Purification and immunochemical detection of a quantitatively major collagen in the dermis of sea cucumber Apostichopus japonicus

Pepsin-solubilized collagen (PSC) was prepared from the dermis of sea cucumber Apostichopus japonicus (green type) by performing pepsin digestion to collagen fiber pretreated with disaggregating solution (0.1 M Tris–HCl, pH 8.0, containing 0.5 M NaCl, 0.05 M ethylenediaminetetraacetic acid (EDTA), and 0.2 M 2-mercaptoethanol) and 0.1 M NaOH. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the PSC clearly showed two alpha bands under phosphate buffer system in the presence of 3.5 M urea. An antiserum was raised against chromatographically purified major molecular species in the PSC, and immunoblot analyses were performed for the soluble fractions at 4 M guanidine hydrochloride treatment and disaggregation as well as the collagen fiber before and after treatment. These fractions and collagen fibers showed quite similar band patterns to that of the PSC, showing mainly two alpha bands. These combined results suggest that the major molecular species of collagen contains at least two distinct alpha components and that the effect of pepsin digestion is relatively small on the structure of this collagen type.

     

Molecular cloning and expression of bottlenose dolphin (Tursiops truncatus) interleukin-8

The bottlenose dolphin interleukin (IL)-8 cDNA was molecularly cloned. The dolphin IL-8 has an open reading frame of 303-bp encoding 101 amino acids. The homology of the amino acid sequence with that of other species was: sheep, 89.1%; cattle, 88.1%; pig, 85.1%; dog, 85.1%; horse, 79.2%; human, 74.5%; and macaque, 72.3%. The amino acid sequence suggested that dolphin IL-8 was a CXC chemokine. The recombinant dolphin IL-8 protein was recognized with anti-ovine IL-8 monoclonal antibody.     

Rapid detection of chitosanase activity in chitosanase gene-transformed strains of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> by lytic infection of specific bacteriophages

 In combination with lytic infection by virulent phages, a simple method for monitoring transgenic strains of Enterobacter cloacae was developed in this study. First, 15 strains of E. cloacae were used as indicator bacteria to isolate virulent phages with different host ranges. Of the phages isolated, five isolates (EcP-22, -35, -45, -55, and -70) were used to construct a set of virulent phages corresponding to all strains of E. cloacae. Using this phage set, a rhizosphere strain (KRM-055E) of E. cloacae was effectively screened from field soil. KRM-055E was transformed with a prokaryotic chitosanase gene csnSM1 and infected with the phage EcP-03, which can lyse the strain most effectively. The lysis of KRM-055E/csn occurred 2 h after inoculation, and the chitosanase activity was simply detected by dropping the lysate onto an agar plate containing glycol chitosan. The positive signal for chitosanase activity was detected in the 2-h lysates, and the signal intensity reached a maximum in the 5-h lysate. The present assay was simple, rapid, inexpensive, easy to perform, and applicable to another strains. Received: August 2, 2002 / Accepted: October 31, 2002 Acknowledgments This work was supported in part by a grant (no. 99L01205) from the “Research for the Future” program of the Japan Society for the Promotion of Science. We are grateful to Dr. M. Sato, National Institute of Agrobiological Science, Dr. H. Okamoto, Fukui Agricultural Experiment Station, and Dr. K. Tsuda, Kyoto Prefectural Institute of Agricultural Biotechnology, for kindly providing E. cloacae strains. We thank Dr. P. Park, Kobe University, for technical support with the electron microscopic observations.     

Contrast‐enhanced computed tomography may be helpful for characterizing and staging canine gastric tumors

