specific genes for soybean and GM soybean in food matrices.     

Conformational Transition of Giant DNA in a Confined Space Surrounded by a Phospholipid Membrane

It has been established that a long DNA molecule exhibits a large discrete conformational change from a coiled state to a highly folded state in aqueous solution, depending on the presence of various condensing agents such as polyamines. In this study, T4 DNA labeled with fluorescent dyes was encapsulated in a cell-sized microdroplet covered with a phospholipid membrane to investigate the conformational behavior of a DNA molecule in such a confined space. Fluorescence microscopy showed that the presence of Mg²⁺ induced the adsorption of DNA onto the membrane inner-surface of a droplet composed of phosphatidylethanolamine, while no adsorption was observed onto a phosphatidylcholine membrane. Under the presence of spermine (tetravalent amine), DNA had a folded conformation in the bulk solution. However, when these molecules were encapsulated in the microdroplet, DNA adsorbed onto the membrane surface accompanied by unfolding of its structure into an extended coil conformation under high concentrations of Mg²⁺. In addition, DNA molecules trapped in large droplets tended not to be adsorbed on the membrane, i.e., no conformational transition occurred. A thermodynamic analysis suggests that the translational entropy loss of a DNA molecule that is accompanied by adsorption is a key factor in these phenomena under micrometer-scale confinement.     

Genotypic differences in root hydraulic conductance of rice (Oryza sativa L.) in response to water regimes

Some plants are more mycorrhizal than others and mycorrhizal colonisation of plants in extreme environments is frequently additionally reduced due to decreased spore density and/or diversity and therefore frequently overlooked. We analysed two plant species from both metal polluted and saline enriched soils with differing mycorrhizal colonisation levels/status using classical and molecular methods. The selected plant species were Sesleria caerulea (L.) Ard. and Thlaspi praecox Wulfen from a metal polluted site, and Limonium angustifolium (Tausch) Degen [Statice serotina Rchb., L. vulgare Mill. subsp. Serotinum (Rchb.) Gams] and Salicornia europaea L. from the Se?ovlje salterns in Slovenia. Despite the high mycorrhizal frequencies (F%) observed, the presence of arbuscules (A%) was at best low in S. caerulea and T. praecox, and undetectable in L. angustifolium and S. europaea. Temporal temperature gradient gel electrophoresis (TTGE) was applied to field-collected samples from both burdened environments and proved to be an effective technique for rapid profiling and identification of arbuscular mycorrhizal fungi (AMF). Sequencing and phylogenetic analysis confirmed the association of AMF of the genus Glomus with roots of all four plant species. This is the first report on the identification and profiling of Glomeromycota in the field-collected Cd/Zn metal hyperaccumulator T. praecox growing at a highly metal polluted site, as well as in L. angustifolium and S. europaea collected in a saline environment. The identification of AMF from both ecosystems only partially resembles previous identifications on the basis of spores.     

Myr-Ric-8A enhances G(alpha15)-mediated Ca2+ response of vertebrate olfactory receptors

The determination of ligand specificities of odorant receptorswill contribute to the understanding of how odorants are discriminatedby the olfactory system. To date, the ways in which some olfactoryreceptors (ORs) pair with their cognate ligands has been studiedusing a Ca²⁺ imaging technique. This approach has been usedto investigate orphan G protein–coupled receptors expressedin heterologous cells; however, most attempts to functionallyexpress ORs on the cell surface of heterologous cells have failed.Recently, receptor-transporting protein 1 and Ric-8B have beenidentified as proteins involved in targeting receptors to thecell membrane and amplifying receptor signals, and thus, theyare able to facilitate cellular responses via ORs in a heterologouscell system. Here, we describe a technique in which we employeda myristoylation sequence–conjugated mutant of Ric-8A(Myr-Ric-8A) as a signal amplifier and show Myr-Ric-8A greatlyenhances G₁₅-mediated Ca²⁺ responses of ORs in HEK293 cells.Coexpression of Myr-Ric-8A enabled us to deorphanize a mouseOR and to determine its molecular receptive range. Our resultssuggest that Myr-Ric-8A should be helpful in functional characterizationof ORs in heterologous cells using Ca²⁺ imaging.     

Acetoxybenzhydrols as highly active and stable analogues of 1′S-1′-acetoxychavicol,a potent antiallergic principal from Alpinia galanga

Through SAR studies on 1′S-1′-acetoxychavicol acetate (1) against Type I antiallergic activity by indexing release of β-hexosaminidase, a marker of antigen-IgE-mediated degranulation in RBL-2H3 cells, more stable and potent analogue, 4-(methoxycarbonyloxyphenylmethyl)phenyl acetate (16), has been developed. The compound 16 also strongly inhibited the antigen-IgE-mediated TNF-α and IL-4 production.