Characterization of Alpha-fetoprotein Levels in Three Dolphin Species: Development of Sensitive Immunoassays for Analysis of the Pregnancy-associated Variations

A single radial immunodiffusion (SRID) assay and a chemiluminescent immunoassay (CLIA) were initially developed for alpha-fetoprotein (AFP) of the striped dolphin. Utilizing these developed assays, we investigated pregnancy-associated changes in the levels of AFP in the sera of fetuses and pregnant females of three dolphin species; samples were either collected from captive individuals or obtained as fishery by-products. The concentrations of AFP in the fetal serum ranged from 419.0 to 2026.3 μg/ml in the striped dolphin, 12.6 to 1218.7 μg/ml (for an AFP equivalent; eqAFP) in the common bottlenose dolphin and 770.6 to 3129.1 μg eqAFP/ml in the Risso''s dolphin. AFP levels decreased with increased fetal size in fetuses over 20 cm in length. The concentrations of AFP in sera of pregnant females ranged from 7.18 to 8068.7 ng/ml in the striped dolphin, 6.6 to 1241.1 ng eqAFP/ml in the common bottlenose dolphin and 3.4 to 2868.7 ng eqAFP/ml in the Risso''s dolphin. The levels in most pregnant females were equal to or lower than those found in males and nonpregnant individuals, although a few pregnant females exhibited extremely high levels (in the range of hundreds to thousands of nanograms per milliliter). Such high levels of AFP were not observed during pseudopregnancy. To our knowledge, this is the first report on basal profiles for serum AFP levels in small odontocetes. The profiles indicated that AFP may play a significant role during embryonic development, although maternal levels do not appear to be a diagnostic biomarker for monitoring pregnancy.     

Survey of mutations of a histidine kinase gene BcOS1 in dicarboximide-resistant field isolates of Botrytis cinerea

Previously, we cloned a putative osmosensing histidine kinase gene (BcOS1) and revealed that a single amino acid substitution, isoleucine to serine at codon 365, conferred dicarboximide resistance in field isolates of Botrytis cinerea. This point mutation (type I) occurred within the restriction enzyme TaqI site of the wild-type BcOS1 gene. Thus, a procedure was developed for detecting the type I mutation of the BcOS1 gene using a polymerase chain reaction (PCR) in combination with restriction fragment-length polymorphism (RFLP). Diagnosis by PCR-RFLP was conducted on the 105 isolates isolated from 26 fields in Japan. All dicarboximide-sensitive isolates (49 isolates) had the wild-type BcOS1 gene, and the 43 isolates with the type I mutation were resistant to dicarboximides without exception. These data indicate that dicarboximide-resistant isolates with type I mutation are widespread throughout Japan. However, other types of dicarboximide resistance were detected among isolates from Osaka; among the 24 resistant isolates from Osaka, 12 had the BcOS1 gene without the type I mutation. BcOS1 gene sequencing of these resistant isolates classified them into two groups, type II and type III. The type II isolates have three amino acid substitutions within BcOS1p (³⁶⁸Val to Phe, ³⁶⁹Gln to His, and ⁴⁴⁷Thr to Ser). The type III isolates have two amino acid substitutions within BcOS1p (³⁶⁹Gln to Pro and ³⁷³Asn to Ser). These amino acid changes are located on the amino acid repeat domain in BcOS1p. The three types of resistant isolates were all moderately resistant to dicarboximides without significant osmotic sensitivity, and their pathogenicity on cucumber leaves was also very similar to that of the wild-type isolate.     

Investigation on the hydrogen abstraction from methyl glucoside by active oxygen species under oxygen delignification conditions IV: appearance of kinetic isotope effect in the reaction between methyl glucoside and deuterated methyl glucoside

In our previous studies, a clear kinetic isotope effect was observed when a pair of carbohydrate model compounds, methyl ??-d-glucopyranoside (MGP??) and a deuterated MGP?? labeled at the anomeric (methyl ??-d-(1-²H)glucopyranoside) or C-2 position (methyl ??-d-(2-²H)glucopyranoside), was reacted with active oxygen species (AOS) generated by reactions between O₂ and a phenolic compound, 2,4,6-trimethylphenol (TMPh), under oxygen delignification conditions. These results indicate that the AOS abstract the anomeric and C-2 hydrogens of MGP??. Contrarily, no clear kinetic isotope effects were observed when AOS were generated by reactions between O₂ and another phenolic compound, 4-hydroxy-3-methoxybenzyl alcohol (vanillyl alcohol, Valc), and hence, the abstraction of the anomeric and C-2 hydrogens by the AOS was not confirmed. In this study, a pair of MGP?? and the deuterated MGP?? labeled at all the positions, (²H₃)methyl ??-d-(1,2,3,4,5,6,6-²H₇)glucopyranoside, was reacted with AOS generated from TMPh or Valc under oxygen delignification conditions to further examine the appearance of a kinetic isotope effect. A clear kinetic isotope effect was observed when either of TMPh or Valc was the origin of AOS, indicating that some AOS abstract at least a hydrogen of MGP?? in either case. The results are further discussed focusing on the type of AOS generated from TMPh and Valc.     