In humans, computed tomography (CT) is a widely performed technique for the diagnosis and staging of gastric tumors. The purpose of this retrospective case series study was to describe CT findings in a group of dogs with confirmed gastric tumors. For each included dog, the following CT parameters were recorded: gastric tumor location, tumor shape, gastric involvement pattern, tumor enhancement pattern in early and late phases, presence and location of lymphadenopathy, gastric tumor attenuation values, attenuation values of enlarged lymph nodes, maximum short‐axis diameter (mm) of enlarged lymph node, maximum long‐axis diameter (mm) of enlarged lymph node, and short‐axis diameter to long‐axis diameter ratio (short axis/long axis). A total of 16 dogs met inclusion criteria and had the following final diagnoses: five lymphoma, six adenocarcinoma, three inflammatory polyps, and two leiomyoma. In the early‐ and delayed‐phase postcontrast images, the mean CT attenuation value for lymphoma was lower than that of other gastric tumors. Lymphadenopathy was widespread in lymphomas and regional in adenocarcinomas. Lymphadenopathy was not detected in leiomyomas. Lymph node measurements in lymphoma were larger than lymph node measurements in adenocarcinoma. Although there were overlapping findings for the different types of gastric tumors, contrast‐enhanced CT provided helpful information for characterizing gastric tumors based on the following criteria: early and late enhancement patterns, the site of origin of the mass lesion, and extent of local invasion and distant metastases. Lymphoma should be considered for canine gastric tumors with low mean attenuation values during early‐ and delayed‐phase postcontrast images, and widespread, bulky, and rounded lymphadenopathy.     

Comparison and characterization of biosorption by Weissella viridescens MYU 205 of periodic group 12 metal ions

Because heavy metals cause various health hazards, we studied biosorption by Weissella viridescens MYU 205. MYU 205 showed high biosorption for Cd (II) and Hg (II) and was low for Zn (II). The Hg (II) biosorption rate was high at about 80%. Different biosorptions were shown for each metal after successive incubation. About 20% of the Zn (II) biosorption was observed after 3 h. Cd (II) biosorption increased in a time‐dependent manner until 3 h, then gradually decreased. Hg (II) was immediately sorbed at 79.6 ± 4.7% and decreased at 3 h to 52.9 ± 2.6%, and then gradually increased to 77.8 ± 3.6%. Using heat‐killed cells, the rate of biosorption of Zn (II) and Cd (II) decreased whereas Hg (II) tended to increase. The metal resistance was high, that is Zn (II) > Cd (II) > Hg (II); while the affinity was opposite where MYU 205 showed high affinity to Hg (II) and low affinity to Zn (II). Our data shows lactic acid bacteria may be powerful heavy metal sorbents for detoxification.     

Characterization of a new hemolytic protein toxin from the sea anemone Anthopleura asiatica

A 20-kDa protein toxin (bandaporin) was isolated from the sea anemone Anthopleura asiatica (Actiniidae) using the precipitation of ammonium sulfate saturation, and ion-exchange and gel-permeation chromatographies. Bandaporin showed potent hemolytic activity (EC₅₀ value: 8.8 ng/ml to 0.8% suspension of sheep red blood cells) and lethal toxicity to crayfish (LD₁₀₀ value: 0.58 mg/kg). The hemolytic activity of bandaporin was inhibited specifically by sphingomyelin. Eight other tested membrane lipids, including cholesterol, and gangliosides GM1 and GM2, did not inhibit bandaporin hemolytic activity. Antimicrobial and phospholipase A₂ (PLA₂) activities were not detected in bandaporin. The complementary DNA (cDNA) encoding bandaporin was sequenced and the deduced amino acid sequence of bandaporin revealed that this actinoporin had not been previously characterized. Actinoporins are cytolytic peptide toxins that are widely distributed in Actiniidae and Stichodactylidae sea anemones. This study was the first characterization of an actinoporin from a sea anemone of the genus Anthopleura.     

Monoblastic leukemia (M5a) with chronic basophilic leukemia in a cat

A cat was presented with depression and anorexia. The complete blood cell count (CBC) revealed non-regenerative anemia (PCV, 8.5%), marked thrombocytopenia (2,400/µl), and leukocytosis (32,090/µl). In the peripheral blood, proliferation of blast cells (85%; 27,276/µl) and basophils (7.7%; 2,460/µl) was observed. Bone marrow aspirate showed hyperplasia with 8.8% blasts and 90.2% basophils of all nucleated cells. The blast cells were negative for myeloperoxidase staining and positive for alpha-naphthol butyrate esterase staining, indicating the agranular blasts are monoblasts. Thus, acute monoblastic leukemia (M5a) with chronic basophilic leukemia was diagnosed. Basophils accounted for more than 40% of the bone marrow, and we diagnosed secondary basophilic leukemia. Secondary basophilic leukemia should be included in the differential list when abnormal basophil increases are observed in feline bone marrow.     

