The purpose of this study was to determine if salivary chromogranin a secretion in dogs exhibits a circadian rhythm. Saliva sampling was performed during three different sessions occurring in three nonconsecutive 24-h periods. Sixteen healthy adult beagle dogs (8 males and 8 females) were moved to a sampling room and housed individually in cages. Saliva samples were obtained every 4 h from 12:00 p.m. to 12:00 p.m. the following day. In the interest of habituation, saliva was obtained hourly from each dog 3 h before the experiment was started. Salivary chromogranin A concentrations were measured using an enzyme-linked immunosorbent assay. No circadian rhythm was detected for salivary chromogranin A secretion, and no differences in salivary chromogranin A concentrations measured every 4 h were demonstrated during the 24-h cycle in dogs.
Animals ; Chromogranin A/*analysis/*metabolism ; *Circadian Rhythm ; Dogs/*physiology ; Saliva/*chemistry

OBJECTIVETo investigate electrophysiology of cardiocytes in ligament of Marshall.

METHODSThe single cardiocytes obtained from ligament of Marshall were direct observed under inverted microscope. The cardiocyte action potential and current density of I(Na), I(Ca), L, I(to), I(K) and I(K1) were researched by whole-cell patch-clamp techniques.

RESULTSThere were two different cardiomyocytes in ligament of Marshall, one was rod shape, the other was short-rectangle shape. The short-rectangle myocyte was short and thick; the rod myocyte was long and thin. The short-rectangle myocyte was more than rod myocyte. The length/width rate of short-rectangle myocyte was less than that of rod myocyte (2.99 +/- 0.95 vs 12.05 +/- 2.41, P < 0.01). The action potential of ligament myocytes was similar to fast responsive cells. The action potential amplitude (APA) and duration (APD) of short-rectangle cells were less than those in rod cells. APA (mV), APD(50) (ms) and APD(90) (ms) were respectively 80.02 +/- 3.68 vs 91.72 +/- 7.56, 69.62 +/- 6.33 vs 83.14 +/- 3.66 and 107.55 +/- 4.25 vs 144.00 +/- 5.15, P < 0.05. The ion current density of I(Na), I(Ca), L, I(to), I(K1) was different between the two kind cells.

CONCLUSIONSThere are two different cardiocytes in ligament of Marshall. The action potential and ion current density of I(Na), I(Ca), L, I(to), I(K1) are different between the two kind cardiocytes.

Action Potentials ; Animals ; Dogs ; Electrophysiology ; Ion Channel Gating ; Ligaments, Articular ; metabolism ; Male ; Myocytes, Cardiac ; metabolism ; physiology ; Patch-Clamp Techniques

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

OBJECTIVETo investigate the role of pulp in the root resorption of primary teeth without permanent tooth germs.

METHODSThe animal model without permanent tooth germs was established by surgery in Beagle dog. The root resorption was observed by taking periapical radiographs periodically. The samples of mandibular bone and pulp at different resorption stages were collected. The distribution of odontoclasts and the activating factor was analyzed by histological staining and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). The role of pulp in the root resorption of primary teeth was tested by early pulpectomy.

RESULTSIn the root resorption of primary molars without permanent teeth germs, a large number of odontoclasts were present on the pulpal surface of the root canal. Semi-quantification RT-PCR showed that the ratios of the expression of receptor activator of NF-κB ligand (RANKL) mRNA and β-actin in the pulp of permanent teeth and primary teeth without permanent teeth germ during different periods of root resorption are 0.1314, 0.1901, 0.2111 and 0.6058 (P > 0.05). The root resorption of primary teeth without permanent teeth germs in test groups was about 5 weeks later than that of control group.

CONCLUSIONSThe pulp of primary tooth played an important role in the root resorption of primary tooth without permanent tooth germ.

