To obtain detailed information about the histological changes occurring in the mouse nipple during the reproductive cycle, we examined and quantified the S-phase of cell by immunohistochemical staining with bromodeoxyuridine (BrdU), and analysed histologically the subepithelial fibrous elements. The nipple markedly increased in size dramatically on days 15-18 of pregnancy. The densities of cells in the epidermis and dermis were very high during the early stages of pregnancy but low during lactation. In the epithelium of the lactiferous sinus, the densities of cells did not differ significantly among stages. The BrdU antibody labeling revealed a number of BrdU-positive cells in the basal layer of the epidermis and epithelium of the lactiferous sinus. The ratios of BrdU-positive cells to total cells in the epidermis and the epithelium of the lactiferous sinus were highest on day 15 and day 10 of pregnancy, respectively. After lactation, however, the ratios were similar to those in the virgin stage. No significant differences were detected in the dermis among all stages. The number of collagen and elastic fibers increased during lactation. These results indicate that cells in the epidermis and lactiferous sinus proliferated actively from day 10 to day 15 of pregnancy. The observation that cellular proliferation in the epithelial system of the nipple was stimulated at the early stage of pregnancy, while the dermis has two growth phases, with cellular proliferation during pregnancy and an increase in extracellular matrix during lacation, suggests that these two phenomena might be regulated by different factors.
The transport of subcutaneously injected retinyl acetate (RA, 100,000 IU/mouse, 105,470 nM) was investigated in male ICR mice (10-week-old) at 0, 3, 6, 12, 18, 24 and 72 hr after a single injection. The retinol and retinyl palmitate levels of liver homogenates, bile in the gallbladder and serum from peripheral blood were measured by high performance liquid chromatography (HPLC) method. Retinyl palmitate in the lipid droplets of hepatocytes and Ito cells was localized by a modified gold chloride staining method. Accumulation of retinyl palmitate peaked at 12 hr post-injection and decreased thereafter until 24 hr post-injection. Fluorescence microscopy revealed many fluorescent vitamin A-containing lipid droplets in hepatocytes around central veins at 12 hr post-injection, but such droplets were not observed in the vehicle control mice or at in the RA-injected mice after 18 hr of injection. Electron microscopic observation also indicated that many retinyl esters-containing lipid droplets were observed in hepatocytes around the central veins at 12 hr post-injection, but no droplets were seen in the controls or 18 hr post-injection. The retinyl palmitate levels in liver homogenates assessed by HPLC decreased from 12 to 24 hr post-injection and increased significantly in bile, while retinol in liver homogenates and serum markedly increased. One of the morphological alterations was intense vacuolization in hepatocyte cell cords from the portal toward the central vein observed at 24 hr post-injection. Transitional lipid droplets between vacuoles and lipid droplets were identified in those hepatocytes. These results of HPLC analysis of retinol and retinyl palmitate in liver homogenates, serum, and bile, together with the results of gold chloride staining suggested that subcutaneously injected RA was first incorporated in hepatocytes at 12 hr and then partially metabolized through vacuoles, transferred into the blood and secreted into the bile over a 24 hr period. Many retinyl esters-containing lipid droplets were visualized in Ito cells at 72 hr post-injection. Most of vitamin A in the liver homogenates measured by HPLC was retinyl palmitate. Therefore, the contents in those lipid droplets might be retinyl palmitate.
This report is to survey elements in frozen or freeze-dried samples of major organs of silky fowls with energy dispersive X-ray fluorescence spectrometer (EDXRF). There was not essential difference between frozen and freeze-dried samples. Of major elements, P, S, K, and Cl were always detected and Mn, Fe, Ca, and Zn were detectable in certain organs. Iodine was detected in thyroid glands. Occasionally Br was detected. The duration of freeze drying, 24 hr or 7 days, did not make significant difference in elemental analysis.
