Characterization of an Alginate Lyase,FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.     

日粮类型对绵羊小肠内代谢性蛋白质的影响

以成年萨福克绵羊为试验对象,分别安装瘤胃瘘管、十二指肠近端T型瘘管和回肠远端T型瘘管,饲喂由鸭茅干草和配合饲料组成的不同瘤胃内可降解有机物/瘤胃内可降解粗蛋白质(OMR/CPR)比例的日粮,研究小肠可代谢性饲料蛋白质(MP)的变化规律.结果表明随着OMR/CPR的提高,小肠内微生物氮的消化量显著降低,而过瘤胃饲料蛋白质氮显著增加;表观MP量随着OMR/CPR的增加呈先增加后减少的变化趋势,表观MP量占CP、DCP摄取量分别为42.2%～59.6%、63.6%～87.8%.     

Oxygen isotopic compositions of asteroidal materials returned from Itokawa by the Hayabusa mission

Meteorite studies suggest that each solar system object has a unique oxygen isotopic composition. Chondrites, the most primitive of meteorites, have been believed to be derived from asteroids, but oxygen isotopic compositions of asteroids themselves have not been established. We measured, using secondary ion mass spectrometry, oxygen isotopic compositions of rock particles from asteroid 25143 Itokawa returned by the Hayabusa spacecraft. Compositions of the particles are depleted in (16)O relative to terrestrial materials and indicate that Itokawa, an S-type asteroid, is one of the sources of the LL or L group of equilibrated ordinary chondrites. This is a direct oxygen-isotope link between chondrites and their parent asteroid.     

A case of canine gastrinoma

The dog of this case was a 10-year-old Shih Tzu with refractory vomiting, diarrhea and anorexia. Endoscopy revealed an unclear at gastric angle, a stenosis at pyloric antrum and congestion in duodenal mucosa. Since abnormal shadows of irregular echo-levels were disclosed by pancreas ultrasonography, serum gastrin level was determined with a suspect of gastrinoma. And an increase of serum gastrin was demonstrated. In addition, postmortem histological examination revealed that the pancreatic cells were positive for gastrin. Based on these findings, the dog was diagnosed as pancreatic gastrinoma.     

Sporadic cases and an outbreak of leptospirosis probably associated with recreational activities in rivers in the northern part of Okinawa Main Island

In the summer of 2003, sporadic cases and an outbreak of human leptospirosis probably related to recreation in rivers occurred in the northern part of Okinawa Main Island. Sixteen of 22 suspected cases were definitely diagnosed as leptospirosis by serological test or isolation. The infective leptospiral serovar in 14 cases was presumed to be Hebdomadis. Transmission was thought to occur by exposure to river water that was contaminated by the urine of infected animals. The findings indicate that recreation in rivers in this area is a significant risk factor for infection with leptospires.     

Anatomical and histological characterization of ileal and jejunal Peyer's patch in lesser mouse deer (Tragulus javanicus)

Tragulidae is a primitive ungulate family within the order Cetartiodactyla, suborder Ruminantia. Domestic ruminants such as cattle, sheep, and goat have two types of Peyer's patches (PP): jejunal and ileal PP, in which there are morphological and functional differences. In this study, lesser mouse deer (Tragulus javanicus) PP was studied by gross anatomical and histological procedures. At the fetal stage, both types of PP were formed in the small intestine. Ileal PP was observed as a single continuous aggregation of lymphatic follicles extending cranially from the ileo-caecal junction. However, jejunal PP was observed as multiple and discrete accumulations of lymphatic follicles. This study showed that the lesser mouse deer has two types of PP in the small intestine. In addition, the anatomical and histological characteristics of jejunal and ileal PP are quite similar to those of other ruminants' jejunal and ileal PP. Further studies are needed to analyze immune function of both PP in lesser mouse deer in order to determine the evolutionary process of Cetartiodactyla.     

