Comparative study of dermal components and plasma TGF-β1 levels in Slc39a13/Zip13-KO mice

Ehlers-Danlos syndrome (EDS) is a group of disorders caused by abnormalities that are identified in the extracellular matrix. Transforming growth factor-β1 (TGF-β1) plays a crucial role in formation of the extracellular matrix. It has been reported that the loss of function of zinc transporter ZRT/IRT-like protein 13 (ZIP13) causes the spondylocheiro dysplastic form of EDS (SCD-EDS: OMIM 612350), in which dysregulation of the TGF-β1 signaling pathway is observed, although the relationship between the dermis abnormalities and peripheral TGF-β1 level has been unclear. We investigated the characteristics of the dermis of the Zip13-knockout (KO) mouse, an animal model for SCD-EDS. Both the ratio of dermatan sulfate (DS) in glycosaminoglycan (GAG) components and the amount of collagen were decreased, and there were very few collagen fibrils with diameters of more than 150 nm in Zip13-KO mice dermis. We also found that the TGF-β1 level was significantly higher in Zip13-KO mice serum. These results suggest that collagen synthesis and collagen fibril fusion might be impaired in Zip13-KO mice and that the possible decrease of decorin level by reduction of the DS ratio probably caused an increase of free TGF-β1 in Zip13-KO mice. In conclusion, skin fragility due to defective ZIP13 protein may be attributable to impaired extracellular matrix synthesis accompanied by abnormal peripheral TGF-β homeostasis.     

Effect of dietary kapok oil supplementation on growth performance,carcass traits,meat quality and sensory traits of pork in finishing‐pigs

Kapok seed and oil from the tropical zone are widely used as pig feed to harden porcine fat in Japan. This study evaluated the effect of dietary kapok oil supplementation on pork quality and sensory traits. Five Duroc pigs each were assigned to an experimental group supplemented with kapok oil and a control group. Dietary kapok oil supplementation had no effect on growth performance and intramuscular fat content in the Longissimus dorsi muscle (LM). Supplemental kapok oil increased saturated fatty acid contents in subcutaneous and intramuscular fat and decreased monounsaturated fatty acid levels (P < 0.05). Off‐flavor detection by a trained panel was higher in the experimental than the control group (P < 0.05), but tenderness, juiciness, texture and flavor intensity of LM chops were similar in both groups. The overall palatability of pork as judged by a consumer panel decreased with kapok oil supplementation (P < 0.01). These results indicate that while growth performance, intramuscular fat contents and carcass characteristics were unchanged, while dietary kapok oil supplementation makes firm fat to prevent inferior soft fat in pork, it can lower the palatability of pork due to a decrease in monounsaturated fatty acids.     

Transgenic Pigs with Pancreas-specific Expression of Green Fluorescent Protein

The development and regeneration of the pancreas is of considerable interest because of the role of these processes in pancreatic diseases, such as diabetes. Here, we sought to develop a large animal model in which the pancreatic cell lineage could be tracked. The pancreatic and duodenal homeobox-1 (Pdx1) gene promoter was conjugated to Venus, a green fluorescent protein, and introduced into 370 in vitro-matured porcine oocytes by intracytoplasmic sperm injection-mediated gene transfer. These oocytes were transferred into four recipient gilts, all of which became pregnant. Three gilts were sacrificed at 47–65 days of gestation, and the fourth was allowed to farrow. Seven of 16 fetuses obtained were transgenic (Tg) and exhibited pancreas-specific green fluorescence. The fourth recipient gilt produced a litter of six piglets, two of which were Tg. The founder Tg offspring matured normally and produced healthy first-generation (G1) progeny. A postweaning autopsy of four 27-day-old G1 Tg piglets confirmed the pancreas-specific Venus expression. Immunostaining of the pancreatic tissue indicated the transgene was expressed in β-cells. Pancreatic islets from Tg pigs were transplanted under the renal capsules of NOD/SCID mice and expressed fluorescence up to one month after transplantation. Tg G1 pigs developed normally and had blood glucose levels within the normal range. Insulin levels before and after sexual maturity were within normal ranges, as were other blood biochemistry parameters, indicating that pancreatic function was normal. We conclude that Pdx1-Venus Tg pigs represent a large animal model suitable for research on pancreatic development/regeneration and diabetes.     

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.     

