Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

Apocrine adenocarcinoma in a golden hamster.

An apocrine adenocarcinoma was observed in the subcutis of the abdomen of golden hamster. Histologically, the tumor cells irregularly formed multiple layers of cysts and some detached cells were presented in the cystic space. PAS stain with alpha-amylase digestion revealed PAS-positive alpha-amylase-resistant granules in the cytoplasm. Immunohistochemically, cytokeratin was demonstrated in the tumor cells. By electron microscopy, the tumor cells had an oval nucleus with invagination, abundant cytoplasmic organelles and microvilli protruding into the intercellular spaces.     

Fluid accumulation in mouse ligated intestine inoculated with the vascular permeability factor produced by Bacillus cereus.

Partially purified vascular permeability (VP) factor (VPF) of Bacillus cereus induced fluid accumulation in the ligated intestinal loops of mouse (MIL) and rabbit (RIL), suggesting that the VP activity may correlate with fluid accumulation in ligated intestinal loops of these animals. Fluid accumulation was observed at 6-8 hr in 55-67% of mouse intestinal loops inoculated with 40-50 immunodiffusion units (IDU) of partially purified VPF, whereas it was found at 2 hr in all loops with 400-600 IDU of partially purified VPF. In rabbit intestinal loops with 120-190 IDU of partially purified VPF, fluid accumulation was observed at 6 hr. From these findings, VPF produced by B. cereus can be easily detected in both MIL and RIL. The intestinal tissue of mouse intestinal loops was histopathologically damaged at different concentrations of the VPF to induce fluid accumulation. With 50 IDU of partially purified VPF, severe edema was found in the laminia proprial layer and submucosa. With 600 IDU of partially purified VPF, on the other hand, severe necrosis in the surface epithelium of villus and laminia proprial layer, and hyperemia in the submucosa were observed, suggesting that partially purified VPF may be cytotoxic and/or intestinecrotic.     

Transgenic cucumber expressing an endogenous class III chitinase gene has reduced symptoms from Botrytis cinerea

A class III chitinase gene (CHI2) is induced in cucumber plants (Cucumis sativa L.) in response to infection by pathogenic microorganisms. Infection of Botrytis cinerea, causal agent of gray mold disease on cucumber, also induces CHI2 expression. To investigate whether CHI2 is involved in resistance to gray mold disease, transgenic cucumber plants were produced to overexpress the CHI2 gene. One line was analyzed in detail in terms of disease resistance. The transgenic cucumber plant (CC2) constitutively expressed CHI2 and reduced the symptoms of B. cinerea for 4 days after inoculation compared with nontransgenic plants. However, this inhibitory effect was not absolute, and CC2 eventually developed serious disease symptoms. Chitinase activity of the crude extract from CC2 leaves was higher than that from nontransgenic plants. A high-molecular-weight fraction containing CHI2 from CC2 leaves had fungistatic activity against B. cinerea. Interestingly, the low-molecular-weight fraction from CC2 leaves with CHI2 removed also had fungistatic activity against B. cinerea. Not only the introduced chitinase activity but also the endogenous defense reactions activated by overexpression of CHI2 may be involved in the enhanced gray mold disease resistance in CC2.     

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH‐Cytochrome B5 Reductase Gene

Background

In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.

Objectives

To analyze the NADH‐cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency.

Animals

Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls.

Methods

Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing.

Results

Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu).

Conclusions and Clinical Importance

This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH‐binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.     

Persimmon breeding in Japan for pollination-constant non-astringent (PCNA) type with marker-assisted selection

Oriental persimmon (Diospyros kaki) originated in Eastern Asia, and many indigenous cultivars have been developed in China, Japan, and Korea. These cultivars are classified into four groups based on their natural astringency loss on the tree and seed formation: pollination-constant non-astringent (PCNA), pollination-variant non-astringent (PVNA), pollination-constant astringent (PCA), and pollination-variant astringent (PVA). PCNA is the most desirable type because the fruit can be eaten without any postharvest treatment; therefore, one of the goals of our persimmon breeding programs is to release superior PCNA cultivars. The PCNA genotype is recessive to the other three non-PCNA genotypes, and PCNA-type F₁ offspring are obtained exclusively from crosses among PCNA genotypes. Moreover, the number of superior PCNA cross-parents have been limited. In the late 1980s, inbreeding depression became obvious, especially in terms of fruit size, tree vigor, and productivity. To mitigate the inbreeding, a backcross program using PCNA [(non-PCNA × PCNA) × PCNA] was started in 1990. This process, however, was inefficient because only 15% of the offspring were PCNA, and all offspring had to be grown to the fruiting stage. Therefore, molecular markers linked to the PCNA locus were developed for discriminating PCNA offspring. A molecular marker linked to Chinese PCNA has also been developed.     

Assessment of water quality in small agricultural ponds in Nara,Japan

Paddy and Water Environment - In Japan, since ancient times, numerous small- to medium-size agricultural ponds have been constructed to irrigate paddy rice fields. Economic growth and urbanization...