Molecular characterization of Cryptosporidium spp. in grazing beef cattle in Japan

Cattle are major hosts of Cryptosporidium spp. Cryptosporidiosis in neonatal calves is associated with retarded growth, weight loss and calf mortality, and zoonotic infections in humans. In many areas, cow-calf glazing system is an important beef cattle rearing method with distinct advantages in terms of cost and the labor required. However, few epidemiologic studies of Cryptosporidium spp. have been conducted in this system, especially using molecular diagnostic tools. To understand the transmission of Cryptosporidium spp. in a grazing system, we followed cryptosporidiosis on a grazing farm in Osaki City, Miyagi Prefecture, in northwest Japan for one year. Fecal samples were collected from Japanese Black and Japanese Shorthorn cattle and examined by PCR-RFLP and sequence analyses. Of 113 fecal samples collected in October 2010, 23 (20%) were positive for Cryptosporidium, including 15 samples (13%) having C. bovis, 6 (5%) having C. ryanae, and 2 (2%) having mixed infections of both species. Additionally, C. bovis or C. ryanae was detected on all other sampling dates involving smaller numbers of animals. The infection rate of C. bovis was significantly different among age groups, and calve-to-calve infection might be the major route of cryptosporidiosis transmission in beef cattle. Interestingly, one animal had C. bovis infection or re-infection for one year. Our results suggest that C. bovis and C. ryanae are distributed in Japan, but might have low level of detection in grazing beef cattle.     

Nutritional values and antioxidative activities of whole peanuts and peanut skins for ruminant feeds

This study was conducted to investigate the nutritive values of two peanut by‐products, nonstandardized whole peanuts and peanut skins, along with their effects on microbial growth and fermentation in the rumen, their roughage values, and their antioxidative activities by a digestion trial using four goats. The experimental rations were alfalfa haycube (basal ration), 85% alfalfa with 15% whole peanuts, and 70% alfalfa with 15% whole peanuts and 15% peanut skins. The ether extracts and crude protein in whole peanuts were 47% and 27% on a dry matter basis (DM) both with over 90% of digestibilities, resulting in total digestive nutrients (TDN) of 140%. Peanut skins also had a high energy value with 91% of TDN. Ruminal concentrations of total volatile fatty acids (VFA) and acetic acid decreased in the rations containing the peanut by‐products, but the NDF digestibility and ruminal microbial protein estimated from urinary purines was not altered by feeding the peanut by‐products. Plasma oxidative stress maker, malondialdehyde, tended to be lower when peanut skins were supplemented. Whole peanuts and peanut skins could be used as high‐energy and high‐protein diets for ruminants, and peanut skins would be expected as a feed having antioxidant functions.     

Occurrence and distribution of cis-7-icosenoic acid in the lipids of Japanese marine fish,shellfish, and crustaceans

This article reports the concentration and composition of the isomers of cis-icosenoic acid (20:1) in the fatty acids of various species of Japanese marine animals. The main purpose of this study is to reveal the distribution of a positional isomer, cis-7-20:1 (c7-20:1), in marine animals. Because this isomer overlaps with the c9-20:1 isomer in gas chromatography (GC) on the commonly used polar capillary columns, less information is available on its occurrence and distribution. In this study, the monounsaturated fatty acids isolated by argentation thin-layer chromatography were analyzed by GC using a highly polar capillary column, SLB-IL111 (100 m long), with the highest polarity among the commercially available GC columns. A clear separation between the c7-20:1 and c9-20:1 isomers enabled the analysis of all the isomers of 20:1 present in marine animals. The results confirmed that the c7 isomer was a minor component in the 20:1 in the pelagic fish, which is rich in the c9 and/or c11 isomers as reported previously. In contrast, the c7 isomer was one of the major isomers in flatfish, shellfish, crabs, and brittle stars. In these samples, the c13 isomer also occurred at higher concentrations rather than the c9 isomer. Because such a specific pattern of the 20:1 positional isomers was generally observed in both benthic fish and its dietary animals, the c7 isomer in the benthic fish probably originated from its diet. These marine animals are used as food in Japan, and therefore the c7-20:1 isomer is consumed by humans.