Induction of reactive oxygen generation and functional changes in mitochondria and their involvement in the development of bacterial rot in lettuce caused by Pseudomonas cichorii

Pseudomonas cichorii is the major causal agent of bacterial rot disease in lettuce, and apoptosis-like programmed cell death is closely associated with disease development. Depletion of cellular ATP and expression of the alternative oxidase gene was observed in lettuce leaves inoculated with P. cichorii suggesting mitochondrial dysfunction. Cytochemical observation showed production of reactive oxygen species in mitochondria of P. cichorii-inoculated lettuce leaf cells. Release of cytochrome c into the cytosol was also induced by inoculation with the bacterium. Superoxide production was observed in the mitochondrial fraction isolated from P. cichorii-infected leaves much more intense than water-treated leaves. Loss of swelling ability was also observed in the mitochondrial fractions following inoculation with P. cichorii. Intriguingly, inhibitors of the mitochondrial respiratory complex III prevented loss of swelling ability, whereas superoxide generation was scarcely affected by inhibitors of mitochondrial permeability transition in the mitochondrial fractions. Inhibitors of the mitochondrial respiratory complex III and mitochondrial permeability transition delayed not only P. cichorii-induced cell death, but also disease development. In contrast, P. cichorii-induced DNA fragmentation was not inhibited in the presence of either type of inhibitor. The findings suggest that mitochondria may play a crucial role in DNA fragmentation-independent cell death pathway(s).     

Reducing effect of dietary anacardic acid on body fat pads in rats

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Toyomizu et al. 2000). In the present study, in order to clarify whether or not anacardic acid could be used advantageously as a special feed/food supplement to reduce fat deposition through the uncoupling action, two sets of experiments were conducted to determine quantitatively the effect of dietary anacardic acid (0.1% w/w) supplementation. More specifically, effects on growth, feed efficiency, fattening, and levels of several constituents of blood serum in rats fed normal and low protein–high carbohydrate (CHO) diets were examined. There were no significant differences in bodyweight gain, feed consumption and feed efficiency among the experimental groups. For the total fat pad content, including inguinal and epididymal fat, significant interaction was shown between both treatments: dietary anacardic acid at 0.1% w/w significantly decreased the total fat pad content in rats fed the CHO diet, but not in rats fed the normal diet. Weights of heart, spleen and brown adipose tissue were not affected by either the dietary treatment or anacardic acid, while both liver and kidney weights decreased with feeding of anacardic acid at 0.1% w/w, but were not affected by the CHO diet. Anacardic acid supplementation in the diet had no effect on serum glutamic oxaloacetic transaminase, alkaline phosphatase or lactate dehydrogenase levels, suggesting that the dysfunction of liver or kidney may not be induced by dietary anacardic acid. The results of the present study reveal a unique function of anacardic acid in that, for dietary conditions enhancing body fat deposition, that is consumption of a diet high in carbohydrates, dietary anacardic acid has the potential to decrease body fat deposition. A possible mechanism for differences observed in anacardic acid‐induced regulation of body fat pad content between rats fed the normal and CHO diets, based on uncoupling action of anacardic acid on the mitochondrial oxidative phosphorylation, is discussed.     

Isoenzyme polymorphism and segregation in isolates of Phytophthora infestans from Japan

Isoenzyme variation of 198 isolates of Phytophthora infestans collected from many locations in Japan during 1987–90, and of four pre-1987 isolates, was examined using starch gel electrophoresis. A previously unreported allele at malic isoenzyme locus/ME (90) was observed. An association between mating types and isoenzyme genotypes at three isoenzyme loci, glucosephosphate isomerase (GPI-1), peptidase (PEP-1) and ME, was found. At PEP-1, the A2 isolates from Japan had a previously unreported genotype (96/96). Normal segregation at the malic isoenzyme locus occurred in a cross of Japanese and Mexican parents. The results provide evidence of a change in the population genetic structure of P. infestans in Japan.     

