The effects of the mixture of crude aqueous extracts from Chinese pangolin and wild pink (C+P), traditional Chinese medicine, on the proliferation and differentiation of mammary gland epithelium in intact and ovariectomized immature mice were investigated by light and electron microscopy and BrdU immunohistochemistry. Although there were no significant differences in mammary gland fat pad and parenchyma areas between the intact experimental groups, the numbers of duct branchings and buds were significantly larger in the C+W treated mice than in the control mice. The ratio of BrdU immunopositive cells to total epithelial cells was higher in C+W treated intact mice. Ultrastructurally, epithelial cells of the mammary buds and ducts possessed an oval and lucent nucleus, and ribosomes increased in number or developed to a greater degree in C+W treated intact mice than in the control mice. Conversely, there were no significant differences in any measurements of mammary gland between the experimental groups of ovariectomized mice. BrdU immunoreactive cells were never seen and the ultrastructure of mammary epihelial cells indicated the inactive cell phase in both ovariectomied mice. In comparison between the intact and overiectomized mice, the mammary fat pad area was larger in the ovariectomized mice than in the intact mice, although another four measurements were larger in the intact groups. These observations suggest that administration with C+W could promote the development of mammary glands via ovary in immature mice.
Capsaicin has effects on the adiposity by increasing energy and lipid metabolism, and decreases appetite and fat intake. In the present study, we investigated changes in food intake and body weight after capsaicin treatment. We also observed changes in orexigenic and anorexigenic neuropeptides-agouti-related peptide (AgRP), α-melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH) and orexin-immunoreactivities in the rat hypothalamus after capsaicin administration. Only one day after capsaicin treatment, the mean food intake was significantly decreased. There was no significant difference in the mean body weight between vehicle- and capsaicin-treated groups. In addition, after capsaicin treatment, numbers of AgRP- and orexin-immunoreactive (+) cells were significantly decreased in the arcuate nucleus (ARC) and lateral hypothalamic area, respectively. In contrast, the number of α-MSH+ and ACTH+ cells in the ARC of the capsaicin-treated rats was higher than in the vehicle-treated rats. These results indicate that capsaicin reduces food intake, not body weight, transiently, and decreases AgRP and orexin immunoreactivities, whereas it increases α-MSH and ACTH immunoreactivities in rat hypothalamic nuclei.
In the present study, we investigated the time-course changes of neuronal degeneration and microglial activation in the gerbil dentate gyrus after transient cerebral ischemia using Fluoro-Jade B histofluorescence staining and immunohistochemistry for Iba-1. Fluoro-Jade B positive cells were observed from 6 hr and markedly increased 1 day after ischemia/reperfusion. Iba-1-immunoreactive microglia were increased and hypertrophied at early time, and Iba-1 immunoreactivity was highest at 2 days after ischemia/reperfusion. These results may be direct evidence on neuronal degeneration and microglial activation in the gerbil dentate gyrus after ischemia/reperfusion.
We have cloned the arginine deiminase (ADI) gene from Mycoplasma hominis PG21 genomic DNA by polymerase chain reaction, and changed four TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The recombinant ADI (rADI) was purified to apparent homogeneity by Ni-affinity chromatography after extraction from inclusion bodies followed by refolding. The rADI expressed in E. coli was estimated to be 50 kDa. Dimeric forms of rADI exerted enzymatic activity. We found that high concentration of potassium dihydrogenphosphate (PDP) and L-arginine addition in refolding reaction increases the enzyme activity. The specific activity of rADl was calculated as 0.618 U/mg. In addition, the enzyme activity of purified rADI remained for at least one month in 100 mM PDP solution (pH 6.5), but diminished within one week in 100 mM PDP solution (pH 7.4). Anti-tumor activity of the purified rADI was estimated to be 0.036 U/ml as 50% growth inhibitory activity against human melanoma cell line G-361.
