OBJECTIVETo observe the expression of receptor activator of NF-κB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) during unloading period of dental implants.

METHODSAn animal model of dental implants was established in Beagle dogs. Bone remodeling was tested at 3, 7, 15, 30, 60 and 90 days after the placement of implants. RANKL and OPG mRNA expression were quantified by real-time PCR. Then mandibular bones were resected and some sections were observed.

RESULTSThe most prominent period of bone remodeling occurred at 7 day after the placement of implants (OPG/RANKL mRNA, 2.15 ± 0.1). The expression of RANKL and OPG increased in a time-dependent manner in both soft and hard tissue. After 7 days they gradually decreased.

CONCLUSIONSBoth OPG and RANKL were expressed in peri-implant tissues, and the changing tendency of RANKL and OPGmRNA was consistent with the change of bone remodeling. The active stage for bone remodelling in peri-implant tissues during unloading period is about 7 days after implantation.

Animals ; Bone Remodeling ; genetics ; Dental Implantation ; Dogs ; Male ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism

The fields of pharmacogenetics and pharmacogenomics have become increasingly promising regarding the clinical application of genetic data to aid in prevention of adverse reactions. Specific screening tests can predict which animals express modified proteins or genetic sequences responsible for adverse effects associated with a drug. Among the genetic variations that have been investigated in dogs, the multidrug resistance gene (MDR) is the best studied. However, other genes such as CYP1A2 and CYP2B11 control the protein syntheses involved in the metabolism of many drugs. In the present study, the MDR-1, CYP1A2 and CYP2B11 genes were examined to identify SNP polymorphisms associated with these genes in the following four canine breeds: Uruguayan Cimarron, Border Collie, Labrador Retriever and German Shepherd. The results revealed that several SNPs of the CYP1A2 and CYP2B11 genes are potential targets for drug sensitivity investigations.
Animals ; Aryl Hydrocarbon Hydroxylases/*genetics/metabolism ; Cytochrome P-450 CYP1A2/*genetics/metabolism ; Dogs/*genetics/metabolism ; P-Glycoprotein/*genetics/metabolism ; *Polymorphism, Single Nucleotide ; Steroid Hydroxylases/*genetics/metabolism

The study was purposed to prepare the recombinant lentiviral vector pTK161 and pTK162 carrying B-domain-deleted canine factor (BDDcFVIII) gene, and to investigate whether the canine FVIII (cVIII) can be expressed in vitro. The BDDcFVIII gene was ligated behind PUB and 2OH1 promotors to create lentiviral vectors pTK161 and pTK162. Meantime lentiviral vectors pTK161' and pTK161' were produced by cloning a green fluorescent protein (GFP) into pTK151 and pTK152, which was driven by PUB and 2OH1 promotors respectively. Vector supernatant were prepared by using transfer calcium phosphate mediated-cotransfection of 293T cells. The virus vector, DeltaNRF packaging-plasmid, and VSV-G envelope-plasmid was assayed by titers and cFVIII activity in cell culture supernatant after infection into 293T cells. pTK161, pTK162, pTK161' and pTK161' were identified by restriction enzyme analyzing. The results showed that the lentiviral vectors pTK161, pTK162, pTK161' and pTK161' were successfully constructed, and the titers of pTK161' and pTK161' reached to 1.54 x 10(6) U/ml and 2.83 x 10(6) U/ml; the activity of cFVIII could be detected at 24 hours after infection of 293T cells by pTK161 and pTK162, and achieved the highest level at 72 hours later. The higher level of cFVIII activity was achieved by transfected with pTK162 than that of pTK161 (p < 0.05), which closed to the cFVIII activity in normal dog plasma. 1/4 of the highest level could be detected 6 weeks later. It is concluded that the prepared HIV1-based lentiviral vectors can infect 293T cells to express cFVIII effectively. The results provide the basis for further studying HIV-1-based lentiviral vector gene therapy for hemophilia A.
Animals ; Dogs ; Factor VIII ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo construct a three plasmids lentiviral vector containing canine coagulation factor IX (cFIX) gene with ubiquinone promoter (PUB) and observe the expression of cFIX gene.

METHODSLentivirus was generated by transient three-plasmid transfection, namely, the VSV-G envelope expression cassette, the delta NRF packaging plasmid and the PTK 164 plasmid. Viral particles were used to infect the target cell, third passage mesenchymal stem cells (MSCs) and 293T cell respectively at MOI 3: 1. The cFIX activity was detected in cultured cells with one-stage clotting assay.

RESULTSThe MSCs were obtained in vitro. The lentivirus infected MSCs and 293T cells all expressed the active factor IX with the activity of (26.30 +/- 2.10)% and (19.70 +/- 1.53)%, respectively, which are significantly higher than that of control (1.00 +/- 0.05)%.

