Multiplex PCR-based Alu insertion polymorphisms genotyping for identifying individuals of Japanese ethnicity

Discrimination of Alu insertions is a useful tool for geographic ancestry analysis, and is usually performed by Alu element amplification and agarose gel electrophoresis. Here, we have developed a new fluorescence-based method for multiple Alu genotyping in forensic identification. Allele frequencies were determined in 70 Japanese individuals, and we selected 30 polymorphic Alu insertions. Three primers were designed for each Alu locus to discriminate alleles using the 3-6 bp differences in amplicon sizes. Furthermore, we classified the amplification primers for the 30 loci into three different sets, and PCR using each set of primers provided 10 loci fragments ranging from 50 to 137 bp. Based on population data, the probability of incorrectly assigning a match was 3.7×10(-13). Three independent amplifications and subsequent capillary electrophoresis enabled the sensitive genotyping of small amounts of DNA, indicating that this method is suitable for identifying individuals of Japanese ethnicity.     

Subepithelial elastic system fibers of oblique mucosal folds in the rat proximal colon.

The proximal colon of the rat is characterized by a 'herring bone' pattern of oblique mucosal folds (OMF) which are arranged in a parallel array. By light and electron microscopy the OMF exhibited rich subepithelial elastic system fibers which bound the epithelial basement membrane and the smooth muscle cells of the lamina propria together. The elastic system fibers usually consist of elastic, elaunin and oxytalan fibers. However, the subepithelial elastic system fibers of the OMF were composed of relatively thin elastic fibers with a few microfibrils, and elaunin and oxytalan fibers which were almost indiscernible. Areas other than the OMF were quite poor in subepithelial elastic system fibers. The interpositions between each of the OMF were composed of typical components: elastic, elaunin and oxytalan fibers. The composition of the subepithelial elastic system fibers of the OMF does not correspond to that of any other organs previously reported. The present study suggests that the OMF of the rat proximal colon might be equipped in such a way to resist to distension or compression.     

Ferric species of the giant extracellular hemoglobin of Glossoscolex paulistus as function of pH: an EPR study on the irreversibility of the heme transitions

The present article is focused on the transitions of ferric heme species of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) induced by successive alterations in pH, involving alkaline and acid mediums. Electron paramagnetic resonance (EPR) is the spectroscopy used to evaluate the transitions that occur in the first coordination sphere of ferric ion as a consequence of ligand changes in a wide range of pH, since this tool is very sensitive to slight changes that occur in the heme pocket of paramagnetic species. This approach is adequate to obtain information regarding the reversibility/irreversibility that involves the heme transitions induced by pH, since the degree of reversibility is associated to the intensity of the changes that occur in the spatial configuration of the polypeptide chains, which is clearly associated to the first coordination sphere. The results demonstrate a significant degree of irreversibility of heme transitions, since the final species, which do not present any change after 6 h of its respective formations, are quite different of the initial species. The results denote that the more stable species are the bis-histidine (hemichrome) and pentacoordinate species, due to the properties of their ligands and to the mechanical influence of the respective subunits. EPR spectra allow to distinguish the types of hemichrome species, depending on the reciprocal orientation between the histidine axial ligands, in agreement with Walker's Classification [Walker, F.A., 1999. Magnetic spectroscopic (EPR, ESEEM, M?ssbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins. Coord. Chem. Rev. 185-186, 471-534]. However, these transitions are not completed, i.e., the appearance of a determined species does not mean the total consumption of its precursor species, implying the coexistence of several types of species, depending on pH. Furthermore, it is possible to conclude that a "pure" EPR spectrum of aquomet ferric species is an important indicator of a high level of conservation referent to the "native" configuration of whole hemoglobin, which is only encountered at pH 7.0. The results allow to infer important physico-chemical properties as well as to evaluate aspects of the structure-activity relationship of this hemoprotein, furnishing information with respect to the denaturation mechanism induced by drastic changes in pH. These data are very useful since HbGp has been proposed as prototype of substitute of blood, thus requiring wide knowledge about its structural and chemical properties.     

Ferric species equilibrium of the giant extracellular hemoglobin of Glossoscolex paulistus in alkaline medium: HALS hemichrome as a precursor of pentacoordinate species

The present work is focused on the complex ferric heme species equilibrium of the giant extracellular hemoglobin from Glossoscolex paulistus (HbGp) in alkaline medium. EPR, UV-vis and CD spectroscopies were used in order to characterize the ferric heme species formed as a consequence of the medium alkalization as well as the oligomeric changes occurring simultaneously with heme transitions. EPR experiments allowed us to characterize the different hemichrome species in equilibrium, illustrating the small difference in spin state of this species and the complexity of the equilibira involving hemoglobin ferric species. The results emphasize the importance of the alkaline oligomeric dissociation, which is decisive to promote the heme ferric species transition as function of the increase in water accessibility to the heme pocket. In fact, the oligomeric dissociation in alkaline medium is a consequence of the intense electrostatic repulsion between anionic charges on the protein surface, since the isoelectric point (pI) of this hemoglobin is acid. This explains the more drastic aquomet-hemichrome-pentacoordinate species transition in alkaline medium as compared with the acid medium. However, these heme species transitions are not completed, i.e., the appearance of new species does not mean the total consumption of the precursor species. This equilibrium complexity is associated to the effective influence of oligomeric arrangement of this whole hemoglobin, which present 144 molecular subunits. The acid pI is probably an important factor to the structure-activity relationship of the giant extracellular hemoglobins.     

Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils

The neutral glycosphingolipid lactosylceramide (LacCer) forms lipid rafts (membrane microdomains) coupled with the Src family kinase Lyn on the plasma membranes of human neutrophils; ligand binding to LacCer activates Lyn, resulting in neutrophil functions, such as superoxide generation and migration (Iwabuchi and Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100, 1454–1464, 2002 and Sato et al. Induction of human neutrophil chemotaxis by Candida albicans-derived beta-1,6-long glycoside side-chain-branched beta glycan, J. Leukoc. Biol. 84, 204–211, 2006). Neutrophilic differentiated HL-60 cells (D-HL-60 cells) express almost the same amount of LacCer as neutrophils. However, D-HL-60 cells do not have Lyn-associated LacCer-enriched lipid rafts and lack LacCer-mediated superoxide-generating and migrating abilities. Here, we examined the roles of LacCer molecular species of different fatty acid compositions in these processes. Liquid chromatography-mass spectrometry analyses revealed that the very long fatty acid C24:0 and C24:1 chains were the main components of LacCer (31.6% on the total fatty acid content) in the detergent-resistant membrane fraction (DRM) from neutrophil plasma membranes. In contrast, plasma membrane DRM of D-HL-60 cells included over 70% C16:0-LacCer, but only 13.6% C24-LacCer species. D-HL-60 cells loaded with C24:0 or C24:1-LacCer acquired LacCer-mediated migrating and superoxide-generating abilities, and allowed Lyn coimmunoprecipitation by anti-LacCer antibody. Lyn knockdown by siRNA completely abolished the effect of C24:1-LacCer loading on LacCer-mediated migration of D-HL-60 cells. Immunoelectron microscopy revealed that LacCer clusters were closely associated with Lyn molecules in neutrophils and C24:1-LacCer-loaded D-HL-60 cells, but not in D-HL-60 cells or C16:0-LacCer-loaded cells. Taken together, these observations suggest that LacCer species with very long fatty acids are specifically necessary for Lyn-coupled LacCer-enriched lipid raft-mediated neutrophil superoxide generation and migration. This study was supported in part by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (16017293) to K.I., by COFIN-PRIN 2004 to A.P., and by “High-Tech Research Center” Project for Private Universities: matching fund subsidy. An erratum to this article can be found at     

Probing the cell peripheral movements by optical trapping technique

Swiss 3T3 fibroblasts cultured on a poly-L-lysine-coated coverslip was stimulated with 0.5 micro M phorbol myristate acetate, and the movements of the peripheral membranes were probed with a 1- micro m polystyrene bead held in an optical trap. The bead brought into contact with the cell edge occasionally moved away from and returned to the original position. The movement ranged over 100 nm and occurred mainly in one direction, suggesting that the protruding cell membrane pushed the bead. The maximum velocities derived from individual pairs of protrusive and withdrawal movements exhibited a correlation, which is consistent with the previous reports. Acceleration and deceleration occurred both in the protrusive and withdrawal phases, indicating that the movements were regulated. Movement of the membrane occurred frequently with an ensemble-averaged maximum speed of 23 nm/s at the trap stiffness of 0.024 pN/nm, but it was strongly suppressed when the trap stiffness was increased to 0.090 pN/nm. Correlation of the protrusive and withdrawal velocities and the acceleration and deceleration both in the protrusive and withdrawal phases can be explained by the involvement of myosin motor at least in the withdrawal process. However, the fact that the movements were suppressed at higher trap stiffness implies a stochastic nature in the creation of the gap between the peripheral cell membrane and the actin network underlying it.     

Saturated fatty acids suppress adrenocorticotropic hormone (ACTH) release from rat anterior pituitary cells in vitro

We studied whether fatty acids modify adrenocorticotropic hormone (ACTH) release induced by stimulation with corticotropin-releasing hormone (CRH) from rat anterior pituitary cells. Stimulation with CRH (0.01-100 nmol/l) significantly and concentration-dependently increased ACTH release, which was synergistically enhanced by the simultaneous stimulation with 1 nmol/l arginine-vasopressin. Addition of saturated fatty acids (butyrate, caprylate, laurate, palmitate and stearate) in a medium at 1 mmol/l, despite effects on the basal release, significantly reduced the ACTH release induced by CRH (1 nmol/l) stimulation. Caprylate suppressed ACTH release in a concentration-dependent manner. However, unsaturated C18 and C20 fatty acids (oleate, linolate, linolenate and arachidonate) at 1 mmol/l significantly increased the basal release, but none of them suppressed CRH (1 nmol/l)-induced ACTH release. In the presence of caprylate (1 mmol/l), CRH (1 nmol/l)-stimulated increase in cellular calcium ion concentration was diminished. From these results we conclude that saturated fatty acids have a suppressing effect on CRH-induced ACTH increase in primary cultured rat anterior pituitary cells.     

