A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death

Programmed cell death (PCD) in animals depends on caspase protease activity. Plants also exhibit PCD, for example as a response to pathogens, although a plant caspase remains elusive. Here we show that vacuolar processing enzyme (VPE) is a protease essential for a virus-induced hypersensitive response that involves PCD. VPE deficiency prevented virus-induced hypersensitive cell death in tobacco plants. VPE is structurally unrelated to caspases, although VPE has a caspase-1 activity. Thus, plants have evolved a regulated cellular suicide strategy that, unlike PCD of animals, is mediated by VPE and the cellular vacuole.     

Mutagenesis, heterogeneous gene expression, and purification and amino acid substitution analyses of plant peroxidase, PrxA3a

We introduced mutations into prxA3a, a peroxidase gene of hybrid aspen, Populus kitakamiensis, to substitute the amino acid residues at the surface of the protein, and analyzed substrate specificities. PrxA3a and mutated enzymes heterogeneously gene expressed in Saccharomyces cerevisiae were purified by Ni affinity chromatography, hydrolysis of sugar chain (Endoglycosidase H_f) and gel filtration. The substrate specificities were altered by substituted amino acid residues. PrxA3a F77Y A165W acquired the substrate specificity to m-chlorophenol. PrxA3a F77Y and PrxA3a F77YA165W could polymerize sinapyl alcohol. In addition, PrxA3a A165W, F77Y, and F77YA165W improved cytochrome c oxidizing activity. These substituted amino acid residues should function as a catalytic site outside of the heme pocket.     

Growth and yield of self-compatible and hybrid common buckwheat lines pollinated with and without flies

Common buckwheat is a self-incompatible, insect-pollinated allogamous plant. This study examined growth and yield of the common buckwheat self-compatible and hybrid lines pollinated with and without flies. Self-compatible ‘IH3’, hybrid ‘IP2/IH3’, and standard self-incompatible ‘Kitawasesoba’ were used in field and pot experiments. Self-compatibility of ‘IH3’ was shown to be of high purity. Approximately 10% segregation of pin plants from ‘IP2/IH3’ was observed. The harvest index of ‘Kitawasesoba’ pollinated without flies was considerably lower than that pollinated with flies in both field and pot experiments. The harvest index values of ‘IH3’ and ‘IP2/IH3’ were hardly affected by the presence or absence of flies. The morphological traits of ‘IH3’ were significantly lower than those of the other two genotypes. Large differences in these traits between ‘IP2/IH3’ and ‘Kitawasesoba’ were not observed in either the field or pot experiments. The seed yield of ‘Kitawasesoba’ pollinated without flies tended to be lower than that pollinated with flies. The seed yield of ‘IP2/IH3’ tended to be higher than that of ‘IH3’. The hybrid line ‘IP2/IH3’ showed a high fertilization rate, which was nearly as high as that of ‘IH3’. Rate of fertilization and percentage of ripe seeds were higher in ‘IH3’ and ‘IP2/IH3’, wherein they were hardly affected by the presence or absence of flies, than in ‘Kitawasesoba’. The ‘IP2/IH3’ hybrid line will be useful for understanding the stable high-yielding ability of self-compatibility common buckwheat.     

Effects of salivary gland extract from Rhipicephalus sanguineus on IgG subclass production and cytokine mRNA expression in mononuclear cells of canine peripheral blood

The effects of salivary gland extract (SGE) from R. sanguineus were examined on the production of IgG1 and IgG2 and the mRNA expression of IFN-gamma, IL-2, IL-4, IL-5 and IL-10 in the mononuclear cells from canine peripheral blood, treated with concanavalin A (ConA) in vitro. SGE suppressed the ConA-induced production of IgG2. It also inhibited the expression of IFN-gamma, IL-2 and IL-5 mRNA in a dose-dependent manner. No dose-dependent suppression was observed of IL-10 mRNA expression although a significant effect was observed at a SGE protein concentration of 25 microg/ml. SGE had no effect on the mRNA expression of IL-4. These results suggest that the suppression of IgG2 production by SGE from R. sanguineus was caused by the suppression of IFN-gamma production.     

