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

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

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

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

OBJECTIVETo observe the regional distribution of propofol in canine spinal cord under noxious stimulation.

METHODSTwelve healthy hybrid dogs (12-18 months old, weighing 10-12 kg) were randomly divided into control group (n=6) and stimulation group (n=6). All the dogs were anesthetized with a single bolus dose of propofol (7 mg/kg) in 15 seconds followed by propofol infusion at a constant rate of 70 mg/kg/h via the great saphenous vein of the right posterior limb. In the stimulation group, the tails of the dogs were clamped for 5 min after 45 min of propofol infusion. Blood samples were taken from the internal carotid artery and internal jugular vein at 50 min after propofol infusion to detect plasma propofol concentrations by high-pressure liquid chromatography (HPLC). The dogs were then immediately sacrificed by decapitation and the frontal horn, posterior horn, intermediate zone, frontal funiculus, posterior funiculus and lateral funiculus of the spinal cord were dissected for determination of propol content by HPLC.

RESULTSThe plasma concentrations of propofol in the internal carotid artery and internal jugular vein were 5.07-/+0.23 and 5.03-/+0.10 microg/ml in the stimulation group, respectively showing no significant differences from those in the control group (5.09-/+0.03 and 5.08-/+0.03 microg/ml, P>0.05). In the control group, the propofol concentration was 5.09-/+0.08 microg/g in the frontal horm, 5.10-/+0.08 microg/g in the posterior horn, 5.05-/+0.19 microg/g in the intermediate zone, 5.06-/+0.14 microg/g in the frontal funiculus, 5.06-/+0.15 microg/g in the posterior funiculus and 5.06-/+0.41 microg/g in the lateral funiculus, showing no significant differences (P>0.05). The propofol concentrations in the frontal horn (7.65-/+0.47 microg/g) and posterior funiculus (7.06-/+0.82 microg/g) in the stimulation group were significantly higher than those in the other spinal cord tissues (P<0.05) and those in the control group (P<0.05).

CONCLUSIONAt 50 min after intravenous injection of propofol at a constant rate of 70 mg/kg/h, plasma propofol concentrations in the internal carotid artery and internal jugular vein reaches equilibrium with a balanced distribution in all the spinal cord regions. Propofol concentration can be higher in the frontal horn and posterior funiculus under noxious stimulation.

Animals ; Dogs ; Female ; Male ; Nociceptors ; drug effects ; physiology ; Pain ; physiopathology ; Physical Stimulation ; Propofol ; administration & dosage ; pharmacokinetics ; pharmacology ; Random Allocation ; Spinal Cord ; metabolism

BACKGROUNDHeart failure (HF) is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging as promising therapeutic modalities in HF patients. Our previous studies have shown that recombinant adeno-associated viral (rAAV) gene transfer of Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) can be effective in treating rats with chronic heart failure (CHF). The aim of this study was to examine the effects of SERCA2a gene transfer in a large HF animal model.

METHODSHF was induced in beagles by rapid right ventricular pacing (230 beats/min) for 30 days. A reduced rate ventricular pacing (180 beats/min) was continued for another 30 days. The beagles were assigned to four groups: (a) control group (n = 4); (b) HF group (n = 4); (c) enhanced green fluorescent protein group (n = 4); and (d) SERCA2a group (n = 4). rAAV1-EGFP (1 x 10(12) microg) and rAAV1-SERCA2a (1 x 10(12) microg) were delivered intramyocardially. SERCA2a expression was assessed by Western blotting and immunohistochemistry.

RESULTSFollowing 30 days of SERCA2a gene transfer in HF beagles its protein expression was significantly higher than in the HF group than in the control group (P < 0.05). Heart function improved along with the increase in SERCA2a expression. Left ventricular systolic function significantly improved, including the ejection fraction, left ventricular systolic pressure, maximal rate of rise of left ventricular pressure (+dp/dt(max)), and the maximal rate of decline of left ventricular pressure (-dp/dt(max)) (P < 0.05). Left ventricular end-diastole pressure significantly decreased (P < 0.05). The expression of SERCA2a in the myocardial tissue was higher in the SERCA2a group than in the HF group (P < 0.05).

CONCLUSIONSIntramyocardial injection of rAAV1-SERCA2a can improve the cardiac function in beagles induced with HF. We expect further studies on SERCA2a's long-term safety, efficacy, dosage and the optimization before using it in humans with HF.

Animals ; Blotting, Western ; Disease Models, Animal ; Dogs ; Echocardiography ; Genetic Therapy ; methods ; Green Fluorescent Proteins ; genetics ; metabolism ; Heart ; physiology ; Heart Failure ; therapy ; Hemodynamics ; Immunohistochemistry ; Myocardium ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; physiology