A comparison of changes in stable isotope ratios in the epidermal mucus and muscle tissue of slow‐growing adult catfish

Although stable isotope analysis is a powerful tool for determining diet, migration patterns and the structure of food webs in aquatic systems, the slow response of isotopic ratios in the widely used muscle tissue often hampers this approach, particularly in slow‐growing or adult fishes. We conducted a diet‐switch experiment to compare the changes in the stable carbon isotope ratio (δ¹³C values) in the epidermal mucus and muscle tissue of five‐year‐old catfish (Silurus asotus). The isotope ratios in noninvasively sampled mucus changed more rapidly than those in the muscle tissue. As isotopic change in mucus was relatively rapid, this technique can be used over a finer timescale than traditional isotopic analyses using the muscle tissue. The isotopic change half‐life in our experimental condition was 200 days, which would not be short enough for some research purposes. Examining mucus along with the muscle would enable food habits of slow‐growing fishes to be determined over different timescales. However, the rate of isotopic change in the mucus was negatively affected by the size of fish and was slower than the previously reported rates in juvenile steelhead. These findings suggest that mucus turnover rates need to be determined prior to the field data interpretation.     

Estimation of the spawning grounds of chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis> and spotted mackerel <Emphasis Type="Italic">Scomber australasicus</Emphasis> in the East China Sea based on catch statistics and biometric data

The spawning grounds of the chub mackerel (Scomber japonicus) and spotted mackerel (Scomber australasicus) in the East China Sea were estimated based on catch statistics of the Japanese large- and medium-type purse seine fishery from 1992 to 2006. Biometric data were obtained from specimens caught by purse seiners in the East China Sea from 1998 to 2006. Gonadosomatic index (GSI) at 50% sexual maturity of chub mackerel and spotted mackerel females was 2.5 and 2.6, respectively. Using this criterion for GSI, chub mackerel larger than 275 mm and spotted mackerel larger than 310 mm in fork length were considered to be mature. Mature chub mackerel was observed in the area of 15–22°C sea surface temperature (SST), and mature spotted mackerel was observed in the area of 17–25°C SST. The spawning period of chub mackerel ranged from February to June, and that of spotted mackerel ranged from February to May in the East China Sea. The spawning grounds were estimated from the distributions of catch per unit effort (CPUE) of spawners and SST. As a result, the spawning ground of chub mackerel was estimated to be in the central and southern part of the East China Sea and the area west of Kyushu in February, March, and April, and in the central part of the East China Sea, the area west of Kyushu and Tsushima Straight in May, and in Tsushima Straight and western part of the Sea of Japan in June. The spawning ground of spotted mackerel was estimated to be in the central and southern part of the East China Sea and southern coastal area of Kyushu in February, March, and April, and the central and southern part of the East China Sea and the area west of Kyushu in May.     

Identification of a 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid Reductase,FlRed, in an Alginolytic Bacterium Flavobacterium sp. Strain UMI-01

In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-d-gluconate (KDG) by a specific reductase, and metabolized through the Entner–Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%–88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.     

CHARACTERIZATION OF CANINE FOCAL LIVER LESIONS WITH CONTRAST-ENHANCED ULTRASOUND USING A NOVEL CONTRAST AGENT—SONAZOID

Contrast-enhanced ultrasound using Sonazoid, a novel contrast medium with a liver-specific Kupffer phase, was evaluated in canine focal liver lesions Twenty-five dogs with a liver mass were given intravenous Sonazoid, and the enhancement pattern in the arterial, portal, and parenchymal phase was characterized. An enhancement defect in the lesion in the parenchymal phase was observed in all malignant lesions, whereas only one of nine benign lesions had a filling defect. The diagnostic value of the presence of a filling defect for malignancy was statistically significant (100% sensitivity, 88.9% specificity, 94.1% positive predictive value, 100% negative predictive value), and was equal to that of hypoenhancement in the portal or delayed phase. The defect pattern (clear or irregular defect) was dependent ( P <0.05) on the types of malignancy (i.e., hepatocellular carcinoma and other types of malignancies). In the arterial phase, five of the six hepatocellular carcinomas had hypervascularity, whereas no other lesion was characterized by hypervascularity. In some dogs, additional lesions that could not be observed with conventional B-mode ultrasonography were detected in the parenchymal phase. The enhancement pattern of Sonazoid, especially in the parenchymal phase, has potential as a diagnostic tool for canine focal liver lesions.