Nonulcerative keratouveitis as a manifestation of Leptospiral infection in a horse

A 2-year-old Thoroughbred filly presented with ocular pain and epiphora of the left eye. The pupil was miotic and the cornea edematous near the ventro-temporal limbus, but did not retain any fluorescein. The topical antibiotics and atropine and diclofenac, and systemic flunixin meglumine and antibiotic therapy did not resolve the condition. A pink and fleshy infiltrate developed near the limbus indicating nonulcerative keratouveitis. The anterior uveitis deteriorated as manifested by the presence of dyscoria, hypopyon, and organized fibrin in the anterior chamber. Ocular signs were improved by topical and subconjunctival corticosteroids, but repeatedly deteriorated as the frequency of medication was reduced. The horse was seropositive to three serovars of Leptospira interrogans. The animal was diagnosed as blind on day 91 by the absence of pupillary light and menace reflexes, and donated for histopathologic diagnosis. The corneal opacity was histologically fibrotic and infiltrated predominantly by lymphocytes with Descemet's membrane partially disrupted by macrophages. The choroid was infiltrated by lymphocytes, eosinophils and basophils, and was positive to IgG and C3. There were filamentous or spiral structures positive to Warthin-Starry stain in the renal cortex. There was also polymerase chain reaction amplification of the leptospiral gene in the kidney. From these findings nonulcerative keratouveitis was believed to be caused by systemic infection with Leptospira.     

Isolation of myxoviruses from migratory waterfowls in San-in district,western Japan in winters of 1997-2000

Between November 1997 and February 2000, winter migratory waterfowls of several species staying in San-in district, western Japan were surveyed for influenza A virus and paramyxovirus at four stations. A total of 18 influenza A viruses was isolated from 1,404 fecal samples of whistling swans, pintails, mallards, and white-fronted geese. Five different hemagglutinins and eight neuraminidases were identified in the viruses isolated, in 11 different combinations, including H7N8 related to a subtype of a highly pathogenic chicken virus. In 2000, five lentogenic (non-pathogenic) Newcastle disease viruses were also isolated from white-fronted geese. These results suggested that possible precursor viruses for highly pathogenic avian myxoviruses are still brought into Japan by migratory waterfowls. The results also support the contention that continued surveillance of wild waterfowl population should be an integral part of control policies for these serious poultry diseases.     

Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

Genotypic and phenotypic characterization of lactic acid bacteria isolated from Italian ryegrass silage

Twenty-three lactic acid bacteria (LAB) isolated from three cultivars (Akiaoba, Nagahahikari and Tachiwase) of Italian ryegrass (Lolium multiflorum Lam.) silage were precisely characterized by a combination of phenotypic tests, genotypic 16S ribosomal DNA sequencing and rapid PCR-based analyses, focusing on their useful phenotypes for silage preparation as inoculants. We successfully identified both at the species and subspecies levels: phenotypically novel Lactococcus lactis subsp. lactis, Lactobacillus brevis, Lactobacillus coryniformis subsp. torquens, Lactobacillus curvatus, Lactobacillus plantarum subsp. plantarum, Lactobacillus sakei subsp. carnosus, Leuconostoc mesenteroides subsp. dextranicum and Pediococcus parvulus. This is the first report to elucidate the presence of Lactobacillus coryniformis ssp. torquens and Leuconostoc mesenteroides subsp. dextranicum in Italian ryegrass silages. Physiological and biochemical tests revealed that phenotypic characteristics are different among the different strains of the same species and subspecies, and that the isolates show unique and diverse phenotypes related to fermentation factors, such as available carbohydrates, optimal growth pH and temperature. These results suggest that, for various well-preserved silage preparations, the isolates may be useful in producing novel inoculants corresponding to their optimally climatic and ecological niches.