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.

     

Purification of serine carboxypeptidase from the hepatopancreas of Japanese common squid Todarodes pacificus and its application for elimination of bitterness from bitter peptides

ABSTRACT:   An acidic serine carboxypeptidase (CPase Tpa) from the hepatopancreas of Japanese common squid Todarodes pacificus was purified. Purified CPase Tpa had a molecular mass of 36 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis, and an isoelectric point of 6.0. The optimum pH of CPase Tpa was pH 4.0. In investigating the specificity of CPase Tpa for several peptide substances, it was found that peptides with hydrophobic or bulky amino acid residues at the P₁ position reacted well. The enzymatic activity was almost completely inhibited by p -chloromercuribenzoic acid, monoiodoacetic acid, diisopropylfluorophosphate and HgCl₂. This is the basis for its grouping in the serine carboxypeptidase family (EC 3. 4. 16. 5). The substrate specificity of CPase Tpa can be used to eliminate the bitterness of bitter peptides. In this study, the bitterness-reductive effect using bitter peptides prepared by hydrolyzing soy protein, casein and corn gluten with pepsin or trypsin was tested. The bitterness of soy peptide digested with pepsin was completely eliminated by treatment with CPase Tpa, whereas the bitterness of casein digested with trypsin and corn peptide digested with pepsin were somewhat less efficient. On the basis of these results, it is anticipated that CPase Tpa would be effective in eliminating the bitterness of some bitter peptides.     

Detection of cyprinid herpesvirus-3 DNA in lake plankton

The disease caused by cyprinid herpesvirus-3 (CyHV-3) severely impacts the natural freshwater ecosystem and damages carp and koi farming, however, the pathway of CyHV-3 transmission remains unclear. It is possible that the virus adheres to plankton, which then facilitate viral movement and transmission, and therefore, it is hypothesised that plankton are involved in the disease dynamics. In this study, plankton were collected at eight sites in the Iba-naiko lagoon; we detected and quantified CyHV-3 DNA from plankton samples. The results of the correlation analysis showed a significant positive correlation between CyHV-3 copies and the number of Rotifera, suggesting that CyHV-3 binds to and/or is concentrated by Rotifera. Our results suggest that plankton affect viral ecology in the natural environment.     

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.     

Effects of urea and trimethylamine-N-oxide on ATPase of requiem shark myofibril and its constituents

ABSTRACT: The effects of trimethylamine- N -oxide (TMAO) on the urea-resistibility of requiem shark myofibrils were investigated, using Ca²⁺- and Mg²⁺-ATPase activities as a parameter. Both activities were hardly changed or activated up to 0.6 M urea. In contrast, the two activities both decreased to less than 50% in the presence of TMAO up to 0.5 M. When measured at a 2 : 1 molar ratio of urea and TMAO, Ca²⁺- and Mg²⁺-ATPase activities were similar to those in the presence of TMAO alone, indicating that TMAO reduced the urea-resistibility of myofibrils. Myosin, the most abundant protein in myofibrils, from requiem shark exhibited the effects of urea and TMAO on its Ca²⁺-ATPase activity, which was primarily similar to those of myofibrils. However, Ca²⁺-ATPase activities in the coexistence of urea and TMAO for actomyosin reconstituted from requiem shark myosin and chicken F-actin were approximately average of those measured independently in the presence of either urea or TMAO alone. Carp myofibrils, reconstituted actomyosin and myosin, which were used as teleost references, all showed a tendency in the effects of urea and TMAO on Ca²⁺-ATPase activities that was similar to those of requiem shark counterparts.     

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.     

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.     

Increase in muscle endurance in mice by dietary Yamabushitake mushroom (Hericium erinaceus) possibly via activation of PPARδ

Skeletal muscle fiber is largely classified into two types: type 1 (slow‐twitch) and type 2 (fast‐twitch) fibers. Meat quality and composition of fiber types are thought to be closely related. Previous research showed that overexpression of constitutively active peroxisome proliferator‐activated receptor (PPAR)δ, a nuclear receptor present in skeletal muscle, increased type 1 fibers in mice. In this study, we found that hexane extracts of Yamabushitake mushroom (Hericium erinaceus) showed PPARδ agonistic activity in vitro. Eight‐week‐old C57BL/6J mice were fed a diet supplemented with 5% (w/w) freeze‐dried Yamabushitake mushroom for 24 hr. After the treatment period, the extensor digitorum longus (EDL) muscles were excised. The Yamabushitake‐supplemented diet up‐regulated the PPARδ target genes Pdk4 and Ucp3 in mouse skeletal muscles in vivo. Furthermore, feeding the Yamabushitake‐supplemented diet to mice for 8 weeks resulted in a significant increase in muscle endurance. These results indicate that Yamabushitake mushroom contains PPARδ agonistic ligands and that dietary intake of Yamabushitake mushroom could activate PPARδ in skeletal muscle of mice. Unexpectedly, we observed no significant alterations in composition of muscle fiber types between the mice fed control and Yamabushitake‐supplemented diets.     

Prevalence of intestinal parasites and genotyping of Giardia intestinalis in pet shop puppies in east Japan

The current study examined the prevalence of intestinal parasites and genotypes of Giardia intestinalis in puppies from nine pet shops in east Japan. Fresh fecal samples from 1794 puppies (≦3 months old) were collected on one occasion. Giardia spp. was examined for specific coproantigen using ELISA kit (SNAP^®Giardia, IDEXX Laboratories, Inc., USA). Other intestinal parasites were detected microscopically using the formalin-ethyl acetate sedimentation technique. Genotyping was determined for the random 29 stool samples identified as Giardia spp. positive using PCR and direct sequencing of the glutamate dehydrogenase (gdh) gene. Overall prevalence of protozoan Giardia spp. and Cystoisospora spp. revealed 23.4% and 11.3%, respectively. Prevalence of ascarids, Strongyloides spp. and hookworms were recorded 1.8%, 1.1% and 0.1%, respectively. Protozoan Giardia spp. and Cystoisospora spp., thus, represent important pathogens among pet shop puppies. All genotyped G. intestinalis isolates were belonged to assemblage C or D, identified as dog-specific genotypes. Zoonotic assemblage A and B were not demonstrated. The result suggests that the risk of zoonotic transmission of G. intestinalis from pet shops puppies to humans may be quite low in Japan.     

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.