Measurement of erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) in racehorses and riding horses

Equine carbonic anhydrase isozymes (CA-I and CA-II) were purified from erythrocytes by several column chromatography. Polyclonal anti-CA-I and anti-CA-II sera were produced in rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISA) were established to determine the developmental changes in CA-I and CA-II levels in equine erythrocytes. Concentrations of CA-I and CA-II in erythrocytes from 150 clinically normal thoroughbreds (123 racehorses and 27 riding horses) were determined by ELISA. Mean (+/- SD) concentrations of CA-I and CA-II in racehorses were 1.70 +/- 0.48 and 0.94 +/- 0.13 mg/g hemoglobin (Hb), respectively. Mean concentrations of CA-I and CA-II in riding horses were 2.34 +/- 0.52 and 0.76 +/- 0.08 mg/g Hb, respectively. When the CA levels in racehorses and riding horses were compared, the CA-I level in riding horses was higher than that in racehorses (p=0.01). The CA-II level in racehorses was higher than that in riding horses (p=0.02). These data suggest that the levels of CA isozymes in erythrocytes of racehorses were influenced by chronic physical stress. The CA-I concentration in erythrocytes of 2-month-old horses was approximately 0.25 mg/g Hb. The CA-I level noticeably increased during the first year of life and approached normal adult levels by 2 years. The CA-II level decreased slightly with age, indicating different regulation of CA-I and CA-II expression during development.     

Transfer of the Rsv3 locus from ‘Harosoy’ for resistance to soybean mosaic virus strains C and D in Japan

Resistance to soybean mosaic virus (SMV) is imperative for soybean (Glycine max (L.) Merr.) production in the Tohoku region. Molecular markers for SMV resistance were previously reported for U.S. SMV strains, but they cannot be applied because of the differences in strain classification between Japan and the U.S. A U.S. variety ‘Harosoy’ has been used mainly as a donor of resistance to SMV strains C and D in a Japanese breeding program, resulting in resistant varieties such as ‘Fukuibuki.’ Because ‘Harosoy’ harbors the Rsv3 gene conferring resistance to the virulent SMV strain groups, G5 through G7, it appears that the Rsv3 gene confers resistance to strains C and D. In this study, we introduced resistance to the two strains from ‘Fukuibuki’ into a leading variety ‘Ohsuzu’ by recurrent backcrossing with marker-assisted selection. All lines selected with markers near Rsv3 showed resistance to the strains, suggesting that the Rsv3 locus is responsible for the resistance. Three years of trials showed that one of the breeding lines, ‘Tohoku 169,’ was equivalent to ‘Ohsuzu’ with respect to agricultural characteristics such as seed size, maturity date, and seed yield, except for the SMV resistance.     

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.     

Pressure-flow relationship and longitudinal distribution of pulmonary vascular resistance in heartworm-infected dogs

The pressure-flow relationships and the longitudinal distributions of pulmonary vascular resistance in normal and heartworm-infected (HWI) dogs were compared in an isolated, blood perfused preparation. The pulmonary circulation was partitioned into pulmonary arterial, middle, and venous segment based on the concept of a five element lumped model. The pulmonary arterial pressure-flow relationships were found to be non-linear and convex to the pressure axis in both normal and HWI lungs. The pressure-flow relationships of the pulmonary arterial and venous segment were linear and these slopes in the HWI lungs were significantly higher than the normal lungs. The pressure gradient of the middle segment was increased as flow increased at lower flow range, however, it was not increased during higher perfusion range in both lungs. At higher flow, the pressure gradient of the middle segment in the HWI lungs was significantly higher than the normal lungs. These results suggest that the ohmic resistance was almost equal to the sum of the two slopes of the pressure-flow relationships of the pulmonary arterial and venous segment because the pressure gradient of the middle segment was not altered as flow increased during higher perfusion rate. Because the slopes of the pressure-flow relationships of the pulmonary arterial and venous segment were increased with heartworm infection, the ohmic resistance of HWI lungs would be higher than normal lungs. The intercept pressure on the pressure axis of the linear portion of the pulmonary arterial pressure-flow relationship, a critical closing pressure, was regarded as pressure gradient of the middle segment during higher perfusing rate because the intercept pressures of pressure-flow relationships of pulmonary arterial and venous segment were almost equal to zero. Therefore, the critical closing pressure of HWI lungs would be higher than normal lungs. The pulmonary hypertension of filariasis appears to be due to an increase in ohmic resistance and elevated critical closing pressure.     

Invasion and colonization of mature apple fruit by Erwinia amylovora tagged with bioluminescence genes

Invasion and colonization of mature apple fruit by a transformant of Erwinia amylovora tagged with bioluminescence genes from Vibrio fischeri was examined. The transformant was deposited on cut surfaces of fruit stems, wounds on the shoulders and calyces, injured fruit-bearing twigs of harvested apple fruit, and cut fruit flesh. After incubation in closed stainless steel or plastic boxes at 25°C, fruit were periodically observed with a two-dimensional luminometer. The presence of the transformant in luminous areas was confirmed by isolating it on selective media. E. amylovora, when deposited in fruit stems: (1) can invade mature as well as immature apple fruit; (2) vertically and horizontally spreads and colonizes along vascular bundles, increasing its population; (3) reaches the calyx end and the flesh just under the exocarp within 3–4 days after inoculation; (4) when deposited on cut fruit flesh, irrespective of its maturity, can easily increase its population and survive 2–4 weeks or more at 25°C; and (5) even at the time of fruit maturation, can migrate within twigs rapidly and reaches the abscission layers between fruit-bearing twigs and fruit stems.     