This report concerns the surface color of major organs of the silky fowl and white leghorn chicken as measured by a color analyser. Although it is obvious that organs of the silky fowl look darker than those of the white leghorn, the color measurement of these organs has not yet been reported. The authors found that color differences between silky fowls and white leghorns were significant in lung, brain, skin, and gluteal muscle. But the surface color of kidney and cardiac muscle of the two groups of fowl was not significantly different. The present data were represented in color charts.
Dehydroepiandrosterone (DHEA) is a steroid hormone which induces the peroxisome proliferation in rodents. The fatty liver induced by orotic acid and a high sucrose diet in male rats was prevented by the administration of DHEA and/or phenobarbital (PB). A significant increase in the liver weight was induced in the DHEA group (relative weight) and the DHEA + PB group (absolute and relative weight). The liver weight increased more conspicuously in the DHEA + PB group than in the DHEA group. The increase in the liver weight was caused by an increase in the cell size and peroxisome number. In addition, the administration of DHEA alone and the combination of DHEA and PB prevented the lipid droplet accumulation in hepatocytes. The administration of PB alone also prevented the accumulation of lipid droplets without any increase in the liver weight.
Avian infectious bronchitis virus (IBV) strain Connecticut A-5968 isolated from respiratory tissue of chickens in USA in the 1960s, is considered as representative of respiratory disease causing IBV strains. Specific pathogen free chicks inoculated with the strain via the cloaca replicated the virus more rapidly in their kidneys than did chicks inoculated with the same virus intratracheally. Virus passaged thirteen-times via the cloaca caused stronger nephrotropism and nephropathogenicity than the parent virus. It is suggested that cloacal inoculation of IBV provides new and interesting information concerning the mechanisms of nephropathogenicity of IBV which has became increasingly important worldwide during the last ten years.
It is known that in rodents and humans the β3-adrenergic receptor (β3-AR) is present primarily in adipocytes and plays a significant role in the adrenergic stimulation of lipolysis. We examined the expression of β3-AR mRNA in the dog and the lipomobilizing effects of β3-AR-selective agonists in vivo. Reverse transcription polymerase chain reaction of RNA extracted from dog adipose tissue produced a cDNA fragment, the nucleotide sequence of which was highly homologous to the corresponding regions of human (86.4%) and mouse (79.5%) β3-AR cDNA. The β3-AR mRNA was present at high levels in subcutaneous and visceral adipose tissues, but undetectable in other organs. When a selective β3-AR agonist, CL316, 243, was infused intravenously into beagle dogs, the plasma level of free fatty acid increased in 30 min and persisted at higher levels for several hours. ICI D7114, another β3-AR agonist, also showed a similar lipomobilizing effect, but with lower potency. β3-AR agonist infusion also increased the plasma insulin level. These results suggested that functional β3-AR is present in adipose tissues of the dog and that it is effective for in vivo lipomobilization.
The aim of this study was to evaluate the effectiveness of β3-adrenergic agonists for the treatment and prevention of obesity in the dog. When a selective β3-adrenergic agonist, CL316, 243 (0.1 mg/kg), was given orally to adult beagles every day for 5-7 weeks, body weight and girth were decreased compared with control placebo-treated dogs. Gross anatomical examinations revealed no noticeable abnormalities in CL316, 243-treated dogs, except an apparent decrease in abdominal fat. Immunohistochemical examination of perirenal adipose tissue showed a remarkable increase in brown adipocytes expressing a thermogenic protein, uncoupling protein (UCP). The increased expression of UCP and its mRNA in CL316, 243-treated dogs was also confirmed by Western blot and reverse transcription polymerase chain reaction analyses. It was concluded that treatment with a β3-adrenergic agonist stimulates UCP expression, which may lead to an increase in energy expenditure, and thereby is useful for the treatment and prevention of obesity in the dog.
