Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens

ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.     

A Comparison of the Histological Structure of the Placenta in Experimental Animals

The primary function of the placenta is to act as an interface between the dam and fetus. The anatomic structure of the chorioallantoic placenta in eutherian mammals varies between different animal species. The placental types in eutherian mammals are classified from various standpoints based on the gross shape, the histological structure of the materno-fetal interface, the type of materno-fetal interdigitation, etc. Particularly, the histological structure is generally considered one of the most useful and instructive classifications for functionally describing placental type. In this system, three main types are recognized according to the cell layers comprising the interhemal area: (1) epitheliochorial type (horses, pigs and ruminants), (2) endotheliochorial type (carnivores) and (3) hemochorial type (primates, rodents and rabbits). The number of cell layers in the interhemal area is considered to modify the transfer of nutrients between maternal and fetal blood and is one of the important factors with respect to the difference in placental permeability between animal species. Therefore, in reproductive and developmental toxicity studies, careful attention should be paid to the histological structure of the interhemal area when extrapolating information concerning placental transfer characteristics to different animal species.     

Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel

ABSTRACT:   In order to find out the role of sodium bicarbonate (NaHCO₃) on the initiation of sperm motility in the Japanese eel Anguilla japonica , interactions were investigated between NaHCO₃ and various reagents (K⁺ channel blocker 4-aminopyridine [4-AP], ammonium chloride [NH₄Cl], sodium acetate and calcium chloride [CaCl₂]) that could regulate internal factors (intracellular K⁺, intracellular pH [[pH]_i] and intracellular Ca²⁺) in sperm motility. Contradictory effects of NaHCO₃ were observed (i.e. an inhibitory effect when 4-AP was absent and a promoting effect when 4-AP was present). Sodium bicarbonate inhibited the initiation of sperm motility in the Japanese eel. However, NaHCO₃ restored the motility of immotile sperm that 4-AP inhibited. The inhibitory effect of NaHCO₃ disappeared with the addition of NH₄Cl, which raised [pH]_i, but the promoting effect was not affected by [pH]_i. Although NaHCO₃ recovered motility in the presence of 4-AP, this recovery was also observed with the addition of CaCl₂ instead of NaHCO₃. In the initiation of sperm motility in the Japanese eel, two roles for NaHCO₃ are suggested: an inhibitory role relating to the regulation of [pH]_i and a promoting role relating to the uptake of another initiation factor, which could be Ca²⁺.     

In vitro development of bovine nuclear transfer embryos reconstructed with mammary gland epithelial cells at different passages

In the present study, the effect of passage of nuclear donor cells on the in vitro development of nuclear transfer (NT) embryos was investigated using colostrum‐derived mammary gland epithelial (MGE) cells at different passages (3–30 passages) to find reliable passages for the efficiency of cloning. Development of NT embryos to the blastocyst stage was affected by the number of passages of MGE cells (P < 0.05). Nuclear transfer embryos reconstructed with MGE cells at 3–7 passages showed a significantly higher blastocyst development (31.3–48.5%) than those with the cells at 10–30 passages (2.5–12.5%, P < 0.05). No difference in the proportion of the MGE cells with normal diploid was observed among passage of 3, 15 and 30 (P > 0.05). The use of MGE cells at early passages for nuclear donor cells may be advantageous for the production of NT embryos.     

Control of rabies: epidemiology of rabies in Asia and development of new-generation vaccines for rabies

Rabies is an enzootic viral disease widespread throughout the world. Although it is a vaccine-preventable disease, the annual number of human deaths caused by rabies is estimated to be 32,000 in Asia. Phylogenetic analysis based on sequence data of the partial N gene of rabies viruses in Asia has shown that the viruses are divided into five genogroups, distributed in Middle East, South Asia, South East Asia, Malay, and Arctic regions. The genetic relationships among these rabies viruses agree basically with the results of previous studies. Meanwhile, new types of vaccines are being developed by applying gene manipulation techniques to rabies virus in order to overcome the disadvantages of current vaccines. This article reviews the molecular epidemiology of rabies in Asia and progress made in the development of new-generation rabies vaccines with the goal of elimination or control of rabies in Asia.     

Uniquely stable 40 kDa subfragment-2 in carp myosin

Digestion of carp myofibrils at 30 degrees C in 0.5 M KCl medium with calcium ion generated unique 135 kDa heavy meromyosin (HMM). The HMM was not produced when digested at 10 degrees C. A further digestion of the 135 kD HMM isolated in the absence of calcium ion generated uniquely short subfragment-2 (S-2) with a size of 40 kDa (40 kDa S-2) together with subfragment-1 (S-1). The 40 kDa S-2 was identified by N- and C-end sequencing, and demonstrated to locate the amino end of the rod portion. The unfolding temperature for the 40 kDa S-2 was around 52 degrees C as studied by circular dichroism measurement. The same unfolding peak was also detected with the intact rod together with a large unfolding peak at around 36 degrees C coming from the rest of the rod portion, light meromyosin. The unfolding peak for the 40 kDa S-2 in myosin was a little lower (48 degrees C) than that in free form, suggesting the involvement of the head portion in the stability of the 40 kDa S-2 in the structure.     

A comparison of regression methods for estimating soil-atmosphere diffusion gas fluxes by a closed-chamber technique

Continuous changes in methane (CH₄) and carbon dioxide (CO₂) concentrations inside a closed chamber were measured on the forest floor at three sites: a deciduous forest and a coniferous forest in Hokkaido, Japan, and a birch forest in West Siberia, Russian Federation. Flux estimations by three types of regression methods, exponential, nonlinear, and linear, were examined using field-collected concentration data. The pattern of change with time of the gas concentration in the headspace differed, mainly according to site but also, to a lesser extent, according to the gas. This was a function of both the chamber height and surface soil property relating to soil gas diffusion and the gas concentration profile. Flux estimations did not differ statistically between the exponential and nonlinear methods for either gas at any site, because both of those regression methods were based on diffusion theory. However, the flux values estimated by linear regression were significantly different from those estimated by the other two methods for both CH₄ and CO₂ at the deciduous forest site and for CO₂ at the coniferous forest site. Shortening the chamber deployment period improved the linearity of the curve, but did not completely eliminate the error. Our results suggest that linear regression is not a good model of the change in headspace concentration with time.