Actins ; metabolism ; Animals ; Dental Pulp ; metabolism ; physiology ; Dental Pulp Cavity ; metabolism ; Dogs ; Male ; Molar ; Osteoclasts ; cytology ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Root Resorption ; metabolism ; Tooth Germ ; Tooth, Deciduous ; physiology

Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.
Animals ; Cattle ; Dogs ; Hemagglutinins, Viral ; chemistry ; metabolism ; Influenzavirus C ; physiology ; Orthomyxoviridae Infections ; metabolism ; virology ; Protein Conformation ; Protein Folding ; Protein Processing, Post-Translational ; Viral Fusion Proteins ; chemistry ; metabolism

To study the transport mechanisms of drugs for transplacental treatment of fetal tachyarrhythmia, MDCKII-BCRP and MDCKII cell models was used. MDCKII-BCRP and MDCKII cell monolayer model was used to investigate the bi-direction transport of sotalol, propranolol, propafenone, procainamide and flecainide. Drug concentrations were measured by HPLC-UV or chemiluminescence. The apparent permeability coefficient (P(app)), efflux rate (R(E)) and net efflux rate (R(net)) were calculated. Drugs with R(net) greater than 1.5 were further investigated using cellular accumulation experiments with or without a BCRP inhibitor. The R(net) of sotalol, propranolol, propafenone and procainamide were less than 1.5, while R(net) of flecainide with concentrations of 20 and 5 μmol x L(-1) were 1.6 and 1.9, respectively. The results showed that the transport of flecainide on MDCKII-BCRP cell monolayer could be mediated by BCRP; and the affinity increased when the concentration of flecainide decreased. Cellular accumulation experiments further suggested that accumulation of flecainide in MDCKII-BCRP cells was significantly lower than that in MDCKII cells in a concentration-dependent manner. BCRP inhibitor quercetin (50 μmol x L(-1)) significantly increased the accumulation of flecainide in MDCKII-BCRP cells (P < 0.05). Our preliminary data showed that flecainide but not sotalol, propranolol, propafenone or procainamide can be a substrate of BCRP. Thus the effect of flecainide may be affected by the BCRP in the maternal placental trophoblast membrane layer when treating fetal tachyarrhythmia.
Animals ; Biological Transport ; Cell Membrane Permeability ; Dogs ; Female ; Flecainide ; metabolism ; Madin Darby Canine Kidney Cells ; metabolism ; Placenta ; physiology ; Pregnancy ; Tachycardia ; drug therapy

OBJECTIVETo construct pseudovirus bearing H5N1 HA based on a lentivirus vector system. Then we study the biological feature of the pseudovirus. With the newly established viral particles, we performed the serological tests.

METHODSH5N1 avian influenza virus that isolated from human case was cloned to construct pLP-HA, then pLP-HA co-transfected with lentivirus vector plasmids pLP1, pLP2 and pEmGFP into 293T cells. The supernatant was harvested 48h post-transfection. Concentrated by super centrifuge, the pseudotyped viruses were analyzed by infection test, HA test and micro-neutralization test. At the same time, optimized HA gene and a Vietnam H5N1 HA gene were used to construct pseudotyped virus for comparison.

RESULTSPseudotyped virus particles can be observed with electronic microscope. Western-blot revealed that HA glycoprotein can be expressed in the virions. Our neutralization assay by using the pseudoviruses was comparable with the conventional microneutralization assay with wild-type viruses. A high degree of correlation was detected.

CONCLUSIONPseudotyped Viruses coated with HA of H5N1 High Pathogenic Avian Influenza were successfully constructed; it can be used to for the microneutralization assay. The HA gene from different sources affect the efficiency of the packaging of the pseudovirus. But the optimized HA gene can not obviously improve packaging efficiency of the pseudovirus.

Animals ; Cell Line ; Dogs ; Gene Expression ; Genetic Engineering ; Genetic Vectors ; genetics ; metabolism ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; physiology ; ultrastructure ; Influenza, Human ; virology ; Lentivirus ; genetics ; metabolism ; Virion ; genetics ; physiology ; ultrastructure

