Improved method for in situ biolistic transformation to analyze barley–powdery mildew interactions

Although the use of stable transformants is indispensable to elucidate mechanisms underlying molecular plant–pathogen interactions, this approach remains difficult to apply to crops. Alternatively, biolistic transformation has often been used as a transient expression method in various plants. In this study, we developed a method for in situ biolistic transformation without separating leaves from barley seedlings by using a hand-held particle bombardment system because unwounded leaves are preferable for analyzing interactions between barley and Blumeria graminis f. sp. hordei which requires healthy living cells. As a result, we found that the infection rate in intact leaves was higher than in separated leaves and that the transformation efficiencies in leaves were higher when plants were grown in vermiculite rather than in culture soil. Furthermore, we determined the appropriate inoculation time after bombardment to analyze the incompatible interaction and successfully monitored the gradual occurrence of cell death over time. Our system was suitable for relatively long-term follow-up analysis of the fate of each single cell during plant–pathogen interactions.     

Three‐dimensional quantitative structure–activity relationship analysis of acyclic and cyclic chloronicotinyl insecticides

The binding activity of chloronicotinyl insecticides, including acetamiprid, nitenpyram and related compounds, to the nicotinic acetylcholine receptors (nAChR) of houseflies was measured. These compounds were defined as ‘acyclic’ compounds. Variations in the binding activity were analysed using comparative molecular field analysis (CoMFA) which is a technique for the analysis of three‐dimensional quantitative structure–activity relationships. The CoMFA results showed that steric interactions were more significant for the acyclic compounds than for imidacloprid and its derivatives (cyclic compounds). It was also shown that the acyclic compounds could bind to housefly‐nAChR in a similar manner to the cyclic compounds, and that the electrostatic natures of the acyclic amino‐ and cyclic imdazolidine‐moieties affected their binding activity. © 2000 Society of Chemical Industry     

Microinsemination with first-wave round spermatids from immature male mice

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.     

Requirement of phospholipase Cdelta4 for the zona pellucida-induced acrosome reaction

Several phospholipase C (PLC) isoforms have been found in male and female mammalian gametes, and splicing isoforms of PLCdelta4 are predominantly expressed in testis. Here we report that male mice in which the PLCdelta4 gene had been disrupted either produced few small litters or were sterile. In vitro fertilization studies showed that insemination with PLCdelta4-/- sperm resulted in significantly fewer eggs becoming activated and that the calcium transients associated with fertilization were absent or delayed. PLCdelta4-/- sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida. These data demonstrate that PLCdelta4 functions in the acrosome reaction that is induced by the zona pellucida during mammalian fertilization.     

Pathological features of arrhythmogenic right ventricular cardiomyopathy in middle-aged dogs

The hearts of four dogs (a 4-year-old Shetland sheepdog, a 4-year-old Labrador retriever, a 5-year-old English Bulldog, and a 6-year-old Dalmatian; three males and one female), that had died suddenly and had been clinically diagnosed as having arrhythmogenic right ventricular cardiomyopathy (ARVC), were studied post mortem. At the cut surface, all four hearts showed mild to moderate hypertrophy of the left and right ventricular free walls and ventricular septum, with grayish-white tissue replacement of the myocardium to various degrees. Histologically, all had typical right ventricular features of ARVC and morphological evidence of left ventricular and ventricular septal involvement. Two main histological patterns were identified: a fatty type (two cases) and a fibrofatty type (two cases). With either type, myocardial replacement by fatty or fibrofatty tissue were detected in both ventricles, but were more severe in the right ventricle, where they usually became transmural. Furthermore, this myocardial replacement was more severely seen in the epimyocardium and midmyocardium; the endomyocardium was less severely affected. On the basis of the present observation, it is evident that, in dogs, the disease process of ARVC affects both the right and left ventricles, although the striking pathological feature is right ventricular involvement. The pathological evidence of biventricular involvement in these canine cases of ARVC may represent a wider spectrum of the disease than has previously been recognized, suggesting that, in dogs, this disease should no longer be considered as limited to the right ventricle.     

Epidemiological and morphological studies of double-chambered right ventricle in dogs

The double-chambered right ventricle (DCRV) is a rare congenital cardiac disease in dogs, and its detailed epidemiological and morphological features are not clearly understood. By investigating the profile, clinical signs, and characteristics of examination findings of eleven dogs with DCRV by means of a retrospective study, we attempted to clarify the epidemiology and morphology of the condition. The study group consisted of nine males and two females. Breeds included Pug (n=3), Miniature Dachshund (n=1), French Bull-dog (n=1), Shiba (n=1), and Retrievers (n=5). The attachment site of the anomalous muscular bundle was continuous with the cardiac apex in nine dogs, and it was attached to the right ventricle free wall in the other two dogs. In dogs with DCRV, at least one of the following conditions was present concurrently: congenital or acquired tricuspid valve regurgitation (TR), ventricular septal defect, and atrial septal defect. Also, the pressure difference between the two chambers increased over time, and progressive right-sided heart failure was observed. In summary, DCRV occurs in small breeds of dog as well as in large breeds of dog and it may be more prevalent in males. The existence of two types of DCRV in dogs was established. Dog with DCRVs will have a high incidence of concurrent cardiac abnormalities. Concurrent TR may be either congenital or acquired. DCRV is a congenital disorder, but the clinical condition progresses as the dog develops.