Notes on beech (Fagus crenata Blume) seed and seedling mortality due to rodent herbivory in a northernmost beech forest, Utasai, Hokkaido

We investigated the mortality of post-dispersal seeds and current-year seedlings of a beech species (Fagus crenata Blume) at the northernmost extent of its geographical range at Utasai (Kuromatsunai), Hokkaido, Japan. Introduced seeds that were protected from rodent predation by wire cages had a higher survival rate from winter to the following spring (mean ± SD: 84 ± 16.7%,n = 10) than controls without cages (mean ± SD: 12 ± 17.9%,n = 10). Unprotected transplanted current-year seedlings with almost fully opened primary leaves never survived more than three days (0%,n = 30), while more than 80% (n = 30) of seedlings within cages survived to the end of the growing season. These results indicate that rodent herbivory is a major mortality factor that strongly prevents the establishment of beech seedlings. In a natural population, we observed that 90% of seedlings (n = 197) disappeared within 10 days after their emergence and rodent herbivory caused this heavy mortality. A herbivorous rodent, the gray-backed vole,Clethrionomys rufocanus bedfordiae, was remarkably abundant in late June (101.5 voles/ha), suggesting that this species strongly affects the formation of the seedling bank. The presence ofC. rufocanus bedfordiae may be one of the reasons for the scarcity of beech seedlings and saplings and the rarity of recruitment in this northernmost beech forest.     

Epizootology and experimental infection of Yokose virus in bats

To reveal whether bats serve as an amplifying host for Yokose virus (YOKV), we conducted a serological survey and experimentally infected fruit bats with YOKV isolated from microbats in Japan. YOKV belongs to the Entebbe bat virus group of vector unknown group within the genus Flavivirus and family Flaviviridae. To detect antibodies against YOKV, we developed an enzyme-linked immunosorbent assay (ELISA) using biotinylated anti-bat IgG rabbit sera. Serological surveillance was conducted with samples collected in the Philippines and the sera supplied from Malaysia. One of the 36 samples from the Philippines (2.7%) and 5 of the 26 samples from Malaysia (19%) had detectable ELISA antibodies. In the experimental infections, no clinical signs of disease were observed. Moreover, no significant viral genome amplification was detected. These findings revealed that YOKV replicates poorly in the fruit bat, suggesting that fruit bats do not seem to serve as an amplifying host for YOKV.     

Histopathological, immunohistochemical and ultrastructural studies of a renal mesenchymal tumor in a young beagle dog

A 15-month-old male beagle dog used in a toxicity study had a primary renal mesenchymal tumor. Macroscopically, the tumor was a gray-white mass which was found in the right kidney, and extended from the capsule to a position slightly compressing the medulla. Microscopically, most of the tumor cells showed a myxoid pattern, in which the matrix was positive for alcian blue staining. In the other parts of the tumor, a fascicular and wavy pattern was observed, and the matrix was full of collagen fibrils. Immunohistochemically, tumor cells were positive for vimentin and fibronectin, and negative for cytokeratin, desmin, α-smooth muscle actin, Von Willebrand factor, cyclooxigenase-2 and myelin basic protein. As a result, we diagnosed this case to be a renal mesenchymal tumor. Based on the microscopic findings, interstitial characteristics and immunohistochemical features, the present case was classified as a congenital mesoblastic tumor.     

DNA profiling of pineapple cultivars in Japan discriminated by SSR markers

We developed 18 polymorphic simple sequence repeat (SSR) markers in pineapple (Ananas comosus) by using genomic libraries enriched for GA and CA motifs. The markers were used to genotype 31 pineapple accessions, including seven cultivars and 11 breeding lines from Okinawa Prefecture, 12 foreign accessions and one from a related species. These SSR loci were highly polymorphic: the 31 accessions contained three to seven alleles per locus, with an average of 4.1. The values of expected heterozygosity ranged from 0.09 to 0.76, with an average of 0.52. All 31 accessions could be successfully differentiated by the 18 SSR markers, with the exception of ‘N67-10’ and ‘Hawaiian Smooth Cayenne’. A single combination of three markers TsuAC004, TsuAC010 and TsuAC041, was enough to distinguish all accessions with one exception. A phenogram based on the SSR genotypes did not show any distinct groups, but it suggested that pineapples bred in Japan are genetically diversed. We reconfirmed the parentage of 14 pineapple accessions by comparing the SSR alleles at 17 SSR loci in each accession and its reported parents. The obtained information will contribute substantially to protecting plant breeders’ rights.     