Lipid and lipoprotein concentrations and apolipoprotein profile were investigated in canine pancreatitis induced by infusion with oleic acid (OA) into the accessory pancreatic duct. Pancreatitis was diagnosed by physical, hematological, biochemical and pathological examinations. In OA-treated dogs, serum triglyceride (TG) concentration was increased; however, there were no changes in serum total cholesterol (TC) and phospholipid (PL) concentrations. Serum concentrations of TG, TC, PL and total lipids (TL) in beta lipoprotein and those of TC, PL and TL in pre-beta lipoprotein were increased and those of TC, PL and TL in alpha1 lipoprotein were decreased. In apolipoprotein profile, the proportions of apolipoprotein B100 in low density lipoprotein fraction and apolipoprotein A-IV in high density lipoprotein fraction were increased. In addition, decreased proportion of apolipoprotein A-I and the appearance of serum amyloid A protein in high density lipoprotein fraction were observed. These results suggest that lipoprotein profiles observed in canine acute pancreatitis are attributed to the alterations in apolipoprotein compositions.
Stimulation and modulation of tumor necrosis factor-α (TNF-α) production during treatment of the murine macrophage-like cell line J774/JA-4 with 25 oligosaccharides were studied. Direct stimulation of TNF-α production by oligosaccharides was measured with a cytotoxic assay using the L929 cell line. Twelve samples showed a significantly higher production (P ≤ 0.01) of TNF-α than the controls. Modulation of TNF-α production by treatment with oligosaccharides, followed by stimulation with lipopolysaccharide (LPS) from E. coli, was examined using the L929 bioassay system. In three samples TNF-α production increased significantly, but in four samples, production was reduced significantly (P ≤ 0.01). No samples showed modulation of growth or viability of L929 cells within the first 26 hr. The present results are useful in the application of these oligosaccharides which is potentially applicable in medical and food technology.
Because CD4+ T cells were considered to be involved in protection against infection with Babesia microti, specific CD4+ T cells were generated in vitro from recovered BALB/c mice and their protective activity was tested in vivo. The cells produced varying amounts of interferon (IFN)-γ in vitro in response to parasite antigen. In passive transfer experiments, three out of eleven T cell clones tested exerted protective activity in the early phase after infection. However, there seemed to be no correlation between this protection and in vitro IFN-γ production by the T cell clones. Although the protection was partial and short-lived, the result provided direct evidence that CD4+ T cells play a crucial role in defense against B. microti.
The in vitro tube feeding technique is used to establish a laboratory colony of Boophilus microplus infected with Babesia bigemina. Pre-fed engorged female ticks offered 2 × 104 and 2 × 10 5/ml of B. bigemina infected bovine red blood cells (RBC) showed sporokinetes in the haemolymph smear sample, and positive signals for B. bigemina in polymerase chain reaction (PCR). Larvae laid from the ticks offered 2 × 10 5/ml of B. bigemina infected RBC showed evidence for B. bigemina infection in microscopic method and PCR. While larvae laid from the ticks offered 2 × 10 4/ml of B. bigemina infected RBC showed positive for B. bigemina in only PCR. The females offered 2 × 103/mlB. bigemina infected RBC and their larvae did not show positive evidences for B. bigemina infection. It is thought that the in vitro tube feeding technique can be a convenient method to study the relationship between ticks and tick-borne pathogens. It is also suggested that the superior sensitivity of PCR compared to the microscopic method in detection of B. bigemina from the tick sample, especially in larvae.
Fecal nematode egg counts and serum pepsinogen concentrations of cattle were determined in six Provinces of the northern (Chiangrai and Lampang), north-eastern (Khonkaen, Mahasarakham and Nakhonratchasima) and eastern (Chonburi) parts of Thailand in the dry season. Nematode eggs were detected in 82% of animals examined. Serum pepsinogen concentrations ranged from 78 to 2,951 (mean 934) mU tyrosine. Animals in Chonburi Province had higher mean values for both egg counts and serum pepsinogen concentrations than animals in the other Provinces. Two calves in Khonkaen and Mahasarakham Provinces were found to be heavily infected with Strongyloides papillosus, with 10,840 and 9,593 eggs/g feces. The source and route of S. papillosus infections remained unknown.
