Production of recombinant extracellular domains of canine desmoglein 1 (Dsg1) by baculovirus expression

The aim of this study was to generate a recombinant protein to represent the entire extracellular domain of canine desmoglein 1 (Dsg1), a desmosomal cell-cell adhesion molecule, by the baculovirus expression system. Cotransfection of a baculovirus transfer vector containing the cDNA for the entire extracellular domain of canine Dsg1 with baculovirus DNA into insect cells resulted in the secretion of soluble canine Dsg1 into insect culture supernatants. Immunoreactivity of 11 human pemphigus foliaceus (PF) sera against the cell surface of canine keratinocytes was completely removed when the sera were preincubated with the canine Dsg1 baculoprotein. This recombinant canine Dsg1 produced by baculovirus shares the major epitopes of the authentic canine Dsg1 recognized by human PF sera, and will be useful in studying the molecular pathophysiological mechanisms in PF and impetigo in canine patients.     

Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro

To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.     

Effect of Figaren, an Antiviral Protein from Cucumis figarei, on Cucumber mosaic virus Infection

Local lesion formation on cowpea leaves was more than 50% inhibited by treatment with a 23 kDa RNase-like glycoprotein from Cucumis figarei, figaren, from 24 hr before to 1 hr after inoculation with Cucumber mosaic virus (CMV). CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5–7 days on most of the tobacco plants, and some plants remained asymptomatic. From fluorescence in situ hybridization, infection sites were present in figaren-treated cowpea or melon leaves after inoculation with CMV, though the sites were reduced in number and size compared with those in water-treated control leaves. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently incubated with figaren similarly increased with time as did that in the control. ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and C. amaranticolor whose lower surfaces had been treated with figaren 5–10 min before CMV inoculation was almost completely inhibited. Figaren did not inhibit CMV infection on the opposite untreated leaf halves of melon, cowpea and C. amaranticolor, whereas it almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. CMV infection was not inhibited in the untreated upper or lower leaves of the four plants. These data suggest that figaren does not completely prevent CMV invasion but does inhibit the initial infection processes. It may also induce localized acquired resistance in host plants. Received 10 October 2000/ Accepted in revised form 6 February 2001     

Purification and Partial Characterization of Figaren, an RNase-like Novel Antiviral Protein from Cucumis figarei

An antiviral protein, designated figaren, was purified from leaves of Cucumis figarei and partially characterized. Column chromatography, SDS-polyacrylamide gel electrophoresis and periodic acid-Schiff staining revealed that figaren is a glycoprotein with a molecular weight of 23 kDa. Figaren was stable at pH 2 to 12 and below 90°C. N-terminal amino acid sequencing indicated that figaren contained the conserved region for the S-allele-associated ribonucleases (RNases). In-gel RNase assay showed that figaren digested yeast RNAs. Figaren also digested double-stranded RNAs extracted from Cucumber mosaic virus (CMV)-infected tobacco tissues. Fluorescence in situ hybridization revealed that figaren and RNases (beef pancrease and RNase T₁ at 1 μg/ml similarly inhibited CMV infection in cowpea leaves. Figaren and the RNases at 5-500 ng/ml had similar inhibitory effect on local lesion formation by CMV. These data suggest that figaren is a novel RNase-like antiviral protein. Received 10 October 2000/ Accepted in revised form 6 February 2001     

Comparison of corn gluten meal and meat meal as a protein source in dry foods formulated for cats

OBJECTIVE: To compare the nutritional value of corn gluten meal (CGM) and meat meal (MM) as a dietary source of protein in dry food formulated for adult cats. ANIMALS: 8 healthy adult cats (4 males and 4 females). PROCEDURE: Diets containing CGM or MM as the main protein source were each fed for a 3-week period in a crossover study. Digestibility and nutritional balance experiments were conducted during the last 7 days of each period. Furthermore, freshly voided urine was obtained to measure urinary pH, struvite crystals, and sediment concentrations. RESULTS: Daily food intake and dry-matter digestibility were significantly higher for the MM diet. Fecal moisture content also was higher for the MM diet. Apparent nitrogen (N) absorption and N retention were higher for the MM diet, even when values were expressed as a percentage to account for differences in N intake. Urinary pH, struvite activity product, number of struvite crystals in urine, and urinary sediment concentrations were not different between diets. Retention of calcium, phosphorus, and magnesium was lower for the CGM diet, and cats lost body calcium and magnesium when fed the CGM diet. CONCLUSIONS AND CLINICAL RELEVANCE: Meat meal was superior to CGM as a protein source in dry foods formulated for cats, because dry-matter digestibility and N utilization were higher for the MM diet. In addition, net loss of body calcium and magnesium for the CGM diet suggests that mineral requirements increase when CGM is used as a protein source.     

