Genetic and geographical differentiation of <Emphasis Type="Italic">Pandaka</Emphasis> gobies in Japan

The mitochondrial DNA (mtDNA) phylogeny of Japanese Pandaka species (Perciformes: Gobiidae) was inferred from partial nucleotide sequences of the mitochondrial 12S and 16S rRNA genes (1083bp). The resultant mtDNA tree showed two major clades (clade I and clade II), which were inconsistent with the present taxonomic classification. One of the major clades was further divided into two geographical groups, distributed on the Japanese Major Islands (clade I-A) and from Amami-oshima Island to Iriomote Island (clade I-B). The mtDNA haplotypes in clade II were found only on Iriomote Island. The mtDNA divergences in clade I indicated that the Japanese Major Island (clade I-A) and Ryukyu (clade I-B) groups have been geographically isolated from each other for millions of years, based on the putative molecular divergence rate. The geographical distributions of mtDNA haplotypes in clade I-A and clade I-B also suggested that Pandaka gobies had not dispersed to distant offshore islands, indicating that their geographical differentiation may be closely associated with the geological history of the Japanese and Ryukyu Archipelagos.This revised version was published online in January 2005 with corrections to the repetition of the 1st authors name.     

Genetic characterization and specific detection of beer-spoilage Lactobacillus sp. LA2 and related strains

AIMS: Lactobacillus sp. LA2 (DSM15502) and related strains (LA2 group) possess strong beer-spoilage ability. The 16S rDNA sequence of LA2 strain is virtually indistinguishable from that of L. collinoides, generally considered to be nonbeer-spoilage bacteria. The aim of this study was to identify the genetic marker to distinguish between Lactobacillus sp. LA2 group and L. collinoides and to provide a rapid means of identifying beer-spoilage strains belonging to Lactobacillus sp. LA2 group. METHODS AND RESULTS: The 16-23S rDNA intergenic spacer (ITS) regions of Lactobacillus sp. LA2 and L. collinoides JCM1123T were sequenced to identify a genetic marker to distinguish between the two groups. As a result, 300 and 500 bp ITS regions of Lactobacillus sp. LA2 were found to be almost identical with those of L. collinoides JCM1123T. Sequence comparison analysis between Lactobacillus sp. LA2 and L. collinoides JCM1123T revealed that the two contiguously located nucleotides are absent in both ITS regions of Lactobacillus sp. LA2. Based on the sequence difference, we have designed specific PCR primers with a minor modification to the primer sequence that can differentiate between beer-spoilage Lactobacillus sp. LA2 group and nonbeer-spoilage L. collinoides. CONCLUSIONS: The PCR-based method has been developed to identify Lactobacillus sp. LA2 group, providing a rapid and sensitive means of determining the beer-spoilage ability of detected bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The substitution of one nucleotide, located at the third position to the 3'-end in the primer sequence, enhanced the specificity of the PCR method while retaining sufficient sensitivity. The nucleotide gap identified in this study appeared to serve as a useful genetic marker that can differentiate 12 beer-spoilage Lactobacillus sp. LA2 group strains from its close relatives that exhibit no beer-spoilage ability.     

Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts

We have evaluated the sizes and lifetimes of rafts in the plasma membrane from the existing literature, with a special attention paid to their intrinsically broad distributions and the limited time and space scales that are covered by the observation methods used for these studies. Distinguishing the rafts in the steady state (reserve rafts) from those after stimulation or unintentional crosslinking of raft molecules (stabilized receptor-cluster rafts) is critically important. In resting cells, the rafts appear small and unstable, and the consensus now is that their sizes are smaller than the optical diffraction limit (250 nm). Upon stimulation, the raft-preferring receptors are clustered, inducing larger, stabilized rafts, probably by coalescing small, unstable rafts or cholesterol-glycosphingolipid complexes in the receptor clusters. This receptor-cluster-induced conversion of raft types may be caused by suppression of alkyl chain isomerization and the lipid lateral diffusion in the cluster, with the aid of exclusion of cholesterol from the bulk domain and the boundary region of the majority of transmembrane proteins. We critically inspected the possible analogy to the boundary lipid concept. Finally, we propose a hypothesis for the coupling of GPI-anchored receptor signals with lipid-anchored signaling molecules in the inner-leaflet raft.     

