Development of a high-throughput screening method using a cell-based, lawn format assay for the identification of novel plant defense activators from combinatorial peptide libraries

Plants respond to attack by pathogens through various defense mechanisms. These defense responses are triggered by a variety of molecules derived from pathogenic microorganisms as well as host plants. In this study, we developed a high-throughput screening method using a cell-based lawn format assay for the identification of novel peptides that can induce plant defense responses from combinatorial peptide libraries. Solid-phase peptide libraries were synthesized using a photocleavable linker and immobilized using agarose gel. The peptides were partially cleaved from beads, and the agarose gel was layered on the tobacco cells. The defense response was then observed by detecting the generated H2O2 using a sensitive H2O2 indicator dye, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium salt (DA-64). Using this assay format, a 6859-member peptide library based on the sequence of flagellin-derived peptides was screened, and several structural features important for the activity were obtained.     

Tyrosinase inhibitory activity of citrus essential oils

Thirteen kinds of citrus essential oils and their volatile flavor constituents were investigated for tyrosinase inhibitory activity. Eureka lemon, Lisbon lemon, Keraji, and Kiyookadaidai significantly inhibited the oxidation of L-dihydroxy phenylalanine (L-DOPA) by mushroom tyrosinase. Citral and myrcene among volatile flavor constituents of citrus essential oils exhibited tyrosinase inhibitory activities with Ki values of 0.318 and 2.38 mM, respectively. The inhibition kinetics analyzed by a Lineweaver-Burk plot indicated that citral is a noncompetitive inhibitor and myrcene is a competitive inhibitor. These results indicated that citral and myrcene are responsible for the tyrosinase inhibitory activity of citrus essential oils.     

Evidence of long distance airborne transport of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae to be transported over long distances via the airborne route was evaluated. A source population of 300 grow-finish pigs was experimentally inoculated with PRRSV MN-184 and M. hyopneumoniae 232 and over a 50-day period, air samples were collected at designated distances from the source herd using a liquid cyclonic collector. Samples were tested for the presence of PRRSV RNA and M. hyopneumoniae DNA by PCR and if positive, further characterized. Of the 306 samples collected, 4 (1.3%) were positive for PRRSV RNA and 6 (1.9%) were positive for M. hyopneumoniae DNA. The PRRSV-positive samples were recovered 4.7 km to the northwest (NW) of the source population. Four of the M. hyopneumoniae-positive samples were obtained at the NW sampling point; 2 samples at approximately 2.3 km and the other 2 samples approximately 4.7 km from the source population. Of the remaining 2 samples, one sample was obtained at the southeast sampling point and the other at the southwest sampling point, with both locations being approximately 4.7 km from the source. The four PRRSV-positive samples contained infectious virus and were ≥ 98.8% homologous to the MN-184 isolate used to inoculate the source population. All 6 of the M. hyopneumoniae-positive samples were 99.9% homologous to M. hyopneumoniae 232. These results support the hypothesis that long distance airborne transport of these important swine pathogens can occur.     

Relative contributions from exposed inshore and estuarine nursery grounds to the recruitment of stone flounder, Platichthys bicoloratus, estimated using otolith Sr:Ca ratios

In Sendai Bay, stone flounder larvae settle and spend their juvenile period in either shallow exposed inshore nursery grounds or estuarine nursery grounds. The purpose of this study is to examine the relative contributions of these two kinds of nursery grounds to the flounder population using otolith strontium:calcium ratios. Stone flounder juveniles were collected from both nursery grounds, and one- and two-year-old flounder were caught deeper in Sendai Bay. Sr and Ca content in the otoliths were measured by electron probe micro analysis. The Sr:Ca ratios in the otolith section corresponding to the early postsettlement period ranged from 3.06 to 3.85 for the exposed inshore areas with stable low temperature and high salinity conditions, and from 3.81 to 5.32 in brackish estuaries with high temperature and low salinity conditions but with large diel and tidal cyclical fluctuations. Values from an estuarine site with stable salinity ranged from 3.58 to 4.15 overlapping with both the above ranges. Rearing experiments supported our inference that the high otolith Sr:Ca ratios of juveniles inhabiting estuarine nursery grounds are attributable to higher temperature and physiological stress caused by the large diel temperature and salinity fluctuations within the estuaries. Estimation of the Sr:Ca ratio of recruited fish using the otolith section formed while in the nursery area showed that at least 20 out of 42 individuals examined originated from estuarine nursery grounds. The present study indicates that estuaries play an important role as nursery grounds for stone flounder, producing about half of the stock in spite of the small and restricted area compared with the wide expanse of the exposed inshore area.     