CONCLUSIONSThe lentiviral vector of three plasmids with ubiquinone promoter (PUB) was constructed and can transfect the MSCs and 293T cells.

Animals ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Dogs ; Factor IX ; genetics ; metabolism ; Genetic Vectors ; Hemophilia B ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; Plasmids ; genetics ; Transfection

The present study is aimed to study the effect of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) gene transfer on the contractile function of isolated cardiomyocytes of canines. The cardiomyocytes were isolated with collagenases. The isolated cardiac cells were divided into untransfected group, empty vector group and SERCA2a-transfected group. Recombinant adenovirus vector carrying enhanced green fluorescent protein gene was used for SERCA2a gene delivery. The expression of SERCA2a protein in cardiomyocytes was determined by Western blot. Contractile function of cardiomyocytes was measured with motion edge-detection system of single cell at 48 h after transfection. The results showed, compared with untransfected group, SERCA2a protein level, percentage of peak contraction amplitude under normal condition, percentages of peak contraction amplitude under Ca(2+) or isoproterenol stimulation, time-to-peak contraction (TTP) and time-to-50% relaxation (R50) in SERCA2a-transfected group all increased significantly. While all the above indices in empty vector group did not show any differences with those in untransfected group. These results suggest that the overexpression of SERCA2a by gene transfer may enhance the contraction function of canine myocardial cells.
Adenoviridae ; genetics ; metabolism ; Animals ; Dogs ; Male ; Myocardial Contraction ; drug effects ; physiology ; Myocytes, Cardiac ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; metabolism ; Transfection

Increasing evidence suggests that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. Additionally, it is becoming apparent that cyclooxygenase (COX) signaling is associated with canine mammary tumor development. The goals of the present study were to investigate COX-2 expression patterns and their effect on CSC-mediated tumor initiation in primary canine mammary tissues and tumorsphere models using immunohistochemistry. Patterns of COX-2, CD44, octamer-binding transcription factor (Oct)-3/4, and epidermal growth factor receptor (EGFR) expression were examined in malignant mammary tumor (MMT) samples and analyzed in terms of clinicopathological characteristics. COX-2 and Oct-3/4 expression was higher in MMTs compared to other histological samples with heterogeneous patterns. In MMTs, COX-2 expression correlated with tumor malignancy features. Significant associations between COX-2, CD44, and EGFR were observed in low-differentiated MMTs. Comparative analysis showed that the levels of COX-2, CD44, and Oct-3/4 expression varied significantly among TSs of three histological grades. Enhanced COX-2 staining was consistently observed in TSs. Similar levels of staining intensity were found for CD44 and Oct-3/4, but EGFR expression was weak. Our findings indicate the potential role of COX-2 in CSC-mediated tumor initiation, and suggest that COX-2 inhibition may help treat canine mammary tumors by targeting CSCs.
Animals ; Antigens, CD44/genetics/metabolism ; Biomarkers, Tumor/genetics/metabolism ; Cell Transformation, Neoplastic/*genetics/metabolism ; Cyclooxygenase 2/*genetics/metabolism ; Dog Diseases/*genetics/metabolism ; Dogs ; Female ; Immunohistochemistry/veterinary ; Mammary Neoplasms, Animal/*genetics/metabolism ; Mammary Neoplasms, Experimental/*genetics/metabolism ; Neoplastic Stem Cells/*metabolism ; Octamer Transcription Factor-3/genetics/metabolism ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Retrospective Studies

To express recombinant Ancylostoma caninum anticoagulant peptide-c2 (AcAPc2), a whole cDNA fragment encoding AcAPc2 was achieved by ligation- PCR and inserted into prokaryotic expression vector pTWIN1 for constructing the specific self-splicing prokaryotic expression vector, pTWIN1-AcAPc2; positive recombinants were transformed into E. coli ER2566 for expression research. The recombinant protein, AcAPc2-intein2-CBD, was soluble and expressed in E. coli ER2566 (about 30.1% fusion protein in total protein). AcAPc2-intein2-CBD was characterized to be 41 KD by SDS-PAGE and identified by Western-blot. The recombinant fusion protein was purified to a efficiently high degree by chitin affinity chromatography. After the process of specific self-splicing induced by beta-Mercaptoethanol, the target protein, AcAPc2, was obtained, characterized to be 21 KD by SDS-PAGE and migrated as a dimmer. Molecular weight of AcAPc2 conformed to native dimmer. Bio-information analysis indicated relationship between secondary construction of AcAPc2 and biologic function. These findings greatly facilitate the purification of AcAPc2 and are very important for the additional studies on its anti-coagulation mechanism and its clinical application as anti-coagulation medicine.
Animals ; Dogs ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genes, Helminth ; Genetic Vectors ; Helminth Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; RNA Splicing ; Recombinant Fusion Proteins ; chemistry ; pharmacology