Malignant melanoma of the eyelid in a red squirrel (Sciurus vulgaris orientis)

A 6-year-old male red squirrel (Sciurus vulgaris orientis) developed bilateral tumors of upper and lower eyelids. The tumors in the left lid recurred despite surgical removal. Necropsy revealed metastasis to the lung. The neoplastic cells were epithelioid and highly pleomorphic, and only a few cells contained melanin granules. Occasionally melanoma cells were immunoreactive for S100, neuron-specific enolase and vimentin, and a small number of cells for cytokeratin. Ultrastructurally, the presence of premelanosomes was confirmed in the cytoplasm. Possible presence of cytokeratin-positive neoplastic melanocytes should be taken into account when differentiating a nonpigmented epithelioid melanoma from other tumors such as anaplastic carcinomas.     

Comparative observation of skeletal-dental abnormalities in wild, domestic, and laboratory rabbits

Dietary habits must be considered as one of the major potential factors resulting in acquired malocclusions in rabbits. Although the dentition of the wild rabbit and the domesticated laboratory rabbit are basically identical, dietary habits are noticeably different. Therefore, the prevalence of tooth problems between these lagomorph species were investigated anatomically and radiographically. Mean measurements of the skull and dental arches suggested that wild rabbits have slightly shorter and wider skulls and dental arches compared with domestic laboratory rabbits. Root elongation of incisors and check teeth, and periodontal disease were more frequently observed in domestic laboratory rabbits. Diagnostic radiographs from domestic pet rabbits showed relatively higher crowns, severe root elongation, and advanced periodontitis. These results do not provide definitive evidence that dietary habits cause malocclusions, however they suggest that diet is a major factor in the initiation of malocclusions in rabbits.     

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively. Received 5 July 2001/ Accepted in revised form 27 September 2001     

Studies on the properties of organic matter in buried humic horizon derived from volcanic ash

Abstract

Humic acids were extracted from the surface horizon Yu 1 and buried humic horizons (Yu 2, 800-864 AD; Yu 4, ca. 4,000 years B.P.; Yu 6, 7,000 years B.P.; Yu 7, ca. 10,000 years B.P.). The humic acids were filtered with ultramembranes with 20, 10, 5, 1 or 0.1 × 10' M.W. in this order and separated into five fractions; (>20), (20-10), (10-5), (5-1) and (1-0.1) × 10⁴ M.W. fractions. The elementary composition, functional groups, optical properties, and hydrolyzable total- and sugar-carbon contents of each humic acid fraction were determined, and the changes in properties of the humic acid with age after burial were discussed.

In horizons Yu 1 to Yu 6, the patterns of M.W. distribution of the >20 × 10⁴ to (1- 0.1) × 10⁴ fractions were rather similar. In contrast, Yu 7 was characterized by the predominance of (1-0.1) x 10' M.W. fraction and the presence of a small amount of <0.1 × 10⁴ M.W. fraction.

The chemical properties of humic acid (HA) fractions of each humic horizon and their changes with age after burial were as folIows:

1) Elementary composition of HA fractions of each humic horizon was rather similar to each other. The C% and C/N ratio increased, and the H%, N% and H/C ratio decreased in all HA fractions with age.

2) Total acidity and carboxyl contents of HA fractions of Yu 1 were higher in the lower M.W. fractions. These values increased with age up to Yu 4 or Yu 6 horizons, then decreased in the Yu 6 or Yu 7 horizon. The contents of carbonyl groups in all the HA fractions which were very low in Yu 1, showed a wide range of variations in Yu 2, intermediate values from Yu 4 to Yu 6 horizons then decreased in the HA fractions of Yu 7, except for the (>20) fraction.

3) The degree of humification of the HA fractions of Yu 1 judging from the RF and \sDlogK values, tended to be higher in higher M.W. fractions, and increased in all the HA fractions with age after burial.

4) Hydrolyzable total- and sugar-carbon contents were high in the HA fractions of Yu 1 and decreased with age.

5) Changes of elementary composition, degree of humification and hydrolyzable total- and sugar-carbon contents were conspicuous from the Yu 1 to Yu 2 horizons, and less significant afterwards.

6) The 0%, C/O ratio and phenolic OH content did not show any consistent change with age after burial.     

Collaborative control initiatives targeting zoonotic agents of alveolar echinococcosis in the northern hemisphere

Alveolar echinococcosis is one of the most important lethal zoonotic helminth infections in the northern hemisphere. Currently, the threat to public health is increasing, as evidenced by the rising prevalence rate of alveolar echinococcosis, as well as the invasion of urban areas by infected wild foxes. This threat is further increased due to the involvement of pet dogs, and probably cats, as emerging sources of infection. These increased threats to public health also have associated economic risks; therefore, there is a need for effective and sustainable methods of control. In this paper, initiatives to control alveolar echinococcosis by targeting its definitive hosts through anthelmintic baiting campaigns initiated by local residents who used local resources for bait production, distribution and collection of fecal samples for diagnosis are described. Further, when such distribution programs are coupled with the use of GIS-based maps, the optimum distribution of bait was obtained. These programs have also included the use of intravital diagnostic analyses of infection rates, which have been overseen by the Forum on Environment and Animals (FEA), and also allowed a nationwide monitoring of echinococcosis in difinitive hosts. In addition, a government initiative requiring mandatory reporting of echinococcosis in dogs to health authorities was recently initiated in Japan. Overall, the results of this study have shown that use of collaborative control initiatives targeting zoonotic agents of alveolar echinococcosis can be an effective method for reducing the threat of lethal echinococcosis in the northern hemisphere.