Metalloproteases with EGF, CUB, and thrombospondin-1 domains function in molting of Caenorhabditis elegans

Functional analysis using RNAi was performed on eleven genes for metalloproteases of the M12A family in Caenorhabditis elegans and the interference of the C17G1.6 gene (nas-37) was found to cause incomplete molting. The RNAi of the C26C6.3 gene (nas-36) also caused a similar molting defect but not so severely as that of the nas-37 gene. Both the genes encode an astacin-like metalloprotease with an epidermal growth factor (EGF)-like domain, a CUB domain, and a thrombospondin-1 domain, in this order. The promoter-driven green fluorescent protein (GFP) expression analysis suggested that they are expressed in hypodermal cells throughout the larval stages and in the vulva of adult animals. In the genetic background of rde-1(ne219), where RNAi does not work, the molting defect caused by the nas-37 interference was observed when the transgenic wild-type rde-1 gene was expressed under the control of the dpy-7 promoter, known to be active in the hypodermal cells, but not under the control of the myo-3 promoter, active in the muscular cells. Therefore these proteases are thought to be secreted by the hypodermal cells and to participate in shedding of old cuticles.     

DNA Microarray for Rapid Detection of Mitochondrial DNA Haplotypes of Chum Salmon

For use in genetic stock identification, we developed an oligonucleotide (DNA) microarray hybridization method for rapid and accurate detection of nucleotide sequence variations in 20 previously identified variable nucleotide sites in about 500 bp within the 5 half of the control region of mitochondrial DNA of chum salmon (Oncorhynchus keta). The method includes immobilization of synthesized oligonucleotides containing respective polymorphic sites on a glass slide precoated with polycarbodiimide resin, a 2-hour hybridization with DNA microarray of biotinylated polymerase chain reaction fragments spanning the 5 variable portion followed by short washing, and visualization of hybridization signals by conventional ABC method and scanner-assisted computation of signal intensity on a computer. The entire process of hybridization and detection was completed within 4 hours. The resulting DNA microarray could detect all of the single nucleotide mutations and therefore could be used to identity the sequence variations defining 30 mtDNA haplotypes of chum salmon as revealed previously by nucleotide sequence analysis.     

Adaptation of sympatric Achromatium spp. to different redox conditions as a mechanism for coexistence of functionally similar sulphur bacteria

Changes in the abundance of sympatric Achromatium spp. in response to the artificial manipulation of redox conditions in sediment microcosms was determined by fluorescence in situ hybridization (FISH). Adaptation to different redox conditions was shown to be one mechanism that supported the coexistence of functionally similar Achromatium spp. In sediment microcosms, in which the overlying water was oxygenated, Achromatium community size and composition remained unchanged over time. However, imposition of anoxic conditions induced changes in community structure. Anoxia caused a reduction in the relative abundance of Achromatium sp. RY8 (72 +/- 4% to 49 +/- 2%) and an increase in Achromatium sp. RY5 (19 +/- 5% to 32 +/- 3%) and a newly identified Achromatium sp., RYKS (14 +/- 4% to 27 +/- 2%). In anoxic microcosms supplemented with a single addition of nitrate at different initial concentrations the relative decline in Achromatium sp. RY8 was dependent on the initial nitrate concentration. In these experiments nitrate was rapidly removed. In contrast, when high levels of nitrate were maintained by periodic replacement of the overlying water with nitrate supplemented anoxic water, the composition of the Achromatium community remained stable over time. This suggested that all of the coexisting Achromatium spp. are obligate or facultative anaerobes, but, Achromatium sp. RY8 was more sensitive to sediment redox conditions than the other Achromatium species. Given the heterogeneous nature of sedimentary environments, redox-related niche differentiation may promote coexistence of sympatric Achromatium spp.     

Non-destructive on-chip cell sorting system with real-time microscopic image processing

Studying cell functions for cellomics studies often requires the use of purified individual cells from mixtures of various kinds of cells. We have developed a new non-destructive on-chip cell sorting system for single cell based cultivation, by exploiting the advantage of microfluidics and electrostatic force. The system consists of the following two parts: a cell sorting chip made of poly-dimethylsiloxane (PDMS) on a 0.2-mm-thick glass slide, and an image analysis system with a phase-contrast/fluorescence microscope. The unique features of our system include (i) identification of a target from sample cells is achieved by comparison of the 0.2-μm-resolution phase-contrast and fluorescence images of cells in the microchannel every 1/30 s; (ii) non-destructive sorting of target cells in a laminar flow by application of electrostatic repulsion force for removing unrequited cells from the one laminar flow to the other; (iii) the use of agar gel for electrodes in order to minimize the effect on cells by electrochemical reactions of electrodes, and (iv) pre-filter, which was fabricated within the channel for removal of dust contained in a sample solution from tissue extracts. The sorting chip is capable of continuous operation and we have purified more than ten thousand cells for cultivation without damaging them. Our design has proved to be very efficient and suitable for the routine use in cell purification experiments.     