Detection and sequence analysis of DNA polymerase and major envelope protein genes in koi herpesviruses derived from Cyprinus carpio in Gunma prefecture, Japan

Koi herpesvirus (KHV), which is believed to be an emerging virus, causes fatal diseases in carps. Since the 1990s, the presence of KHV has been confirmed in several countries. In Japan, from 2003 to 2004, large outbreaks of KHV infection in farmed carps resulted in the death of numerous fishes. From April to May 2004, we collected 43 dead or dying carps exhibiting typical symptoms of KHV infection in Gunma prefecture. To conduct a molecular epidemiologic study of KHV in our prefecture, we amplified DNA polymerase and the major envelope protein genes of KHV derived from carp gills using newly designed primers. We also performed sequence analysis of both genes of KHV. Sensitivity of our PCR method for amplification of DNA polymerase and the major envelope protein genes of KHV was 3 x 10(2) (100 fg) and 3 x 10(3) (1000 fg) copies of KHV genome, respectively. We detected both DNA polymerase and major envelope protein genes in 37 of 43 carps (86%). No mutation was found in both the genes sequenced from 11 strains, which included two foreign strains and one domestic strain. The results suggested that KHV strains derived from carps in our prefecture were closely related genetically to the other KHV strains.     

Genetic and antigenic analyses of bovine respiratory syncytial virus detected in Japan

Genetic and antigenic analyses of bovine respiratory syncytial virus were conducted on 12 field strains from Tohoku and Hokuriku districts in Japan during from 2002 to 2004. On the phylogenetic tree of the nucleotide sequences of the glycoprotein region, the examined strains fell in the same cluster as the strain isolated in Nebraska and were classified as the subgroup III. The examined strains were subdivided into 2 lineages (A, B). Isoleucine 200 of the epitope domain was replaced by threonine as a feature of the lineage B strains. The examined strains showed the nucleotide sequence homologies of 88.3-93.3% with the known Japanese strains classified as the subgroup II and of 86.1-96.6% with those in the subgroup III. No significant difference was found on the neutralization index between the examined strain and the 52-163-13 phylogenetically similar to the Japanese vaccine one. The results suggest that the subgroup III strains have existed in Japan and that epidemics of the strains could be protected due to the present vaccination.     

Changes of serum alkaline phosphatase activity in dry and lactational cows

Serum alkaline phosphatase (ALP) activities were measured during dry and lactational periods to investigate the influence of lactation on serum ALP activity in cows. Higher levels of serum ALP activity were seen in lactational periods than in dry periods. The serum activities of bone-specific ALP (BALP), liver ALP (LALP), tartrate resistant acid phosphatase (TRAP) and aspartate aminotransferase also increased in lactational periods. ALP activities in the bone extract and in whey were decreased at similar rates by the addition of lectin. Moreover, since the ALP band in whey was observed to have the same migration in polyacrylamide gel (PAG) disk electrophoresis as that of the bone extract, analysis of ALP isoenzymes by lectin affinity or PAG disk electrophoresis could not distinguish ALP originating from the mammary gland from that of bone. In this study, it was clear that the increased level of serum ALP activity was due to increases of BALP and LALP in lactational periods. However, the extent of the influence of ALP originating from the mammary glands on serum ALP activity was unknown. Judging from changes of BALP and TRAP activities in the serum and the correlation between the both, it was guessed that ALP originating from the mammary glands influenced serum ALP activity.     

Caprine enteritis associated with Yersinia pseudotuberculosis infection

Yersiniosis was prevalent among a caprine herd during the late autumn of 2003 in Iwate Prefecture, Japan. The disease affected 29 of about 100 lactating goats, but not dried or nonparous goats, mature male goats or kids. Four animals died within an epidemic period of 20 days. Affected animals developed decreased milk production with subsequent watery diarrhea, neutrophilia with increased band forms and multiple microabscesses characteristic of yersiniosis in the intestinal mucosa from the jejunum to caecum as well as in the mesenteric lymph nodes. Y. pseudotuberculosis serotype III was isolated from intestinal contents and mesenteric lymph nodes. The organism was also cultured from clinically normal dried animals. The outbreak might have been precipitated by multiple stress factors, such as lactation, cold weather, Corynebacterium pseudotuberculosis infection resulting in abscess formation and tapeworm and coccidium parasitisms.     

Attempt to produce nuclear transferred primordial germ cells using electrofusion in domestic chicken

As a step to develop a somatic nuclear transfer technique for avian species, an attempt to produce somatic nuclear transferred primordial germ cells (PGC) in the domestic chicken was carried out. Primordial germ cells and embryonic blood cells (EBC) were collected from 2‐day‐old embryos and the nuclei were transferred from EBC into PGC by electrofusion. The most efficient pearl chain was developed when a 350‐V/cm AC field was applied for 60 s. Cell fusion between PGC and EBC was most effective when 4‐kV/cm DC pulses, 60 µs pulse width, were applied three times to a cell suspension dispersed in 0.2 or 0.25 mol/L saccharose solution. The present results provide basic information for the production of somatic cell nuclear transferred chickens using PGC as the nuclear recipient.