Sequence of the S 9-RNase cDNA and PCR-RFLP system for discriminating S 1- to S 9-allele in Japanese pear

A new S ₉-allele was discovered in 6 Japanese pear cultivars, ‘Shinkou’, ‘Shinsei’, ‘Niitaka’, ‘Amanogawa’, ‘Nangetsu’ and ‘Nansui’. cDNA encoding S ₉-RNase, a stylar product of S ₉-allele, was cloned from pistils of ‘Shinkou’ and ‘Shinsei’ by 3' and 5' RACE. The S ₉-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S ₉-RNase had a hypervariable (HV) region different from S ₁- to S ₈-RNase and shared higher similarity (95.2%) with apple S ₃-RNase than with 8 Japanese pear S-RNases (from 61.0% to 70.7%). Genomic PCR with primers ‘FTQQYQ’ and ‘anti-(I/T) IWPNV’ provided S ₁- to S ₉-amplicon (product), but could not discriminate the S ₂ from the S ₉ of ca. 1.3 kb. The S ₂ and S ₉ were distinguished by digestion with AflII and BstBI, respectively. The digestion with nine S-allele-specific restriction endonucleases, SfcI, AflII, PpuMI, NdeI,AlwNI, HincII, AccII, NruI and BstBI, distinguished S ₁ to S ₉, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S ₁- to S ₉-allele. ‘Shinkou’, ‘Shinsei’, ‘Nangetsu’ and ‘Nansui’ assigned as S ₄ S ₉ were determined to be cross incompatible. This revised version was published online in July 2006 with corrections to the Cover Date.     

alpha-Klotho as a regulator of calcium homeostasis

alpha-klotho was identified as a gene associated with premature aging-like phenotypes characterized by short lifespan. In mice, we found the molecular association of alpha-Klotho (alpha-Kl) and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and provide evidence for an increase of abundance of Na+,K+-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca2+]e) rapidly induce regulated parathyroid hormone (PTH) secretion in an alpha-Kl- and Na+,K+-ATPase-dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca2+ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of alpha-Kl in the regulation of calcium metabolism.     

KISS1 Gene Expression in the Developing Brain of Female Pigs in Pre- and Peripubertal Periods

Puberty is associated with an increase in gonadotropin secretion as a result of an increase in gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is considered to play a key role in puberty onset in many mammalian species, including rodents, ruminants and primates. The present study aimed to determine if changes in hypothalamic expression of the KISS1 gene, encoding kisspeptin, are associated with the onset of puberty in pigs. The animals (n=4 in each group) were perfused with 4% paraformaldehyde at 0, 1, 2, 3 and 4 months old, as prepubertal stages, and at 5 months old, as the peripubertal stage, following each blood sampling. KISS1 gene expressions in coronal sections of brains were visualized by in situ hybridization. Plasma luteinizing hormone (LH) was measured by radioimmunoassay. KISS1 mRNA signals were observed in the arcuate nucleus (ARC) at all ages examined without any significant difference in the number of KISS1-expressing cells, indicating that the KISS1 gene is constantly expressed in the ARC throughout pubertal development in pigs. The plasma LH concentration was the highest in 0-month-old piglets and significantly decreased in the 1- and 2 month-old groups (P<0.05), suggesting a developing negative feedback mechanism affecting gonadotropin release during the prepubertal period. Considering the potent stimulating effect of kisspeptin on gonadotropin release in prepubertal pigs, kisspeptin secretion rather than kisspeptin synthesis may be responsible for the onset of puberty in pigs.     

