Isolation and characterization of tri‐ and tetra‐repeat microsatellite loci in the white‐spotted charr Salvelinus leucomaenis (Salmonidae)

Tri‐ and tetra‐motif repeat microsatellite marker loci were developed for the white‐spotted charr Salvelinus leucomaenis. The 454 pyrosequencing was used to discover repeat arrays, and eight microsatellite‐primer sets, available for the estimation of polymorphisms, were identified. The number of alleles in a wild population ranged from two to four and the observed and expected heterozygosities were 0·180–0·600 and 0·188–0·599, respectively.     

A Nitrate Reductase Inactivator Protein from Spinach. Purification, Molecular Weight and Subunit Composition

A nitrate reductase-inactivator protein has been purified 16,000-foldfrom spinach leaves by pH 5 treatment, chromatography on SE53,Con A-Sepharose, and chromatofocusing. The yield was 12%, thespecific activity was 115 units mg^–1. Polyacrylamide gelelectrophoresis of the final purified inactivator fraction yielded2 major protein bands and both bands exhibited nitrate reductase-inactivatoractivity. Analysis of this inactivator protein by gel filtrationand SDS-gel electrophoresis revealed protein stainable materialonly in a molecular weight range of 110,000–115,000. SDSgel electrophoresis under reducing conditions yielded 2 proteinbands corresponding to molecular weights of 51,000 and 53,000.The proteolytic mapping for the two separated subunits appearedsimilar and possibly identical. (Received October 28, 1991; Accepted February 24, 1992)     

Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.     

Application of high-performance liquid chromatography with a chemiluminescence detection system to determine catecholamines in urine

Chemiluminescence generated with the reaction of bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide was applied to a detection system for high-performance liquid chromatography to determine fluorescamine-labeled catecholamines. The sensitivity of the chemiluminescence detection system with 25 fmol of detection limit was approximately 20 times higher than that of a conventional fluorescence detection system. Norepinephrine and dopamine in human urine were determined by the use of the new high-performance liquid chromatography detection system with the coefficient of variation of less than 4.0%. Good correlations (r = 0.998 for norepinephrine and r = 0.999 for dopamine) were obtained between the values by the present method and the conventional method.     

Location,size and age at onset of metamorphosis in the Japanese eel Anguilla japonica

This study clarifies the location, size and age at the onset of metamorphosis in Japanese eels Anguilla japonica through oceanic surveys, rearing experiments and analyses of the morphology and otoliths of leptocephali and glass eels. Twenty‐eight metamorphosing leptocephali were collected in the mesoscale eddy region to the east of Taiwan during research expeditions in 2004. Rearing experiments showed that the total length (L_T) of leptocephali decreased by an average of 12·5% during metamorphosis and 13·9% during the 2–12 h after death. Thus, the mean back‐calculated L_T at the onset of metamorphosis for 630 glass eels from Taiwan and Japan was estimated at 67·8 ± 2·7 mm (mean ± S.D.). The estimated mean ante‐mortem size of the fully grown pre‐metamorphic leptocephali collected in 2004 was 64·6 ± 3·4 mm, which was consistent with the L_T estimate for glass eels. Otolith analysis showed that the mean age at the onset of metamorphosis was 137 ± 15 days and indicated that Japanese eels may have a recruitment route through the mesoscale eddies to the east of Taiwan in addition to the direct transfer route from the North Equatorial Current to the Kuroshio Current.     

Light, nutrients and primary productivity in Lake Biwa: An evaluation of the current ecosystem situation

Simple correlation and multiple regression analyses were performed to examine the relationship between primary productivity and environmental factors in the north basin of Lake Biwa. The primary production rates used in the analyses were estimated monthly or bimonthly during the growing season (April–November) in 1992, 1996 and 1997 with the ¹³C method. Elemental (C, N and P) contents of seston were used to assess nutrient conditions. Analyses revealed that 86% of variance in depth-integrated primary production rates (areal PP) can be explained by changes in light intensity, and sestonic C, N and P concentrations. Water temperature had no effect on areal PP. To assess relative effects of light and nutrients on PP, the P:B ratio was estimated by normalizing PP with sestonic C. The areal P:B ratio correlated most significantly with the sestonic N:P ratio, followed by light intensity. When regression analyses were made at each depth, however, the P:B ratio correlated significantly only with the sestonic N:P ratio at 0 and 1 m depths, while light intensity was also incorporated into the regressions at deeper than 2.5 m. In these regressions, the P:B ratio was negatively correlated with sestonic N:P ratio but positively with light intensity. The results suggest that the primary production rate in this lake was mainly limited by P relative to N supply rates, but was not free from light limitation in a large part of the epilimnion. In Lake Biwa, the vertical water mixing regime as well as the nutrient supply seem to be important in determining the growth and composition of primary producers, since the surface mixing layer extends into 10–15 m depths during most of the growing season.     