This study was performed to evaluate the osteogenic effect of allogenic canine umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) mixed with beta-tricalcium phosphate (beta-TCP) in orthotopic implantation. Seven hundred milligrams of beta-TCP mixed with 1 x 10(6) UCB-MSCs diluted with 0.5 ml of saline (group CM) and mixed with the same volume of saline as control (group C) were implanted into a 1.5 cm diaphyseal defect and wrapped with PLGC membrane in the radius of Beagle dogs. Radiographs of the antebrachium were made after surgery. The implants were harvested 12 weeks after implantation and specimens were stained with H&E, toluidine blue and Villanueva-Goldner stains for histological examination and histomorphometric analysis of new bone formation. Additionally, UCB-MSCs were applied to a dog with non-union fracture. Radiographically, continuity between implant and host bone was evident at only one of six interfaces in group C by 12 weeks, but in three of six interfaces in group CM. Radiolucency was found only near the bone end in group C at 12 weeks after implantation, but in the entire graft in group CM. Histologically, bone formation was observed around beta-TCP in longitudinal sections of implant in both groups. Histomorphometric analysis revealed significantly increased new bone formation in group CM at 12 weeks after implantation (p < 0.05). When applied to the non-union fracture, fracture healing was identified by 6 weeks after injection of UCB-MSCs. The present study indicates that a mixture of UCB-MSCs and beta-TCP is a promising osteogenic material for repairing bone defects.
Animals ; Biocompatible Materials/metabolism/therapeutic use ; Bone Substitutes/*therapeutic use ; Calcium Phosphates/*therapeutic use ; Dogs ; Fetal Blood/*cytology ; Fracture Fixation/methods/veterinary ; Mesenchymal Stem Cells/*physiology ; Osteogenesis/*physiology ; Tissue Engineering/methods ; Wound Healing/physiology

This review briefly describes the origin, chemistry, molecular mechanism of action, pharmacology, toxicology, and ecotoxicology of palytoxin and its analogues. Palytoxin and its analogues are produced by marine dinoflagellates. Palytoxin is also produced by Zoanthids (i.e. Palythoa), and Cyanobacteria (Trichodesmium). Palytoxin is a very large, non-proteinaceous molecule with a complex chemical structure having both lipophilic and hydrophilic moieties. Palytoxin is one of the most potent marine toxins with an LD50 of 150 ng/kg body weight in mice exposed intravenously. Pharmacological and electrophysiological studies have demonstrated that palytoxin acts as a hemolysin and alters the function of excitable cells through multiple mechanisms of action. Palytoxin selectively binds to Na(+)/K(+)-ATPase with a Kd of 20 pM and transforms the pump into a channel permeable to monovalent cations with a single-channel conductance of 10 pS. This mechanism of action could have multiple effects on cells. Evaluation of palytoxin toxicity using various animal models revealed that palytoxin is an extremely potent neurotoxin following an intravenous, intraperitoneal, intramuscular, subcutaneous or intratracheal route of exposure. Palytoxin also causes non-lethal, yet serious toxic effects following dermal or ocular exposure. Most incidents of palytoxin poisoning have manifested after oral intake of contaminated seafood. Poisonings in humans have also been noted after inhalation, cutaneous/systemic exposures with direct contact of aerosolized seawater during Ostreopsis blooms and/or through maintaining aquaria containing Cnidarian zoanthids. Palytoxin has a strong potential for toxicity in humans and animals, and currently this toxin is of great concern worldwide.
Acrylamides ; chemistry ; isolation & purification ; toxicity ; Animals ; Anthozoa ; pathogenicity ; physiology ; Dinoflagellida ; pathogenicity ; physiology ; Dogs ; Guinea Pigs ; Haplorhini ; Humans ; Lethal Dose 50 ; Marine Toxins ; chemistry ; isolation & purification ; toxicity ; Mice ; Rabbits ; Rats ; Seaweed ; pathogenicity ; physiology ; Shellfish Poisoning ; physiopathology ; Sodium-Potassium-Exchanging ATPase ; metabolism

Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton's jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with beta-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.
Adipocytes, White/cytology/physiology ; Alkaline Phosphatase/metabolism ; Animals ; Biocompatible Materials/metabolism/*therapeutic use ; Bone Diseases/*therapy ; Bone Marrow Cells/cytology/physiology ; Calcification, Physiologic ; Calcium/metabolism ; Calcium Phosphates/metabolism/therapeutic use ; Cell Proliferation ; Dogs ; Female ; Fetal Blood/cytology/physiology ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells/cytology/*metabolism ; *Osteogenesis ; Polyesters/metabolism/therapeutic use ; Tissue Engineering/*methods ; Vascular Endothelial Growth Factor A/metabolism