A total of 184 resistant Salmonella enterica isolated from poultry and swine were investigated for the distribution of and associations between various antimicrobial resistance genes. All the isolates were screened for the presence of class 2 and 3 integrase genes and 18 resistance genes corresponding to their resistance phenotypes. None of the isolates carried class 2 and 3 integrons. The investigated resistance genes were responsible for resistance in 78% of the isolates. All the strains harboring more than one resistance gene were resistant to three or more antimicrobials. The blaTEM, cmlA, tetA, dfrA12, sul3, aadA1 genes were detected in the majority of strains resistant to ampicillin (87%), chloramphenicol (63%), tetracycline (60%), trimethoprim (42%), sulphonamides (42%) and streptomycin/spectinomycin (61%), respectively. Two ciprofloxacin-resistant isolates had a single point mutation leading to Ser-83-Phe in GyrA or Thr-57-Ser in ParC. Statistical analysis revealed good correlation between the presence of antimicrobial resistance genes and corresponding resistance phenotype (p<0.01). The results indicated that the resistance genes play a major role in conferring resistance among the Salmonella isolates investigated.
Vibrio vulnificus hemolysin (VVH), a pore forming toxin, is thought to be a virulence factor of this bacterium. It is well known that VVH induces apoptosis as well as cell lysis in susceptible target cells. Although pore formation is an essential step in cell lysis, it is unknown whether this step is necessary for VVH-induced apoptosis. In this study, Chinese hamster ovary (CHO) cells were exposed to non-oligomerized mutant F334I, in which phenylalanine 334 was replaced by isoleucine. The rate of apoptosis caused by the wild type VVH (VVH wt) was 41.5 ± 6.4 %, whereas that caused by F334I was 0.4 ± 0.8% at the same concentration. Our results clearly showed that oligomerization is essential for the cell lytic activity as well as apoptotic activity of this toxin.
A comparison was performed between preoperative ultrasonographic and macroscopic findings for gallbladders and their contents in eleven dogs that were found to have any content in the gallbladder and then underwent cholecystectomy. In addition, correlation was considered between prognosis and age, hematologic analysis, serum biochemical analysis and ultrasonographic findings. Ultrasonographic findings of the gallbladder were classified into 3 patterns: 1) hyperechoic content filling the entire gallbladder and precipitated immobile content, 2) a somewhat thinner hypoechoic area in the exterior layer with a less distinctive border adjacent to the internal hyperechoic area than pattern 3 with moth-eaten or mosaic-form hypoechoic portions scattered within the internal hyperechoic area and 3) a thick hypoechoic area in the exterior layer with a distinctive border adjacent to a prominent internal hyperechoic area. The macroscopic findings of the contents mainly consisted of biliary sludge and concentrated bile in pattern 1, a softer mucous mass in pattern 2 and an elastic mucous mass in pattern 3. Pattern 2 seemed to be in the stage before the kiwi fruit-like pattern that characterizes gallbladder mucocele, suggesting disorder of the gallbladder and bile excretion as well as onset of cholangiohepatitis associated with such disorders. In regard to association with prognosis, the mean ages were 11.8 ± 1.5 years in death cases and 8.4 ± 2.8 years in surviving cases, while the total WBC counts were 46,600 ± 11,912 /μL in the death cases and 18,266 ± 9,411 /μL in the surviving cases, showing significant differences (p<0.05) in age and total white blood cell count between the two groups.
A 6-month-old miniature Schnauzer presented with hypernatremia and clinical signs of vomiting, diarrhea, inappetence, and lethargy. The dog did not consume water on its own. Hypernatremia and the related clinical signs were resolved by fluid administration. Endocrinological investigations and urinalysis excluded the possibility of diabetes insipidus and hyperaldosteronism. Therefore, the dog was diagnosed with hypodipsic hypernatremia. Magnetic resonance imaging revealed dysgenesis of the corpus callosum and other forebrain structures. On the basis of these findings, congenital brain malformation associated with failure of the osmoreceptor system was suspected.
A 10-year-old, neutered male, Maltese dog presented with a three week history of intention tremor, right hind limb rigidity, poor coordination, and occasional circling to the left. On magnetic resonance imaging (MRI) of the brain, a mass was identified in the right occipital lobe and cerebellum. Three weeks after the initial MRI scan, we performed an 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) of the brain. The FDG-PET demonstrated areas of hypermetabolism in the right occipital lobe, cerebellum, pons, and medulla oblongata. When the standardized uptake value was calculated, the hypermetabolic lesion was higher than the gray matter values. The anatomical location of the hypermetabolic lesion was more precisely identified by the PET-MRI fusion images. The dog was definitively diagnosed as a primary histiocytic sarcoma of the brain. This is the first report of PET findings of an intracranial histiocytic sarcoma in a dog.
