Molecular cloning of canine membrane-anchored inhibitor of matrix metalloproteinase, RECK

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5% and 91.9% homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation, migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.     

Differential expression of neurofilament 200-like immunoreactivity in the main olfactory and vomeronasal systems of the Japanese newt, Cynops pyrrhogaster

Expression of neurofilament 200 (NF200)-like immunoreactivity was examined in the main olfactory system and the vomeronasal system of the Japanese newt, Cynops pyrrhogaster, using anti-porcine NF200 monoclonal antibody (clone N52) to investigate the differences in phenotypical characteristics between these systems. The entire nasal cavity was a flattened single chamber consisting of the main nasal chamber (MNC) and the lateral nasal sinus (LNS) communicating with each other. The olfactory epithelium (OE) was present in the MNC, and the vomeronasal epithelium (VNE) was in the LNS. The OE possessed only a small number of NF200-like immunoreactive receptor neurons. The olfactory nerve and the olfactory nerve layer of the main olfactory bulb also contained a small number of NF200-like immunoreactive axons. In contrast, the VNE possessed many NF200-like immunoreactive receptor neurons. The vomeronasal nerve and the vomeronasal nerve layer of the accessory olfactory bulb contained many NF200-like immunoreactive axons. These findings in the Japanese newt indicate that NF200-like immunoreactive receptor neurons constitute a major subpopulation in the VNE and a minor subpopulation in the OE. In addition, NF200-like immunoreactivity seems to be a useful marker to distinguish the vomeronasal system from the other nervous systems including the main olfactory system in the Japanese newt. The localization of a few NF200-like immunoreactive receptor neurons in the OE might indicate that pheromone-sensitive receptor neurons are intermingled in the OE of the Japanese newt.     

Distribution of type VI collagen in the cartilaginous tissue of the proximal tibia in the domestic cat

To investigate the distribution of the early stage chondrocytes during the formation and closure of epiphyseal growth plate (EGP) of the domestic cat, we examined the EGP of proximal tibiae by immunohistochemistry for type VI collagen. In the epiphyseal cartilage without the secondary ossification center (SOC) and EGP in newborn cats aged 1 and 10 days, type VI collagen-positive chondrocytes were located around the cartilage canals and articular surface. In the epiphyseal cartilage with the SOC and EGP in young cats aged 1 to 3 months, type VI collagen-positive chondrocytes were located in the upper resting zone of the EGP, and then increased throughout the resting zone along with maturation. In the adult cats with the partially closed EGP, type VI collagen-positive chondrocytes were distributed throughout the remaining EGP. These findings indicate that the early stage chondrocytes characterized with type VI collagen are continuously located in the EGP during maturation. In addition, the increase of the early stage chondrocytes and the decrease of the reserve chondrocytes in the EGP along with maturation may cause the cessation of the longitudinal growth of the EGP, and finally bring about the EGP closure.     

The dynamic expression of extracellular matrix in the bovine endometrium at implantation

Remodeling of uterine endometrial extracellular matrix (ECM) is pivotal to successful implantation and placentation, and has been well described in the rodents and humans. However, bovine endometrial ECM remodeling is still vaguely defined, especially at the time of implantation. Therefore, this study investigated the distribution of four ECMs namely, types I and IV collagen, laminin and fibronectin, from days 0 to 30 of gestation in bovine endometrium by immunofluorescence microscopy. A change in the distribution pattern of ECMs was evident by day 14 of gestation as features at this stage were clearly different from those of day 14 of the estrous cycle. The immunoreactivity of type I collagen, fibronectin and laminin decreased from day 14 of gestation and was obscured by day 24 of gestation. The type I collagen fibers formed were of thinner consistency than those of the estrous cycle and showed a coarser meshwork within the epithelium sites during the implantation period. In addition, the type IV collagen and laminin immunoreactivities of epithelial basement membrane also remarkably declined at exactly the same time. By day 30 of gestation, the four ECMs had regenerated with the formation of the placentome. In conclusion, this study reveals that remodeling of ECM is essential for the successful establishment of pregnancy in the bovine.     

Evaluation of the field application of PCR in the eradication of contagious equine metritis from Japan

The effectiveness of the polymerase chain reaction (PCR) as a field application test for the eradication of contagious equine metritis (CEM) was evaluated. Seven-thousands five-hundred and thirty-four genital swabs were collected from 4,026 Thoroughbred broodmares and stallions in Japan to test "high risk" horses as well as for general surveillance testing from 1998 to 2001. Bacterial isolation as well as PCR testing of original specimens and cultured specimens was performed for detection of Taylorella equigenitalis from genital swabs. As a result, T. equigenitalis was detected in 12 mares and 1 stallion by PCR, although the bacteria were isolated from only 2 of the PCR-positive mares. CEM-infected and carrier horses were treated by a combination of chemotherapy and surgery. Subsequent follow-up testing over a 3-year period did not detect T. equigenitalis. It was demonstrated that PCR testing was more sensitive than isolation as a method for the detection of T. equigenitalis from genital swabs of horses in the field. It was therefore suggested that a combination of PCR testing and treatment were useful measures in the eradication of CEM from Japan.     

Investigation of contributors to zinc protoporphyrin IX formation at optimum pH 5.5 in pork

We investigated zinc protoporphyrin (ZnPP) formation in pork at pH 5.5, identified the contributors to ZnPP formation, and verified the involvement of myoglobin in this process. When pork homogenate was separated into two water‐soluble fractions (>10 and <10 kDa) and an insoluble fraction, ZnPP formation was suppressed. ZnPP formation was rescued after mixing of all three fractions. Heating of the soluble <10 kDa fraction did not suppress the formation of ZnPP as opposed to heating of the soluble >10 kDa fraction, suggesting that protein(s) presents in the >10 kDa fraction contributed to ZnPP formation. Components of the soluble 10–30 kDa fractions separated by ultrafiltration were important in ZnPP formation. Exogenous myoglobin was not essential for ZnPP formation. A gel filtration study showed that soluble protein(s) with molecular weight higher than that of myoglobin was involved. Therefore, it was suggested that the soluble <10 kDa fraction, the insoluble fraction, and the soluble 10–30 kDa fraction (excluding myoglobin) are essential for ZnPP formation in pork at pH 5.5.