Thermally induced gelation of paramyosin from scallop adductor muscle

ABSTRACT:   Thermally induced gelation of paramyosin from scallop smooth adductor muscle was investigated by dynamic rheological measurements under various conditions. The paramyosin thermal gel was produced at pH 6.5 and 7.2 at temperatures above 30°C through a two-step increase in storage (G') and loss (G') moduli; these values were higher than in gels produced from actomyosin at a high temperature. The thermal gel properties were very firm and brittle. In contrast, one main peak of G' was observed during gelation at pH 8.0. The gel produced at pH 8.0 was more transparent and less soluble in a 6 M urea−0.5 M NaCl solution than those formed either at pH 6.5 or 7.2. These differences in the thermal gel properties are presumed to derive from the pH dependence of the gel matrix-forming process, such as oxidative cross-linking between cysteine residues, rather than from the thermal unfolding of the paramyosin molecules. The thermal gelation profile of chymotrypsin-digested paramyosin showed marked depression of G' at high temperature.     

Thermal stress responses of a sterile mutant of Ulva pertusa (Chlorophyta)

SUMMARY: The thermal stress responses of a sterile mutant of the marine alga Ulva pertusa were investigated at 20°C and 30°C. The amounts of the photosynthetic pigments, β-carotene, chlorophylls a and b , lutein, neoxanthin, and violaxanthin, were 1.4–2.4 times higher in the 30°C-cultivated alga than in the 20°C-cultivated alga. The free amino acids, asparagine, aspartic acid, glutamine, glutamic acid, glycine, and serine, were abundant in the 20°C-cultivated alga, and increased 1.9–10.5-fold in response to thermal stress (30°C). Total carbon and nitrogen contents also increased in the 30°C-cultivated alga. Sodium dodecylsulfate-polyacrylamide gel electrophoretic patterns of total proteins extracted from both temperature-treated algae showed the increases of 20, 25, and 90 kDa proteins in the 30°C-cultivated alga. Isozyme assays for 20 enzymes showed a different banding pattern only in the case of glutamate dehydrogenase (GDH). Although it was observed that both temperature-treated algae possessed NAD⁺- and NADP⁺-specific GDH, the 30°C-cultivated alga had an additional NADP⁺-specific GDH (NADP-GDH). These results suggest that NADP-GDH compensates for the thermally induced decreases in nitrogen assimilation efficiency and thereby regulates nitrogen metabolism under conditions of temperature stress.     

Hypoxic conditions induce centrum defects in red sea bream Pagrus major (Temminck and Schlegel)

A previous study elucidated that an extreme hypoxia during somitogenesis induced the most frequent skeletal malformation centrum defects in red sea bream (RSB), Pagrus major. In this study, details of the hypoxic conditions to induce them in RSB, dissolved oxygen (DO) concentration and exposure time to hypoxia, were investigated. Fertilized eggs were exposed to seawater of six DO concentrations (0%, 10%, 25%, 50%, 75% and 100% of saturation) for seven different periods (5, 10, 30, 60, 120, 240 and 360 min) during somitogenesis. Somitic disturbances in newly hatched larvae were induced by exposure to 0% and 10% DO concentration for 10 and 120 min and longer respectively. Rearing eggs exposed to hypoxic condition of 10% DO for 240 min for 40 days post‐hatch showed that the location and the frequency of somitic disturbances in larvae and centrum defects in juveniles were significantly correlated (P<0.01). Dissolved oxygen concentration of the interstitial water in the egg high density layer formed at the water surface in a stationary state abruptly decreased to 3.7% within 7 min. Centrum defect induction by exposure of eggs to extreme low DO concentrations for a short period, which is the probable situation in the practical juvenile production, suggests that careful maintenance of DO concentration is important in the incubating water of fertilized eggs during egg sorting and transportation, where eggs are made into a pile and undergo hypoxia, for the prevention of centrum defects.     