This study assessed the effects of pregnancy and lactation on the morphology of the dentine tubules and external enamel surface of rat incisor teeth using a scanning electron microscope (SEM) equipped with an energy dispersive x-ray (EDX) system. Twenty-four female Wistar rats were divided into three groups; group A rats were at the end of pregnancy, group B rats were in the post-lactation period and group C rats, the control group, were unmated. The outer enamel surface and openings of the dentine tubules at the neck regions of the incisors were observed under the SEM and photographed. Examination of the incisor teeth of pregnant and post-lactation rats revealed scratches on the enamel surface. There were few eroded areas and slight changes and the dentine tubules of the pregnant group were fully or partially occluded on the entire surface of the enamel in the lactating rats. Almost all dentine tubules of the rats in this group were open. During the study, EDX analysis of calcium, phosphate and magnesium was also performed at 20 kV and 0 degree tilt. The results of EDX analyses of magnesium were significantly lower in the pregnant group compared with the lactation and control groups for the dentine in the neck region (p<0.05). The calcium values increased in the lactation group compared with those of the rats in the other two groups (p<0.05). These results might indicate that changes during pregnancy and lactation affect the content and morphology of mineralized dental hard tissue.
The aim of this study was to investigate the effect of exposure period and dose of diethylstilbestrol (DES), which has strong estrogenic activity, on pregnancy in rats. All rats with observed vaginal plugs or sperm in vaginal smear tests after mating were divided into 3 groups: those fed a normal diet, a diet mixed with DES throughout pregnancy and a diet mixed with DES from day 13 of pregnancy. DES was mixed into the diet at 0.1, 1, 10 and 100 ppm. All bred rats fed the normal diet and 0.1 ppm DES from day 13 of pregnancy delivered pups, while none of the rats treated with 1-100 ppm DES during pregnancy and 100 ppm DES from day 13 of pregnancy delivered any pups. The number of male and female pups born decreased in rats treated with 10 ppm DES from day 13 of pregnancy. These results suggest that DES could affect pregnancy and that the exposure period and dose may result in sterility, abortion, poor fetal growth and reduced number of pups born.
The recombinant human erythropoietin (rhEPO) is used for the treatment of patients with renal anemia. However, rhEPO should be administered subcutaneously or intravenously three times a week. The repetitive injections of rhEPO result in burdens to patients. To resolve this problem, we investigated the sustaining release methods using an rhEPO-hydroxyapatite (HAp) made by spray-drying technique as the drug delivery system. Two types of rhEPO-HAp formulations were prepared; zinc (Zn) formulation and Zn and poly-L-lactic acid (PLA) formulation. These formulations were examined in genetically anemic model, ICGN (ICR-derived glomerulonephritis) mice. According to in vivo release test of rhEPO from HAp in ICGN mice, elevated plasma concentration of rhEPO could be maintained for more than 7 days. These mice showed the amelioration of anemia for more than 3 weeks post-administration without causing any side effect. In conclusion, Zn or Zn/PLA formulation of HAp was considered to be one of the useful carriers of rhEPO for long-term improvement of anemia.
To evaluate the efficacy of intracerebral (IC) immunization, mice were immunized with a rabies vaccine by the subcutaneous (SC), intramuscular (IM) or IC route, and 10-fold the 50% lethal dose of rabies virus was inoculated into the hindleg of the immunized or non-immunized mice. The antibody titer in serum was elevated and boosted by additional immunization via all routes, but highest after the IC immunization followed by the IM and SC routes, in this order. Intracerebrally immunized mice were completely protected from death and the neurological signs of infection, whereas the IM or SC immunization only partly protected the mice. In mouse models, IC immunization is more effective at inducing a protective immune response against the transneural spread of rabies virus than IM or SC immunization.