Effects of ketamine on the sodium (INa) and L-type calcium currents (ICa) were examined by using whole-cell patch clamp techniques in guinea pig single ventricular myocytes. The mode of action of ketamine was compared with those of quinidine, a sodium channel blocker, and verapamil, a calcium channel blocker. Ketamine (30-300 μM) inhibited both INa and ICa in a concentration-dependent manner. Quinidine (30 μM) and verapamil (0.1 μM) produced use-dependent depression of INa and ICa, respectively. The amplitude of INa elicited by the first depolarizing pulse after a long quiescent period was slightly decreased by quinidine. During a train of depolarizing pulse the current amplitude decreased gradually, and reached a steady state level in the quinidine-treated cells (use-dependent block, UDB). Verapamil produced a similar mode of inhibition of ICa, i.e., UDB. In contrast, ketamine produced significant decrease in INa and ICa elicited by the first depolarizing pulses and the decreases of both currents were not augmented during a train of depolarizing pulses. From these results, it can be concluded that ketamine produces tonic block of the cardiac sodium and calcium channels and the mode of inhibition is clearly different from UDB by quinidine and verapamil.
The decrease of plasma 5-methyltetrahydrofolic acid (5-MF) levels, postulated as an indicator of folate status, was studied following the administration of both methotrexate (MTX) alone and MTX with folic acid (FA) using rats as our experimental model. Blood and urine samples were serially collected over a 9 hr period after the administration of MTX, MTX with FA and from a control group to examine the plasma kinetics and the renal clearance of 5-MF. The pharmacokinetics of MTX and the plasma protein binding of 5-MF were also examined. The concentrations of these analytes were assayed using high performance liquid chromatography (HPLC). MTX administration produced decreased plasma 5-MF levels. This observed decrease was potentiated by oral FA administration, suggesting that the folate status was more severely altered by the coadministration of FA. The renal clearance of 5-MF also increased dose-dependently with FA (0.05-5 mg/kg) coadministration. The plasma protein binding of 5-MF was not affected by the FA administration, which indicates that the fraction of 5-MF that was filtered through the glomerular apparatus appeared to be unchanged. In addition, the pharmacokinetic profiles of MTX also appeared not to be affected by the addition of FA. We conclude that the inhibition of reabsorption of 5-MF in the renal tube by concurrent administration of MTX and FA must be one of the causal factors for the demonstrated decrease in the plasma 5-MF levels in rats.
We produced two recombinant Borna disease virus (BDV) proteins, p40 and p24, by using a baculovirus vector as a diagnostic antigen. Antigenicities of these recombinant proteins were evaluated by immune rabbit sera. Recombinant p40 was a more sensitive antigen than p24 for the detection of antibodies in infected rats. Rats inoculated with BDV within 24 hr after birth showed higher detection rates of viral RNA and viral proteins from the brain than rats inoculated at 4 weeks-old. Depending on the age of infection and the time postinfection, the detection of BDV RNA, protein, or anti-BDV antibody did not always correlate in individuals. We suggest both serological and molecular biological methods are needed in the diagnosis of BDV infection.
The efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with poly D, L lactic-co-glycolic acid (PLGA)/gelatin sponge complex (PGS) as a carrier on the repair of segmental long-bone defects was evaluated using an ulnar model in dogs. The defect was 2 cm in length and was fixed with bone plating. After implantation of PGS with or without rhBMP-2, the repair process of the defect was evaluated by serial radiography until 16 postoperative weeks. All defects treated with 160 μg or 640 μg of rhBMP-2/PGS revealed bone union radiographically by 12 postoperative weeks, whereas all defects treated with PGS alone revealed no radiographic evidence of healing throughout the experimental period. In defects treated with 40 μg of rhBMP-2/PGS, new bone appeared partially at the defects but did not accomplish union. Bone mineral contents at the defect sites after harvest at 16 weeks postoperatively were significantly (p<0.05) higher in those treated with 160 μg or 640 μg of rhBMP-2 than in those treated with 40 μg of rhBMP-2 or PGS alone. Histologically, defects radiographically diagnosed as having achieved union showed the appearance of cortical bone and bone marrow cells. These findings suggest the use of rhBMP-2/PGS as a potential bone graft substitute in reconstructive surgery in dogs.