Site-level model–data synthesis of terrestrial carbon fluxes in the CarboEastAsia eddy-covariance observation network: toward future modeling efforts

Based on the model–data comparison at the eddy-covariance observation sites from CarboEastAsia datasets, we report the current status of the terrestrial carbon cycle modeling in monsoon Asia. In order to assess the modeling performance and discuss future requirements for both modeling and observation efforts in Asia, we ran eight terrestrial biosphere models at 24 sites from 1901 to 2010. By analyzing the modeled carbon fluxes against the CarboEastAsia datasets, the strengths and weaknesses of terrestrial biosphere modeling over Asia were evaluated. In terms of pattern and magnitude, the carbon fluxes (i.e., gross primary productivity, ecosystem respiration, and net ecosystem exchange) at the temperate and boreal forest sites were simulated best, whereas the simulation results from the tropical forest, cropland, and disturbed sites were poor. The multi-model ensemble mean values showed lower root mean square errors and higher correlations, suggesting that composition of multiple terrestrial biosphere models would be preferable for terrestrial carbon budget assessments in Asia. These results indicate that the current model-based estimation of terrestrial carbon budget has large uncertainties, and future research should further refine the models to permit re-evaluation of the terrestrial carbon budget.     

Use of batch leaching tests to quantify arsenic release from excavated urban soils with relatively low levels of arsenic

Purpose

In Japan, the excavated soils produced from constructions projects, which contain relatively low levels of arsenic (As), are considered as a potential concern if they could release significant amount of As to the environment. The aim of this study was to investigate the As leaching from excavated alkaline soils and, in particular, the influence of drying methods, pH of extracting solution, and consecutive washing on As leaching from these soils.

Materials and methods

Four excavated alkaline soils obtained from different construction sites in Tokyo, Japan, were used in this study. The soils were pretreated by three drying methods (air-dried, 40 °C-dried, and freeze-dried). Sequential extraction procedure was applied to partition As into five operationally defined chemical fractions. Batch leaching tests (initial pH-controlled leaching test and consecutive washing test) were conducted to investigate the As release under different leaching conditions.

Results and discussion

The As contents in the four soils were 9.22, 79.4, 6.75, and 11.7 mg kg^?1, respectively, and As was primarily associated with the residual phase. Arsenic leaching was strongly dependent on the extracting solution pH values. Strongly acidic extracting solution (pH 2) led to circumneutral leachates and limited As mobility, whereas the strong alkaline-extracting solution (pH 12) greatly enhanced the As release from these soils. The consecutive washing test results revealed a long-term release of As from these excavated soils. The pollution potential indices (PPIs) were successfully used to evaluate the pollution threat of As leaching from excavated soils. In addition, different drying methods resulted in variations in the short- and long-term release of As from these excavated soils.

Conclusions

The results revealed that the soil pretreatment and the leaching conditions should be considered if we want to use batch tests for the contamination assessment of excavated urban soils from construction projects. Different drying methods and single extraction may lead to misestimation of the As pollution level. High extraction efficiency with strong alkaline-extracting solution (pH 12) reveals that it could potentially be used to wash As from excavated alkaline soils.

     

Development and application of West Nile virus subgenomic replicon RNA expressing secreted alkaline phosphatase

We have developed a West Nile virus (WNV) subgenomic replicon harboring the secreted alkaline phosphatase (SEAP) reporter gene instead of viral structural genes (designated repWNV/SEAP). The repWNV/SEAP allowed easy evaluation of viral replication efficiency by direct measurement of SEAP secretion in the cell culture medium in physical containment level 2 facilities. Furthermore, we validated the availability of this system using a known anti-flavivirus gene, mouse oligoadenylate synthetase 1b (Oas1b). The Oas1b-transfected cells were more resistant to repWNV/SEAP replication than the original cells. Thus, this system not only affords a useful tool for identification/evaluation of anti-flavivirus genes/drugs in terms of safety, ease of use and reliability, but should be able to reduce or replace the bioassay using laboratory animals.     

Effect of methotrexate on cerebellar development in infant rats

Six-day-old rats were treated intraperitoneal injections with methotrexate 1 mg/kg, and the cerebellum was examined. Both the length and width of the vermis decreased in the methotrexate-treated group instead of the control from 4 day after treatment (DAT) onward. A significant reduction in the width of the external granular layer was detected on 2 and 3 DAT in the methotrexate group. By 4 DAT, the width of the external granular layer of the methotrexate group was indistinguishable from the control, and by 8 DAT, it was greater than that of the control. The molecular layer of methotrexate group on 8 and 15 DAT was thinner than that of the control. On 1 DAT, in the methotrexate group, there were many TUNEL and cleaved caspase-3-positive granular cells throughout the external granular layer, and they decreased time-dependently. On 1 DAT, in the methotrexate group, phospho-histone H3-positive cells in the external granular layer were fewer than in the control and tended to increase on 2–4 DAT. The p21-positive-rate of the external granule cells in the MTX group was higher than in the control on 1–4 DAT. These results suggested that methotrexate exposure on postnatal day 6 induces a delay, slowing in the migration of external granular cells to the inner granular layer, attributed to decrease or inhibition in the production of external granular cells that had arisen from apoptosis and the decrease in cell proliferative activity, resulting in cerebellar hypoplasia.