Two (1 adult and 1 young bird) of 4 H5N1-highly-pathogenic-avian-influenza (HPAI)-virus-infected whooper swans in Akita, Japan, in 2008 were investigated pathologically. Macroscopically, white spots with hemorrhages were scattered in the pancreas in the adult bird. Histologically, the adult bird had severe necrotizing pancreatitis and mild nonpurulent encephalitis. The young bird had severe nonpurulent encephalitis and nonpurulent enteric ganglionitis, and intestinal venous wall thickening. Virus antigens were detected in the lesions of pancreatitis in the adult bird and of encephalitis in adult and young birds. These findings suggest that the swans died or became moribund due to neurological disorders and necrotizing pancreatitis caused by H5N1 HPAI virus infection.
An 18-month-old, Japanese Black heifer had ananastasia over a two-week period. The heifer was euthanized and necropsied. Grossly, a brown and soft mass was found in the left cerebellar hemisphere, which originated from the fourth ventricle in the pons. Multiple papillary masses were also found on the wall of the fourth ventricle. There was massive dilatation of the cerebral aqueduct, and also in the third and lateral ventricles. Histologically, the neoplastic cells formed numerous ependymoblastic rosettes and perivascular pseudorosettes. In the fourth ventricle, the normal ependymal cells were replaced by papillary proliferation of the neoplastic cells. Ultrastructually, the neoplastic cells had a feature differentiating to the ependymal cell showing a few basal bodies in the cytoplasm and connecting to adjacent cells with junctional complexes. We diagnosed the present case as ependymoblastoma with its origins in the fourth ventricle.
The antimicrobial susceptibilities of 2,205 isolates of Escherichia coli and 1,181 isolates of Enterococcus faecalis (n=610) and E. faecium (n=571) from apparently healthy cattle, pigs and broiler and layer chickens collected from 2000 to 2003 were examined using an agar dilution method. Overall, the isolates from cattle and layer chickens showed a lower incidence of resistance to almost all antimicrobials studied compared with those from pigs and broiler chickens. Fluoroquinolone resistance was found at a low level in isolates of E. coli from four animal species and in E. faecalis from pigs and broiler and layer chickens. Resistance to cephalosporin was identified in isolates of E. coli from broiler chickens in 2000-2002 and from four animal species in 2003. Incidence of antimicrobial resistance in the bacteria did not vary from year to year during the investigation period.
Apx toxins have been identified as important virulence factors of Actinobacillus pleuropneumoniae, the etiologic agent of porcine pleuropneumonia. In some A. pleuropneumoniae serotypes, Apx toxins are secreted by the cell membrane proteins encoded by apxIIIB and apxIIID genes. In an effort to develop a live vaccine strain against A. pleuropneumoniae, we inactivated the apxIIIB and apxIIID genes in A. pleuropneumoniae 1536, a serotype 2 strain, resulting in the ΔapxIIIB/DapxIIID mutant strain (1536ΔBΔD). Immunization of pigs with live 1536ΔBΔD A. pleuropneumoniae conferred protection against homologous challenge with wild-type A. pleuropneumoniae 1536. Thus, impaired Apx toxin secretion may decrease the virulence of A. pleuropneumoniae and may be an effective strategy for the development of a live-attenuated A. pleuropneumoniae vaccine.
In the present study, the efficacy of a nerve stimulator in performing brachial plexus block (BPB) in dogs was investigated. The nerve blocking effects of bupivacaine and ropivacaine for BPB were also compared. Twelve beagles were allocated to groups based on the following treatments: conventional BPB with 0.5% bupivacaine (0.5% BupiM group) or BPB with 0.5% bupivacaine, 0.5% ropivacaine or 0.75% ropivacaine and a nerve stimulator (the 0.5% BupiS, 0.5% RopiS and 0.75% RopiS groups, respectively). After BPB, nerve blocking effects were assessed based on sensory blockade in several cutaneous areas and knuckling. The ratio of full block (blockade in all cutaneous areas) for 0.5% BupiM was 25%, and that for 0.5% BupiS was significantly higher, 75% (p<0.05). For the 0.5% BupiS, 0.5% RopiS and 0.75% RopiS groups, the average duration of full block was 387, 184 and 275 min, respectively, and the average duration of knuckling was 703, 460 and 421 min, respectively. The duration of full block and knuckling for the two ropivacaine groups was shorter compared with that of the 0.5% BupiS group. In conclusion, when using bupivacaine and ropivacaine for BPB in dogs, it is worth noting that there are differences in onset time and duration and that effective perioperative analgesia can be achieved depending on the intended use.