This is the first report on successful treatment of spontaneous nonunion fracture of a dog by use of recombinant human bone morphogenetic protein-2 (rhBMP-2). A 4-year-old Pomeranian dog with a 2-year history of femoral nonunion fracture was treated with implantation of rhBMP-2. The dog had received surgical correction twice prior to the admission but radiography of the affected limb revealed a typical figure of nonunion fracture. Glossly, the fractured ends were sclerotic and the area between the ends was filled with fibrous tissue. After debridement the femoral shaft was fixed by an 8-hole plate. rhBMP-2 at a total dose of 256 μg was implanted with a synthetic carrier into the 8-mm defect formed by the debridement. Callus formation responding to rhBMP-2 was radiographically observed at 2 weeks after implantation and the defect reached radiographic union by 8 weeks after implantation. The plate was removed at 9 months after implantation. Any complications were not observed for 5 months after removal of the plate.
The effects of protein supplements and culture dish type on in vitro fertilization (IVF) and embryo development in culture were examined in the domestic cat. In Experiment I, follicular oocytes were fertilized and cultured in either 1) modified Earle's balanced salt solution, designated MK-1, supplemented with one of the following: 10% human serum (HS), 10% FCS or 0.4% BSA, or 2) Medium 199 (M-199) supplemented with 10% FCS. Fertilization rates were lower (P<0.01) in MK-1 + BSA (74.4%), MK-1 + FCS (56.1%), and M-199 + FCS (51.4%) than in MK-1 + HS (94.7%). A greater (P<0.01) percentage of blastocysts was obtained in MK-1 + HS (50.0%) than in other treatment groups (range, 4.3-17.2%). In Experiment II, the effect of dish type (tissue culture dish, TCD, versus suspension culture dish, SCD) on embryo development was evaluated in MK-1 supplemented with either HS or BSA. Significantly higher proportions of IVF-derived embryos developed to blastocysts at 120 and 144 hr post-insemination, respectively, when cultured in HS/SCD (47.2 and 71.7%) than in BSA/SCD (11.4 and 27.3%) or BSA/TCD (10.4 and 25.0%). At 120 hr post-insemination, there was a lower (P<0.01) percentage of blastocysts in HS/TCD (22.2%) than in HS/SCD. In Experiment III, six embryos per cat were transferred to the uterine horns of 17 recipients at 144 hr after hCG treatment. Five of 7 recipients which received late morulae cultured in MK-1 + BSA (SCD) for 120 hr became pregnant (71.4%). Eight of 10 recipients which received early blastocysts cultured in MK-1 + HS (SCD) for 120 hr became pregnant (80.0%). We conclude that MK-1 containing HS is highly beneficial for overcoming the in vitro developmental block of IVF-derived feline embryos and increasing the success rate of IVF/ET.
The effects of glutamine, glycine and taurine on the development of bovine zygotes derived from IVM/IVF were determined using a protein-free chemically defined medium. After the cumulus cells were removed at 18 hr post-insemination, the presumptive zygotes were cultured for 4 or 6 days (about 104 or 154 hr) under a gas atmosphere of 5% O2: 5% CO2: 90% N2. A modified synthetic oviduct fluid medium supplemented with 20 amino acids (1 mM glutamine, essential amino acids for basal medium Eagle and non-essential amino acids for minimum essential medium), insulin, and PVA was used as a basic medium (mSOFai). Omitting 1 mM glutamine from mSOFai did not affect the embryonic development after 4 and 6 days of culture. After 4 days of culture, no significant effects of glycine and taurine on the development of zygotes to the morula stage were observed. However, supplementation with glycine or taurine significantly (P<0.05) affected, with no interaction, the embryonic development to blastocysts after 6 days of culture. Addition of 5 mM glycine and 2 or 10 mM taurine significantly (P<0.05) increased the percentage of blastocysts. The mean cell number in the blastocysts was affected by the glycine level, and was increased by the addition of 10 mM glycine (P<0.001). These results demonstrate that glycine and taurine in a chemically defined medium containing a group of essential and non-essential amino acids improve the development of bovine zygotes to the blastocyst stage under 5% O2.