Correlation between plasma leptin concentration and body fat content in dogs

OBJECTIVE: To evaluate the relationship between plasma leptin concentration and body fat content in dogs. ANIMALS: 20 spayed female Beagles that were 10 months old at the start of the experiment. PROCEDURE: Dogs were kept under regulated feeding and exercise conditions for 21 weeks, resulting in a wide range of body weights, body condition scores (BCS), and subcutaneous thicknesses. Plasma leptin concentration was measured by use of a canine leptin-specific ELISA test to evaluate its correlation to body fat content estimated by the deuterium oxide dilution method. Plasma concentrations of glucose, cholesterol, triglycerides (TG), and nonesterified fatty acids (NEFA) were also measured. RESULTS: Body fat content (9 to 60% of body weight) was positively and closely correlated (r = 0.920; n = 20; P < 0.001) to plasma leptin concentration (0.67 to 8.06 ng/ml), compared with other variables (ie, glucose, cholesterol, TG, and NEFA; r = 0.142, 0.412, 0.074, and 0.182, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: The positive relationship between plasma leptin concentration and body fat content in dogs was similar to correlations reported for humans and rodents, suggesting that plasma leptin is a quantitative marker of adiposity in dogs.     

High level expression of C-terminal truncated recombinant chicken interferon-gamma in baculovirus vector system

Chicken interferon-gamma (ChIFN-gamma) was expressed by baculovirus in a C-terminal truncated form, namely ChIFN-gammaT, to accelerate the secretion of the expressed protein. It is also expressed as ChIFN-gammaT bearing poly His tag, ChIFN-gammaTHis, for easy purification. The expressed proteins were detected by SDS-PAGE analysis with Coomassie brilliant blue staining. The purified ChIFN-gammaTHis with nickel chelated column showed anti-viral activity in vitro and stimulation of the secretion of nitrogen intermediates such as nitric oxide in chicken peripheral blood mononuclear cells. Antiserum against ChIFN-gammaTHis recognized the 15 kDa, 16 kDa, and 32 kDa bands that seemed to be an unglycosylated monomer, a glycosylated monomer, and a homodimer of ChIFN-gammaTHis in the culture supernatant, respectively. The anti-serum also recognized around 14 kDa and 28 kDa bands in the sera of chickens or concanavalin A stimulated spleen cell culture supernatants that seemed to be monomeric and dimeric forms of a natural ChIFN-gamma, respectively.     

Phylogenetic relationships between the lettuce root rot pathogen Fusarium oxysporum f. sp. lactucae races 1, 2, and 3 based on the sequence of the intergenic spacer region of its ribosomal DNA

The genetic relationship between the vegetative compatibility groups (VCGs) and between physiological races of Fusarium oxysporum f. sp. lactucae (FOL), the causal pathogen of lettuce root rot, was determined by analyzing the intergenic spacer (IGS) region of its ribosomal DNA. A total of 29 isolates containing a type strain were tested: 24 Japanese isolates, 2 Californian isolates, and 3 Italian isolates. Three races (races 1, 2, and 3) were found in Japan, and race 1 was also distributed in California and Italy. Races 1, 2, and 3 each belonged to a distinct VCG: VCG-1, VCG-2, and VCG-3 (VCG-3-1, VCG-3-3), respectively. Phylogenetic (neighbor-joining) analysis of the IGS sequences revealed that races 1, 2, and 3 coincided with three phylogenetic groups (PG): PG-1, PG-2, and PG-3, respectively. These results indicate that the three races are genetically quite different and have a strong correlation with VCGs and phylogenetic groupings. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession no. AB195218