Differential stereoselective pharmacokinetics of pantoprazole,a proton pump inhibitor in extensive and poor metabolizers of pantoprazole—a preliminary study

Pantoprazole (PAN) is a proton pump inhibitor that is administered as a racemic mixture. The pharmacokinetics of PAN enantiomers were investigated in extensive metabolizers (EMs) and apparent poor metabolizers (PMs) of PAN who received a single 40, 60, or 80 mg oral dose of racemic PAN as enteric-coated formulation. In the EMs, the serum concentrations of (−)-PAN were slightly higher than those of (+)-PAN at each dose level. The (+)/(−) ratios for the area under the concentration-time curve (AUC) and the half-life were 0.58–0.89 and 0.62–0.88, respectively. In the PMs, the serum concentrations of both enantiomers were much higher than those in the EMs at each dose level and significant differences in pharmacokinetics of (+)- and (−)-PAN were observed. The half-lives for (+)-PAN were 2.67–3.77 times longer than those for (−)-PAN. The AUCs for (+)-PAN were 2.65–3.45 times greater than those for (−)-PAN. Therefore, the metabolism of (+)-PAN is impaired to a greater extent than (−)-PAN in the PMs, which resulted in the stereoselective disposition of PAN in the PMs. It has been suggested that the EMs and the PMs of PAN could be differentiated by determining the (+)/(−) enantiomer ratio in serum at one time point, possibly 2–6 h after oral dosing, because the (+)/(−) enantiomer ratios in the PMs were opposite those in the EM subjects. Chirality 9:17–21, 1997 © 1997 Wiley-Liss, Inc.     

Genomic Evidence for Speciation with Gene Flow in Broadcast Spawning Marine Invertebrates

How early stages of speciation in free-spawning marine invertebrates proceed is poorly understood. The Western Pacific abalones, Haliotis discus, H. madaka, and H. gigantea, occur in sympatry with shared breeding season and are capable of producing viable F₁ hybrids in spite of being ecologically differentiated. Population genomic analyses revealed that although the three species are genetically distinct, there is evidence for historical and ongoing gene flow among these species. Evidence from demographic modeling suggests that reproductive isolation among the three species started to build in allopatry and has proceeded with gene flow, possibly driven by ecological selection. We identified 27 differentiation islands between the closely related H. discus and H. madaka characterized by high F_ST and d_A, but not high d_XY values, as well as high genetic diversity in one H. madaka population. These genomic signatures suggest differentiation driven by recent ecological divergent selection in presence of gene flow outside of the genomic islands of differentiation. The differentiation islands showed low polymorphism in H. gigantea, and both high F_ST, d_XY, and d_A values between H. discus and H. gigantea, as well as between H. madaka and H. gigantea. Collectively, the Western Pacific abalones appear to occupy the early stages speciation continuum, and the differentiation islands associated with ecological divergence among the abalones do not appear to have acted as barrier loci to gene flow in the younger divergences but appear to do so in older divergences.     

Imaging activity of neuronal populations with new long-wavelength voltage-sensitive dyes

We have assessed the utility of five new long-wavelength fluorescent voltage-sensitive dyes (VSD) for imaging the activity of populations of neurons in mouse brain slices. Although all the five were capable of detecting activity resulting from activation of the Schaffer collateral-CA1 pyramidal cell synapse, they differed significantly in their properties, most notably in the signal-to-noise ratio of the changes in dye fluorescence associated with neuronal activity. Two of these dyes, Di-2-ANBDQPQ and Di-1-APEFEQPQ, should prove particularly useful for imaging activity in brain tissue and for combining VSD imaging with the control of neuronal activity via light-activated proteins such as channelrhodopsin-2 and halorhodopsin.     

Dissolved organic carbon and fluorescence in Lake Hovsgol: factors reducing humic content of the lake water

 Lake Hovsgol is a large tectonic lake located in northern Mongolia, which has extremely transparent lake water. In our survey, the dissolved organic carbon of the lake water was 80–100 μM-C, and the fluorescence intensity in an excitation and emission matrix was very low. The brown color and high content of humic substances in river water flowing from a watershed consisting of grassland and forests rapidly declined in the coastal area of the lake. The decrease in humic content may be due not only to dilution by the lake water but also to flocculation and photobleaching. Among tectonic lakes in Asia, Lake Hovsgol would appear to have unique biological and hydrological features that reduce humic content and help to maintain water transparency. Received: June 25, 2002 / Accepted: January 10, 2003 Acknowledgments The authors acknowledge helpful discussion with Dr. J. Urabe. We thank Dr. T. Galbaatar, Mongolian Academy of Science, Mongolia, for his arrangements on the expeditions in 1999. We are also indebted to Mr. D. Hadbaatar, B. Ganbat, and the cruise staff of the R/V Suchbaatar for their assistance in the course of the study. This study was supported by Grant-in-Aid No. 09041159 and 13575034 for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Correspondence to:K. Hayakawa