Adhesion through microbial surface components that recognize adhesive matrix molecules is an essential step in infection for most pathogenic bacteria. In this study, we report that LigB interacts with fibronectin (Fn) through its variable region. A possible role for LigB in bacterial attachment to host cells during the course of infection is supported by the following observations: (i) binding of the variable region of LigB to Madin-Darby canine kidney (MDCK) cells in a dose-dependent manner reduces the adhesion of Leptospira, (ii) inhibition of leptospiral attachment to Fn by the variable region of LigB, and (iii) decrease in binding of the variable region of LigB to the MDCK cells in the presence of Fn. Furthermore, we found a significant reduction in binding of the variable region of LigB to Fn using small interfering RNA (siRNA). Finally, the isothermal titration calorimetric results confirmed the interaction between the variable region of LigB and Fn. This is the first report to demonstrate that LigB binds to MDCK cells. In addition, the reduction of Fn expression in the MDCK cells, by siRNA, reduced the binding of LigB. Taken together, the data from the present study showed that LigB is a Fn-binding protein of pathogenic Leptospira spp. and may play a pivotal role in Leptospira-host interaction during the initial stage of infection.
Animals ; Antigens, Bacterial/*genetics/metabolism ; Cell Line ; Dogs ; Enzyme-Linked Immunosorbent Assay ; Fibronectins/*metabolism ; Immunoglobulin Variable Region/genetics/*metabolism ; Leptospira/*genetics/metabolism ; Microscopy, Confocal ; Protein Binding/*genetics ; *Protein Structure, Tertiary ; RNA, Small Interfering/genetics

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.
Animals ; Animals, Genetically Modified ; Cloning, Organism/methods/*veterinary ; Dogs/*genetics ; Female ; Gastrointestinal Tract/metabolism ; Gene Expression Regulation ; Kidney/metabolism ; Liver/metabolism ; Luminescent Proteins/*genetics/metabolism ; Lung/metabolism ; Male ; Myocardium/metabolism ; Nuclear Transfer Techniques/veterinary ; Spleen/metabolism ; Trachea/metabolism

OBJECTIVETo investigate the expressions of gamma aminobutyric acid transporter 1 (GAT-1) and glutamate decarboxylase 65 (GAD65) mRNA in different brain regions at brain propofol uptake equilibrium in dogs.

METHODSEighteen 12- to 18-month-old healthy hybrid dogs were randomized equally into control group (group C), low dose group (group L), and high dose group (group H). In groups L and H, anesthesia was administered by intravenous injection of 5.5 and 7.0 mg/kg propofol followed by propofol infusion at a constant rate of 55 and 70 mg·kg(-1)·h(-1) for 50 min, respectively. Blood samples were taken from the internal carotid artery and jugular vein to measure plasma propofol concentrations, and the brain tissues of the hypothalamus, sub thalamus, dorsal thalamus, hippocampus, pons, parietal lobe and frontal lobe were examined for GAT-1 and GAD65 mRNA expressions using quantitative real-time PCR.

RESULTSIn groups L and H, propofol infusion at a constant rate for 50 min resulted in comparable plasma propofol concentrations between the internal carotid artery and jugular vein (P>0.05), but the concentrations differed significantly between the two groups (P<0.01). GAT-1 mRNA levels in the hypothalamus and hippocampus were significantly higher in groups L and H than in group C (P<0.05 and P<0.01), but comparable between the former two groups. The variations of GAT-1 mRNA levels between the hypothalamus and hippocampus were similar in both group L [(61.26∓7.17)% and (79.34∓39.95)%, P>0.05] and group H [(74.64∓19.63)% and (97.12∓32.31)%, P>0.05]. GAT-1 mRNA levels in other brain regions showed no significant difference among the 3 groups. GAD65 mRNA levels were similar between group L and group H, but both significantly higher than that in group C (P<0.01). GAD65 mRNA in other brain regions had no significant difference among the 3 groups.

CONCLUSIONGAT-1 mRNA in the hypothalamus and hippocampus and GAD65 mRNA in the dorsal thalamus are upregulated when propofol uptake reaches an equilibrium in the brain of dogs.

Animals ; Brain ; drug effects ; metabolism ; Dogs ; GABA Plasma Membrane Transport Proteins ; genetics ; metabolism ; Glutamate Decarboxylase ; genetics ; metabolism ; Hippocampus ; drug effects ; metabolism ; Hypothalamus ; drug effects ; metabolism ; Propofol ; pharmacology ; RNA, Messenger ; genetics ; Thalamus ; drug effects ; metabolism