Migration history of Sakhalin taimen <Emphasis Type="Italic">Hucho perryi</Emphasis> captured in the Sea of Okhotsk,northern Japan,using otolith Sr:Ca ratios

Sakhalin taimen Hucho perryi populations have decreased drastically to near extinction. It is urgent to establish an effective conservation strategy based on an understanding of the characteristics of migration and habitat use of this species. We examined the migration history of anadromous Sakhalin taimen captured off the Sarufutsu coast, northern Hokkaido, Japan, using otolith Sr:Ca ratios and also examined the relationship between their otolith Sr:Ca ratios during freshwater and seawater residence in a rearing experiment. Otolith Sr:Ca ratios of some fish from the Sarufutsu coast showed freshwater levels (0.5–4.0 × 10⁻³) near the core, which thereafter increased to brackish water levels (4.0–6.0 × 10⁻³), and then to seawater levels (6.0–10.0 × 10⁻³) in the outermost regions. Those findings indicate that specimens from the Sarufutsu coast migrated to the brackish water region or the sea and spent most of their lives there. The anadromous migration pattern including the timing of downstream migration seems to be flexible among individuals in the species. They migrate between freshwater and seawater or brackish water several times during their lives, showing extensive habitat use. It is essential to secure the continuity among the freshwater, brackish water, and seawater areas for their effective conservation.     

Potential use of otolith microchemistry for field studies of temperature-dependent sex determination and gonadal degeneration in fish

This paper describes a novel approach to study the expression of temperature-related reproductive phenomena such as thermolabile sex determination (TSD) and heat-induced germ cell loss in wild fish populations. The proposed approach is based on the reconstruction of the past thermal history of individual fish through the microchemical analysis of the otoliths (mineralized structures responsible for the sense of balance). As an example, we outline the preliminary results of an investigation on the natural occurrence of TSD in pejerrey Odontesthes bonariensis, an atherinid fish in which all-female to all-male populations can be produced by experimental manipulation of the rearing temperature during the critical time of sex differentiation, from Lake Kasumigaura, Japan.     

Effective use of a reservoir for paddy irrigation in tropical monsoon Asia—a case study of the Ngamoeyeik Project,Lower Myanmar

Irrigation projects with reservoirs have contributed to the stabilization of traditional rainfed rice and to the introduction of double-rice in monsoon Asia. This type of irrigation also has the capability of shifting the traditional rice-cropping season. This paper discusses the influences of the shifted cropping season on water resources and the effective use of the reservoir in the Ngamoeyeik Irrigation Project Area in Lower Myanmar. Synthesized streamflow data were applied to simulate water storage in the reservoir. The main results are as follows: (1) shifting the cropping season to avoid inundation problems can increase water availability, (2) the amount of evaporation loss in the reservoir water budget can be as high as 16%, which decreases water efficiency in the reservoir operation during the dry season, (3) a shifted cropping season is preferable under the special hydrological conditions found in Lower Myanmar.     

Mechanical transmission of porcine reproductive and respiratory syndrome virus throughout a coordinated sequence of events during cold weather

Using a field-based model, mechanical transmission of porcine reproductive and respiratory syndrome virus (PRRSV) was assessed throughout a coordinated sequence of events that replicated common farm worker behavior during cold weather (< 0°C). The model involved fomites (boots and containers), vehicle sanitation, transport, and the movement of personnel. A field strain of PRRSV was inoculated into carriers consisting of snow and water, and carriers were adhered to the undercarriage of a vehicle. The vehicle was driven approximately 50 km to a commercial truck washing facility where the driver's boots contacted the carriers during washing, introducing the virus to the vehicle interior. The vehicle was then driven 50 km to a simulated farm site, and the driver's boots mechanically spread virus into the farm anteroom. Types of containers frequently employed in swine farms (styrofoam semen cooler, metal toolbox, plastic lunch pail, and cardboard animal health product shipping parcel) contacted drippings from footwear on the anteroom floor. The truck wash floor, vehicle cab floor mats, boot soles, anteroom floor, and the ventral surface of containers were sampled to track the virus throughout the model. Ten replicates were conducted, along with sham-inoculated controls. At multiple sampling points PRRSV nucleic acid was detected in 8 of 10 replicates. In each of the 8 PCR-positive replicates, infectious PRRSV was detected on the surfaces of containers by virus isolation or swine bioassay. All sham-inoculated controls were negative. These results indicate that mechanical transmission of PRRSV can occur during coordinated sequence of events in cold weather.