Seasonal changes and controlling factors of gross N transformation in an evergreen plantation forest in central Japan

We conducted a year-round measurement of gross N transformation rates using the ¹⁵N dilution method, and analyzed seasonal changes and the mechanisms regulating gross N transformation in the Kiryu Experimental Forest in central Japan. While soil microbial biomass C (SMB-C) decreased from the dormant to growing seasons at the organic (O) horizon, no significant trend was observed in SMB-N. This resulted in SMB-C/N being high in the dormant season and low in the growing season, and suggests that the microbial composition changed seasonally. No clear seasonal trend was found in gross NH₄ ⁺ production rates at either the O or surface mineral soil horizons. In contrast, the NH₄ ⁺ consumption rate varied seasonally, with high values in January and April during the dormant season and low values in July and October during the growing season. There was no clear trend in seasonal fluctuation of net NH₄ ⁺ production rates. Gross NH₄ ⁺ production and gross NH₄ ⁺ consumption rates were 10 times greater than the gross nitrification rate. Almost all of the produced NH₄ ⁺ was immobilized, indicating that N tightly cycles at this study site. Considered together with results of the gross N transformation rates, the dominance of high SMB-C/N microbes might stimulate immobilization in the dormant season. At this study site, the change in microbial composition likely influences gross N transformation through immobilization efficiency.     

Effects of NO3- availability on NO3- use in seedlings of three woody shrub species

We determined the effects of short-term cultivation with various amounts of available nitrate nitrogen (NO3-) on NO3- use by woody shrub species. Nitrate concentration ([NO3-]) and nitrate reductase activity (NRA) were measured in leaves and roots of seedlings of Hydrangea hirta (Thunb.) Siebold, Lindera triloba (Sieb. et Zucc.) Blume and Pieris japonica (Thunb.) D. Don. Root [NO3-] increased with increasing NO3- supply in all species, whereas leaf [NO3-] remained low. There were significant correlations between [NO3-] in roots and leaves in all species, but no correlation was found between root NRA and leaf NRA. The low proportion of leaf NO3- assimilation to total NO3- assimilation in all species can be ascribed to the lack of NO3- transport from roots to leaves. In all species, root NRA increased with increasing NO3- supply until reaching a plateau. Species ranking based on maximum root NRA was H. hirta > L. triloba > P. japonica. Root NRA in P. japonica was low, even though root [NO3-] increased with NO3- supply, indicating that NO3- was not an effective N source for this species. The ranking also suggested that H. hirta depended more on NO3- as an N source than L. triloba. The increase in root NRA with increasing NO3- supply was greater in H. hirta than in L. triloba, possibly indicating that a change in NO3- availability has a stronger influence on NO3- use in H. hirta than in L. triloba.     

Dissolved inorganic nitrogen cycling in Banzu intertidal sand-flat,Japan

The sediment-water exchange of nitrogen, nitrification, denitrification and sedimentary oxygen production were simultaneously measured in the Banzu intertidal sand-flat, Tokyo Bay, Japan. The exchange flux across the sediment-water interface showed a sink of dissolved inorganic nitrogen (DIN) in the light. However, in the dark, the intertidal sediment acted as a source. The diffusive flux obtained by the ammonium profile explained only 22% of the source flux directly measured in the dark, suggesting that bioturbation or excretion by macrofauna greatly contributed to the exchange flux. The total microphytobenthic uptake of DIN estimated from O2 productivity and the Redfield ratio was 573.4 ± 64.4 mol N m2 h-1, 31% (175.9 ± 64.4 mol N m-2 h-1) of which was assumed to be derived from the overlying water. The release rate of DIN from the sediment to the water column (1.1 mmol N m-2 day-1) was balanced with the removal rate of DIN from the water column by benthic microalgae on a diurnal basis. This result suggests that DIN was recycled within the sediment, and the microalgae on the sediment surface played a significant role in suppressing the release of mineralized DIN from the sediment. The measured denitrification rate using an acetylene inhibition technique was 99.6 ± 23.5 mol N m-2 h-1. Since the direct supply of nitrate or nitrite from the water column only accounted for 27% of the total denitrification at the highest estimate, nitrogenous oxide in the sediment pool was the major contributor to sedimentary denitrification.