Isolation of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> from Open Ocean and Comparison with Freshwater,Clinical, and Animal Isolates

Pseudomonas aeruginosa is an opportunistic pathogen responsible for morbidity and mortality in humans, animals, and plants. This bacterium has been regarded to be widely present in terrestrial and freshwater environments, but not in open ocean environments. Our purpose was to clarify its presence in open ocean, and their genotypic and physiological characteristics were compared with those of isolates from clinical, animal, and freshwater sources. Water samples were collected from freshwater, bays, and offshore environments in Japan. Sixty-two isolates, including 26 from the open ocean, were identified as P. aeruginosa by phenotypic characteristics and the BD Phoenix System. Pulsed-field gel electrophoresis (PFGE) was performed on all strains, together with 21 clinical and 8 animal strains. The results showed that open ocean strains are composed of a few genotypes, which are separated from other strains. Although some clinical isolates made a cluster, other strains tended to mix together. Different antibiotypes were observed among marine isolates that had similar PFGE and serotyping patterns. Some were multidrug-resistant. Laboratory-based microcosm study were carried out to see the responses of P. aeruginosa toward increased NaCl concentrations in deionized water (DW). Marine strains showed better survival with the increase, whereas river and clinical strains were suppressed by the increase. These findings illustrate the potential significance of open ocean as a possible reservoir of P. aeruginosa, and there may be clones unique to this environment. To our knowledge, this is the first report on the presence and characterization of P. aeruginosa in the open ocean.     

Effects of micro-environmental factors on photosynthetic CO2 uptake and carbon fixation metabolism in a spring ephemeral, Erythronium japonicum, growing in native and open habitats

Microenvironmental factors and physiological parameters (such as water potential, activity of ribulose 1,5-bisphosphate carboxylase (RuBPcase), levels of ribulose 1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA) and sucrose in leaves) affecting photosynthetic processes of the typical vernal species Erythronium japonicum Decne. were examined on the floor of a deciduous broad-leaved Quercus mongolica forest (Q.m. stand) and on bare land left undisturbed for 7 years after forest clearing (bare stand). Daytime solar radiation and the air and leaf temperatures at the bare stand were significantly higher than those at the Q.m. stand. The relative air humidity was very low and did not differ much between the stands, whereas the leaf–air vapor pressure difference (VPD) at the bare stand was twice as high as that at the Q.m. stand. The water potential in leaves at the bare stand was lower than two times that at the Q.m. stand. Therefore, the aboveground parts of the plants at the bare stand were subjected to much more severe heat stress than those at the Q.m. stand. When these environmental factors observed at the bare stand were reproduced in an assimilation chamber, the rate of photosynthetic CO₂ uptake, stomatal conductance and water potential in leaves were significantly low in comparison with those when the factors at the Q.m. stand were simulated. The internal CO₂ partial pressure in leaves at the bare stand was considerably lower than that at the Q.m. stand. Consequently, the decrease in the photosynthetic rate of the plants at the bare stand was caused mainly by a decrease in stomatal conductance through a lowering of water potential due to subjection of the aboveground parts to much more severe heat stress than that at the Q.m. stand. The possibility that an inhibition of the photosynthetic carbon fixation metabolism induced by the decrease in water potential contributes to the reduction in photosynthetic CO₂ uptake in the plants at the bare stand is also discussed in light of physiological characteristics such as the activity of RuBPcase and levels of PGA, RuBP and sucrose in the leaves.     

Role of bottom sediments in sustaining plankton production in a Lake ecosystem — Experimental demonstration using enclosed water bodies in a shallow eutrophic Lake

Using large enclosures set in Lake Suwa, experimental studies were conducted to examine the effects of bottom sediments on plankton production in a natural lake ecosystem. Successive changes in biotic and abiotic components in two types of enclosure with and without bottom sediments were surveyed throughout a one-month period in the early fall of 1985. Remarkable differences were found between the two types of enclosure with regard to the time courses of abundance of chlorophyll, PON, PP, TN, TP and zooplankton as well as those of POC/PON and POC/Chl ratios and primary production rate in terms of mg N m⁻² day⁻¹. Quantitative examination of the major causes of these differences revealed that the release of nitrogen and phosphorus from the bottom sediments was significantly effective for sustaining the active production of phytoplankton cells with a high nitrogen and phosphorus content, subsequently resulting in active zooplankton growth.