Acoustic stiffness and change in plug cartilage over time after autologous osteochondral grafting: correlation between ultrasound signal intensity and histological score in a rabbit model

We investigated quantitative changes over time in ultrasound signal intensity (an index of stiffness), signal duration (an index of surface irregularity), and interval between signals (an index of thickness) of plug cartilage in an animal model of autologous osteochondral grafting. A full-thickness osteochondral plug was surgically removed and replaced in male Japanese white rabbits (n = 22). Specimens obtained at day 0 and weeks 2, 4, 8, 12 and 24 postoperatively were assessed using an ultrasound system and by macroscopic and histological evaluation (modified Mankin's score). Histology revealed that the plug sank until 2 weeks postoperatively, and that newly formed cartilage-like tissue covered the plug, but at 24 weeks the tissue detached. The plug itself survived well throughout the period of observation. Although the signal intensity at the plug site was same as that in the sham operated contralateral knee at day 0, from 2 to 24 weeks postoperatively it was less than that in the sham knee. At 8 weeks, this difference was significant (P < 0.05). Modified Mankin's score revealed early degenerative changes at the site, but macroscopic examination did not. Signal intensity correlated significantly with score (both at day 0 and at the five postoperative time points [P < 0.05, r = -0.91] and as a whole [P < 0.05, r = -0.36]). Signal intensity also significantly correlated with the individual subscores for 'cartilage structure' (P < 0.05, r = -0.32) and 'cartilage cells' (P < 0.05, r = -0.30) from the modified Mankin's score, but not significantly with subscores for 'staining' and 'tidemark'. Signal duration correlated significantly with total score (as a whole [P < 0.05, r = 0.34]), but not significantly with the score for cartilage structure (P = 0.0557, r = 0.29). The interval between signals reflected well the actual thickness of the plug site. The significant relationships between ultrasound signal intensity and scores suggest that early degenerative changes in plug cartilage and cartilage-like tissue, especially in the superficial layer, are detectable by high-frequency ultrasound assessment.     

Imaging gene expression in regional brain ischemia in vivo with a targeted [111in]-antisense radiopharmaceutical

The gene encoding glial fibrillary acidic protein (GFAP) is downregulated 24 hr after reversible brain ischemia, such as with a middle cerebral artery occlusion (MCAO). The in vivo imaging of decreased GFAP gene expression in cerebral ischemia was examined in the present studies using a targeted peptide nucleic acid (PNA), which was labeled with (111)In, and which hybridized to nucleotides 20-37 of the rat GFAP mRNA. The PNA was monobiotinylated, and was attached to a monoclonal antibody (MAb) to the transferrin receptor (TfR) via a biotin-streptavidin linkage. The TfR MAb enables trans-membrane transport of the PNA antisense radiopharmaceutical from blood to the cytosol of brain cells. The decreased GFAP gene expression at 24 hr after a 1-hr reversible MCAO was confirmed by immunocytochemistry. The [(111)In]-labeled PNA - MAb conjugate was administered intravenously to anesthetized rats at 24 hr after the 1-hr reversible MCAO, and the brain uptake of the targeted antisense imaging agent was decreased relative to brain regions outside of the infarct zone. These studies provide evidence that decreased expression of a target gene in brain can be imaged in vivo with a sequence-specific PNA, provided the antisense radiopharmaceutical is delivered across cell membranes with a receptor-specific targeting agent.     

Synthesis and antibacterial activity of novel and potent DNA gyrase inhibitors with azole ring

The 4-piperidyl moiety and the pyrazole ring in 1-(3-chlorophenyl)-5-(4-phenoxyphenyl)-3-(4-piperidyl)pyrazole 2, which has previously shown improved DNA gyrase inhibition and target-related antibacterial activity, were transformed to other groups and the in vitro antibacterial activity of the synthesized compounds was evaluated. The selected pyrazole, oxazole and imidazole derivatives showed moderate inhibition against DNA gyrase and topoisomerase IV with similar IC(50) values (IC(50)=9.4-25 microg/mL). In addition, many of the pyrazole, oxazole and imidazole derivatives synthesized in this study exhibited potent antibacterial activity against quinolone-resistant clinical isolates and coumarin-resistant laboratory isolates of Gram-positive bacteria with minimal inhibitory concentration values equivalent to those against susceptible strains.