The effect of hypothyroidism on adrenals and gonads in adult female rats was investigated throughout the estrous cycle. Hypothyroidism was induced by administration of 4-Methyl-2-Thiouracil (Thiouracil) in the drinking water. The weight of ovaries and adrenals, and the plasma levels of corticosterone decreased in hypothyroid rats as compared with euthyroid rats throughout the estrous cycle. Hypothyroidism resulted in decreased concentrations of plasma LH on the day of diestrus and proestrus, whereas the plasma concentrations of prolactin and progesterone increased as compared with euthyroid rats. The weight of uteri and plasma concentrations of estradiol decreased during the day of diestrus and proestrus in hypothyroid rats as compared with euthyroid rats. To further clarify the dysfunction of hypothalamo-hypophysial-adrenal axis in hypothyroid rats, animals were stressed by immobilization for 3 hr. In hypothyroid rats, a marked increase in plasma levels of ACTH in response to immobilization stress was observed compared to euthyroid control, whereas increases in plasma concentrations of corticosterone were much smaller in hypothyroid than euthyroid rats. These results clearly indicate that hypothyroidism causes both gonadal and adrenal disturbances in adult female rats. The increased concentrations of plasma progesterone may be due to hypersecretion of prolactin during the day of proestrus and estrus, which in turn result in disruption of the estrous cycle.
This study examined the timing of completion of meiosis I of bovine oocytes in which meiotic resumption had been inhibited by cycloheximide (CHX), and also determined the optimum interval of maturation in culture for subsequent fertilization and development. Most oocytes treated with CHX reached metaphase II at 16 hr in the maturation culture, while control oocytes did at 20 hr. CHX-treated oocytes cultured for 16 hr were normally fertilized but failed to develop into blastocysts. Maturation in culture for 20 hr resulted in comparable development for control oocytes. The results indicate that nuclear maturation of CHX-treated oocytes was completed 4 hr faster than for control oocytes, however the same interval of maturation as that of control oocytes is necessary for subsequent development to blastocysts.
Heparin inhibited hemagglutination (HA) by equine arteritis virus (EAV) as well as did HA by Aujeszky's disease virus (ADV), but failed to inhibit HA by parainfluenza virus type 3 (PIV-3). The minimal concentration of heparin required to inhibit 8 HA U of EAV was 0.1 U/ml. In addition, most EAV hemagglutinin was retained by heparin acrylic beads, as was ADV hemagglutinin, but was not PIV-3 hemagglutinin. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. However, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by EAV. All these findings suggest that a heparin-like molecule on the surface of mouse erythrocytes serves as the virus-cell receptor.
A crane herpesvirus (CrHV) grown in chickn embryo fibroblast (CEF) cells was tested for hemagglutination (HA) with erythrocytes from a variety of species at 4°C, room temperature and 37°C. HA was observed at all temperatures with erythrocytes from mouse, ddY and BALB/c strains, but not with those from cattle, sheep and chicken. Mice, ddY strain, showed an individual variation in agglutinability of their erythrocytes and erythrocytes from BALB/c gave a higher HA titer. The HA activity was inhibited by the sera obtained from naturally infected cranes, experimentally infected duck and immunized rabbit with CrHV. HI antibody titers of these sera showed a closely positive correlation with their neutralizing antibody titers.
The glycoprotein E2 sequences of classical swine fever virus (strain p97) were cloned, sequenced and expressed in E. coli. Result from SDS-polyacrylamide gel electrophoresis analysis of expressed proteins revealed the presence of a prominently stained band corresponding to a molecular mass of 61 kDa, which is in agreement with the predicted size from the DNA sequence. The recombinant E2 protein contained an aminoterminal tag of six histidines that could be used for purification by the nickel chelate affinity chromatography. The elution fractions of the expressed protein also contain additional bands of 40 and 35 kDa proteins, indicating proteolytic cleavages might occur. Our Western blotting result also supported that the expression of the recombinant E2 protein of the classical swine fever virus were accomplished.