Expression of CC chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion

CC chemokine receptor 4 (CCR4) is a G protein-coupled seven transmembrane receptor that is selectively expressed on Th2 cells and plays an important role in the trafficking of Th2 cells into inflammatory sites. In this study, a full-length canine CCR4 cDNA was cloned and characterized in order to examine the potential role of CCR4 in allergic responses that produce skin lesions in canine atopic dermatitis (AD). The canine CCR4 cDNA reported in this study contained an open reading frame of 1083 nucleotides encoding 360 amino acids. The predicted amino acid sequence of canine CCR4 showed 91.9, 85.3 and 84.5% similarity with those of the human, mouse and guinea pig counterparts, respectively. Expression of CCR4 mRNA was detected in various tissues including thymus, spleen, heart, small intestine and lymph node. Furthermore, it was found that CCR4 mRNA was preferentially expressed in lesional skin of dogs with AD, together with the mRNA of thymus and activation-regulated chemokine (TARC), which is a ligand for CCR4. The present study demonstrates that CCR4 contributes strongly to the immunopathogenesis of canine AD.     

Morphological and cytological characteristics of protoplast-derived plants of statice (Limonium perezii Hubbard)

Morphological characteristics of 56 protoplast-derived plants (protoclones) of statice (Limonium perezii Hubbard) were compared with meristem-derived plants (controls). Some protoclones had smaller leaf shape index (length/width) and shorter length of flower stalk and calyx than the controls. However, no distinct somaclonal variations were observed in other characteristics such as color of calyx and number of petals and stamens, except for protoclone No. 1–7, which had flowers with abnormal petals and low pollen fertility (25%). Microscopic observation showed that all of the 17 protoclones examined, including clone No. 1–7, had the normal diploid chromosome number of 14.     

D-Alanine-D-Alanine Ligase Gene (ddl) of Erwinia chrysanthemi Strain EC16 I. Isolation and Gene Dosage Effect on Pectate Lyase Synthesis

New regulatory gene for pectate lyase (Pel) production of Erwinia chrysanthemi strain EC16 was searched by observing the gene dosage effect of each cosmid library in Pel production. From this survey, a cosmid clone, p5A, had reduced in Pel production under both inducing and non-inducing conditions and caused less tissue maceration of potato tubers. The 2.64-kb HindIII fragment from p5A was found to be responsible for this phenotype and to contain one major ORF consisting of 1107 nucleotides, which had homology with ddlA (49.6%) and ddlB (49.6%) of Escherichia coli and with ddlA (44.6%) of Salmonella typhimurium. These genes had been shown to encode D-alanine-D-alanine ligase (Ddl), an enzyme which is involved in the synthesis of peptidoglycan. This ORF encodes 368 amino acid residues and its molecular weight is estimated to be 43 kDa. This ORF of EC16 could complement ddl deficient mutant of Escherichia coli. Received 15 May 2002/ Accepted in revised form 20 June 2002     

Influence of feeding a grass hay diet during the early stage of the fattening period on growth performance,carcass characteristics,and meat production in Japanese Black steers

The present study investigated the influence of feeding a large amount of grass hay to steers from the early to middle fattening period on growth, carcass characteristics, and meat characteristics. Steers were randomly divided into grass hay‐fed (GHF, n = 6) and concentrate‐fed (CF, n = 6) groups. The dressed weight of the GHF steers was lower than that of the CF steers, but the final body weight was not significantly different between the groups. The GHF steers had decreased subcutaneous fat and rib thickness compared with the CF steers. Lipid content, monounsaturated fatty acids, and drip loss in the muscles were lower in the GHF steers than in the CF steers. Furthermore, n‐3 polyunsaturated fatty acids were higher in the GHF steers compared with the CF steers. The GHF steers had lower body weight during the middle fattening stage, which may have occurred as a result of muscle growth suppression caused by increased Myostatin expression; an increase in daily gain during the finishing period may have occurred as a result of muscle growth activation caused by decreased Myostatin expression. Feeding steers a grass hay‐based diet during the early fattening period possibly maintains the quantitative productivity of beef similarly to feeding a concentrate‐based diet.     

Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization

Electroporation is the technique of choice to introduce an exogenous gene into embryos for transgenic animal production. Although this technique is practical and effective, embryonic damage caused by electroporation treatment remains a major problem. This study was conducted to evaluate the optimal culture system for electroporation‐treated porcine embryos by supplementation of chlorogenic acid (CGA), a potent antioxidant, during in vitro oocyte maturation. The oocytes were treated with various concentrations of CGA (0, 10, 50, and 100 μmol/L) through the duration of maturation for 44 hr. The treated oocytes were then fertilized, electroporated at 30 V/mm with five 1 msec unipolar pulses, and subsequently cultured in vitro until development into the blastocyst stage. Without electroporation, the treatment with 50 μmol/L CGA had useful effects on the maturation rate of oocytes, the total cell number, and the apoptotic nucleus indices of blastocysts. When the oocytes were electroporated after in vitro fertilization, the treatment with 50 μmol/L CGA supplementation significantly improved the rate of oocytes that developed into blastocysts and reduced the apoptotic nucleus indices (4.7% and 7.6, respectively) compared with those of the untreated group (1.4% and 13.0, respectively). These results suggested that supplementation with 50 μmol/L CGA during maturation improves porcine embryonic development and quality of electroporation‐treated embryos.     

Molecular screening of canine GM1 gangliosidosis using blood smear specimens after prolonged storage: detection of carriers among shiba dogs in northern Japan

Molecular screening of GM1 gangliosidosis in Shiba dogs was carried out in northern Japan using blood smear specimens after prolonged storage. Of 125 specimens obtained from 3 veterinary teaching hospitals for this screening, 68 specimens (54%) were adequate for direct amplification in a polymerase chain reaction (PCR)-based DNA test, and the percentage of adequacy was different at each hospital (34%, 73%, and 100%), suggesting that the amount of blood on the smear and the storage condition of specimens may affect adequacy. Of the 68 dogs examined, 2 dogs (2.9%) were heterozygous carriers for this disease and the other dogs were all genotypically normal. The results suggest blood smear specimens can be useful for PCR testing after prolonged storage provided specimens contain a generous amount of blood and have been adequately stored. The study also suggests that GM1 gangliosidosis may be widely prevalent in the Shiba dog population in northern Japan.     

Nucleotide sequence polymorphisms of beta1‐, beta2‐, and beta3‐adrenergic receptor genes on Jinhua,Meishan, Duroc and Landrace pigs

The full amino acid coding sequences of adrenergic receptor genes beta1, beta2, and beta3 (ADRB1, ADRB2, and ADRB3)were determined for Jinhua, Meishan, Duroc and Landrace pigs. Non‐synonymous substitution of Arg458Pro was found in the porcine ADRB1 gene, resulting in a 469 amino acid sequence. Continuous substitutions of Asn29Asp and Glu30Gln were found in the porcine ADRB2 gene, resulting in a 418 amino acid sequence. Additionally, a Lys30 polymorphism of the ADRB2 gene was found in the Jinhua pigs. There were three non‐synonymous substitutions of Asn24Thr, Arg264Gln and Asn398Asp on the porcine ADRB3 gene. A thymine insertion in the ADRB3 gene, resulting in a protein with two fewer amino acids, was found in the Jinhua and Meishan pigs. To assess the effect of ADRB polymorphisms on porcine subcutaneous fat layer thickness, we calculated the genetic frequency of the variants in fatty and lean groups, each consisting of 24 pigs that were crossbreds of Duroc and Jinhua pigs. The effect of the ADRB3 gene polymorphism was not evaluated, because there was insufficient variation on the ADRB3 gene in the examined groups. Although Fisher's exact test showed no significant difference in the frequency of ADRB1 and ADBR2 variants between the two groups, the Arg458 variant of ADRB1 was higher (P = 0.11) in the lean group, and pigs in that group had a thinner fat layer than did those with the Pro458 variant. These results imply a possibility of ADRB1 polymorphism as a minor factor in porcine fat layer thickness. The Asp29 variant of ADRB2 was higher in the lean group (P = 0.11), and the Glu30 variant was higher in the fatty group (P = 0.15), but the Asp29 variant was found only in the Chinese pigs. Thus, the effect of ADRB2 polymorphisms was not clear in this study.     

Identification of an antihypertensive peptide derived from chicken bone extract

A novel angiotensin‐converting enzyme (ACE) inhibitory peptide was isolated and purified from chicken bone extract by enzymatic digestion. The peptide was defined as an ACE inhibitor, and it demonstrated antihypertensive activity following oral administration to spontaneously hypertensive rats (SHRs). The results of this study suggest that peptides derived from an extract of chicken bones, administered orally, have the ability to reduce the blood pressure of SHRs significantly over a short period of time (3 h). Moreover, the blood pressure then remains low for 3 h. This peptide derived from chicken bones may therefore have great value as a short‐term remedy for chronic conditions such as high blood pressure. The amino acid sequence of the peptide was YYRA (Tyr‐Tyr‐Arg‐Ala), which was the origin of the Ig heavy chain V region (27–30 position). The IC₅₀ value of its synthetic peptide was 33.9 μg/mL. We suggest that the ACE inhibitory and antihypertensive peptides derived from chicken bone extract may contribute to develop physiologically functional foods or improve food functionality.