High susceptibility to zymbal gland and intestinal carcinogenesis in diabetic Otsuka long-evans Tokushima Fatty rats

Diabetes mellitus (DM) and obesity are believed to be risk factors for colorectal cancer in humans. In experiment 1, male nondiabetic Long-Evans Tokushima Otsuka (LETO) rats and Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model animal of type 2 DM, were whole-body X-irradiated (4 Gy) at 6 and 8 weeks of age and euthanized at 78 weeks of age (n=15, respectively). The incidences of small intestine adenocarcinoma in LETO and OLETF rats were 0% and 30%, respectively. In experiment 2, male LETO and OLETF rats (n=24, respectively) were given s.c. injections of 15 mg/kg azoxymethane (AOM) once weekly for 3 weeks and euthanized at 36 weeks of age. The incidences of Zymbal gland tumors in LETO and OLETF rats were 0% and 67%, respectively (P<0.001), whereas those of small intestine adenocarcinoma were 0% and 43% (P<0.001) and those of cecum/colon adenocarcinoma were 46% and 79% (P<0.05), respectively. Fatty change of hepatocytes was common in OLETF rats (63%) but not in LETO rats. Serum triglyceride and free fatty acid levels in OLETF rats were significantly higher than in LETO rats at sacrifice, whereas serum insulin levels in OLETF rats were very diverse. These data suggest that hyperlipidemia plays a significant role in high susceptibility to lower intestinal tract carcinogenesis in OLETF rats; this strain is susceptible to AOM-induced Zymbal gland carcinogenesis.     

Possible origin of CSF antibodies induced by intrathecal immunization and prophylactic effects against intracerebral rabies virus infection

Intrathecal (IT) immunization involves injecting antigens directly into the intraventricular or subarachnoid spaces, or brain, to induce antigen-specific antibodies (Ab) in the cerebrospinal fluid (CSF). In the present study, rabbits were immunized IT with inactivated rabies virus to investigate the origins of CSF Ab. The time course of Ab induction and tumor necrosis factor-alpha expression suggested the possibility that the CSF Ab originated in the serum. In addition, Ab-producing cells infiltrated around the blood vessels of the brain, suggesting local production of Ab within the central nervous system (CNS). Furthermore, subcutaneous (SC) immunization prior to IT immunization induced a rapid and magnified Ab response in the CSF compared with IT immunization alone. These results were confirmed by the fact that mice immunized SC prior to IT were more resistant to intracerebral challenge with rabies virus than mice immunized via the IT route alone. Taken together, these results suggest that combined SC and IT immunization is a more effective vaccination protocol for prophylaxis and treatment of rabies.     

Contraceptive effect of a gonadotropin-releasing hormone vaccine on a captive female African Lion (Panthera leo): a case study

Lions (Panthera leo) breed well under captivity, so contraception has been commonly conducted for population management, leading to a demand for a less invasive and reversible contraceptive approach in lions. In this study, we examined the efficacy of a commercial gonadotropin-releasing hormone vaccine as a method of suppressing reproductive activity in a sexually matured female lion. Under behavioral restraint, the vaccine was injected twice (days 0 and 109). After the initial vaccination, ovarian activity is still observed. After the second vaccination, contraceptive effect was confirmed for 246 days until restart of estrous cycles. We confirmed only a slight swelling around the injection site after the second vaccination. This study may suggest an alternative option for a contraceptive method in lions.     

Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by In Vitro Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.     

Genetic and antigenic characterization of bovine viral diarrhea viruses isolated from cattle in Hokkaido,Japan

In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5′-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-1a (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.     

Structural gene and strain specificity of a novel cysteine protease produced by Staphylococcus aureus isolated from a diseased chicken

Recently whole genome sequencing of Staphylococcus aureus has revealed the genes encoding cysteine proteases such as staphopain and SspB. In this study, we cloned and sequenced the structural gene (ScpA) encoding a cysteine (thiol) protease of S. aureus strain CH-91 from a chicken with dermatitis using polymerase chain reaction (PCR) and inverse PCR methods. The sequence information revealed a coding sequence (CDS) of 1200 nucleotides encoding the ScpA preproenzyme of 399 amino acids with a molecular mass of 45,071 Da. The deduced amino acid sequence of the ScpA differed at many positions from those of staphopain and SspB with identities of 64 and 42%, respectively. In the Southern blot analysis with a total DNA of S. aureus strain CH-91, the ScpA probe hybridized with a single 7.7 kb XbaI fragment or 2.8 and 0.8 kb EcoRI fragments, whereas the staphopain and SspB probes did not hybridize with these DNA fragments. These results suggest that this ScpA gene is a single-copy gene and is a novel gene, which is not found in the published whole genome sequences of S. aureus. In immunoblot, PCR, and Southern blot assays, the ScpA or its gene was detected in high protease-producing strains from chickens, but was not recognized in bovine and porcine strains or low protease-producing avian strains. These results indicate that the ScpA of CH-91 type may be specific to the high protease-producing strains of S. aureus from chickens, namely, there is a strain specificity of the ScpA.