Changes in acute-phase proteins and cytokines in serum and milk whey from dairy cows with naturally occurring peracute mastitis caused by Klebsiella pneumoniae and the relationship to clinical outcome

This study was conducted to evaluate the changes in acute-phase proteins and cytokine concentrations in dairy cows with naturally occurring peracute Klebsiella pneumoniae (K. pneumoniae) mastitis and their association with the outcome of the disease. Seventeen Holstein cows with K. pneumoniae mastitis from 8 dairy farms were divided on the basis of outcome after local and systemic therapy into 2 groups comprising 8 euthanized cows and 9 that recovered. Changes in acute-phase proteins and cytokine concentrations in cows with K. pneumoniae mastitis were evaluated at the onset of the disease (day 0) and at days 3, 7 and 14 after therapy and compared with those of 13 healthy dairy cows. The concentrations of haptoglobin (Hp) and interleukin (IL)-6 in serum and α(1)-acid glycoprotein and IL-1β in serum and whey on day 0 were significantly (P<0.05) higher in the euthanized cows than in those that recovered and the healthy cows. A correlation (r=0.90, P<0.01, n=17) was found between IL-6 and Hp concentrations in sera from recovered and euthanized cows at day 0. This indicated that serum concentrations of Hp and IL-6 at the initial examination were prognostic factors for survival, and the cutoff values were 2,020 μg/ml and 32 ng/ml, respectively. These results suggest that IL-6 and Hp concentrations are involved in the manifestation of K. pneumoniae mastitis and may be possible indicators of the prognosis of peracute K. pneumoniae mastitis.     

Size-exclusion chromatography of tea tannins and intercepting potentials of peptides for the inhibition of trypsin-caseinolytic activity by tea tannins

Molecular-weight distribution and characterization of tea tannin were investigated by high-performance liquid chromatography and the equivalent preparative exclusion gel chromatography using Sephadex G-25. The characteristics of the fractions were studied regarding the amounts of terminal catechin, sugar, and gallic acid, the color reaction of the Folin-Chiocalteu reagent, the UV absorbance, and the inhibition activity for the trypsin-caseinolytic activity per weight. Furthermore, we investigated the intercepting activities of the inhibition by the amino acids, peptides, their analogues, poly(ethylene glycol)s (PEGs), and histatin 5 using the inhibition of trypsin-caseinolytic activity by tea. Arg, Lys, and their peptides had strong intercepting activities for the inhibition, but only a weak activity was detected in the Pro peptides or gelatin-like peptides of (Pro-Pro-Gly)(n) (n = 5 or 10). The guanidyl group of Arg and the amino methylene group of Lys were important for the intercepting activity, but the activity was weakly dependent upon the peptide bond formation. The intercepting activity of the peptides or PEG exponentially increased with the number of polymerizations. Histatin 5 did not have a remarkably strong intercepting activity considering the peptide length. The activity of the synthetic histatin 5 in which all of the Lys and Arg were substituted by Ala was at the same level as histatin 5.     

High prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 from cattle in selected regions of Japan

The prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 was examined in bovine faeces. EHEC O157 was isolated from the faeces of 42 (13.0%) of 324 cattle. Of the 4 farms and the facilities tested, the 3 farms and the facilities were found positive for EHEC O157. The highest isolation rate among the farms was 33.7%. The prevalence of EHEC O157 in heifers was higher than that in calves and other cattle. No cattle positive for EHEC O157 showed any clinical signs except 2 calves with diarrhea in a veterinary hospital. Almost all isolates possessed the stx gene, and Stx-positive strains carrying both stx(1) and stx(2) genes were predominant. These results indicate that EHEC O157 are distributed in bovine faeces, and that dairy and beef farms in selected regions of Japan are heavily contaminated with the organisms.     

Stimulatory effect of cold acclimation on skeletal muscle branched-chain α-keto acid dehydrogenase in rats

The effects of cold-acclimation (3 weeks at 4°C) on the actual activities of branched-chain α-keto acid dehydrogenase (BCKDH) and pyruvate dehydrogenase (PDH) complexes in the skeletal muscle and liver of normal 6-week-old rats were studied. The activities of BCKDH and PDH complexes were assayed using [1–¹⁴C] α-ketoisocaproate and [1-¹⁴C]pyruvate, respectively. Cold-acclimation markedly stimulated the activity of BCKDH, and slightly but significantly increased the activities of PDH and citrate synthase (CS) in the skeletal muscle, but did not alter PDH, BCKDH or CS activities in the liver. The increase in muscle BCKDH activity (control rats, 1.45 ± 0.54 mU/g wet wt; cold-acclimated rats, 2.54 ± 0.50 mU/g wet wt; 75% increase: P  = 0.001) was much greater than the increases in PDH activity (84 ± 16 mU/g wet wt and 111 ± 25 mU/g wet wt; 32% increase: P  = 0.020) or CS activities (27 ± 3 μmol/min per g wet wt and 31 ± 2 μmol/min per g wet wt; 14% increase: P  = 0.046). These results suggest that, unlike PDH, BCKDH is not directly associated with the mitochondrial oxidative activity of the skeletal muscle of cold-acclimated rats. This study provides the first evidence that BCKDH in skeletal muscle responds selectively to cold-acclimation.     

Enzymatic high digestion of soybean milk residue (okara)

The objective of this study was to digest okara in high yield by food-processing enzymes. Autoclaving of okara was effective in increasing cellulase digestion for the primary cell wall, and the digestion was accelerated by the formation of single cells by stirring. Most of the residual okara after autoclaving and cellulase digestion was found to be the secondary cell walls compared with the cellulase-treated soybean single cells. The secondary cell wall was found to be composed of galacturonic acid, neutral sugars, and protein and was considered to be a complex of these compositions. Many cellulolytic and proteolytic enzymes could not digest the secondary cell wall; however, it was found that two pectinases could digest the secondary cell wall. A series of digestions resulted in yields of 83-85% from the raw okara, and the final residues were identified as oil body complexes in the soybean cells and fiber-like organ between the cells.     

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.