Twelve healthy Beagles were divided into 3 groups of 4 dogs each. To compare 2 methods of total hepatic vascular exclusion (THVE), we investigated the hemodynamic changes during THVE and assessed the influences on hepatic, renal and pancreatic biochemistry and the complications after THVE. In Group A, the thoracic aorta, hepatic pedicle and prehepatic and posthepatic caudal vena cava were occluded for 20 min, while in Group B, the celiac and cranial mesenteric arteries, hepatic pedicle and prehepatic and posthepatic caudal vena cava were occluded for 20 min. In Group C, a sham operation was performed. The arterial, venous and portal venous pressures and serum biochemistry parameters were measured before and during THVE and for 30 min after reperfusion. The carotid arterial pressure did not change in Group A during THVE, but decreased in Group B. The femoral and portal venous pressures in Group B increased significantly during THVE compared with those in Group C. With the exception of alanine aminotransferase, the serum biochemical profiles remained unchanged after the operation. For 7 days after the operation, no complications were observed in any of the dogs. In conclusion, occlusion of thoracic aorta, hepatic pedicle and prehepatic and posthepatic caudal vena cava is a feasible and safe method of hepatic vascular occlusion. This technique appears to be effective for canine hepatic surgery, such as removal of a large right-divisional hepatic tumor and attenuation of intrahepatic portosystemic shunt.
The purpose of this study was to evaluate the effect of an autogenous free fat graft (FFG) on hemilaminectomy defects. Hemilaminectomy was performed at three levels in three beagles, and each defect was designated as a control site or a site treated with one of two different sizes of FFG. Subsequently, longitudinal computed tomography scanning and histopathological examination were performed. As a result, no postoperative dural adhesion was recognized at the sites where FFGs were placed regardless of the size of the FFG. Moreover, there was no compression of the spinal cord by the FFG. Therefore, it appears that postoperative dural adhesion was controlled by the FFG and that the possibility of FFG migration into the spinal canal is low in hemilaminectomy. In conclusion, placement of an FFG over the defect was considered useful for preventing complications in hemilaminectomy.
A Beagle with a low plasma testosterone (T) level and azoospermia was given 10 subcutaneous injections of 1 μg gonadotropin-releasing hormone analogue (GnRH-A) per head at intervals of 3 days (Experiment 1), and 6 months after the final injection was given, 15 subcutaneous injections of 2 μg GnRH-A were given at intervals of 2 days (Experiment 2). The plasma T level increased and peaked at 8 weeks after the first injection of GnRH-A in both Experiment 1 and Experiment 2. Motile sperm were detected in the semen collected 8 weeks and 7 weeks after the first injection in Experiment 1 and Experiment 2, respectively. The total number of sperm peaked 9 weeks after the first injection in both Experiment 1 (4.5 × 106) and Experiment 2 (72.8 × 106).
Novel cytochrome P450 1A (CYP1A) cDNA fragments were isolated from the liver of nine Japanese amphibian species using reverse transcription polymerase chain reaction (RT-PCR). Degenerate PCR primers were used to amplify 122-bp fragments of CYP1A cDNAs. Construction of a phylogenetic tree revealed that urodele and anuran amphibians formed two branches. Within the anuran species, three branches were formed: 1) Ranidae and Rhacophoridae, 2) Bufo japonicus formosus and Hyla japonica, and 3) Xenopus laevis. The cDNA nucleotide sequence of these CYP1A fragments showed identities ranging 72-98% (all), 72-78% (Anura vs. Urodela), 75 to 98% (Anura), 81% (Urodela), 74-80% (Xenopus laevis vs. nine Japanese amphibians).
The distribution of anti-hantavirus antibodies in humans and rodents in northern Vietnam was examined. In total, 837 serum samples from healthy humans (617) and patients with fever (220), living in six different areas were screened for IgG antibodies against Hantaan or Seoul virus (SEOV) by ELISA, IFA, and Western blot analysis. Antibody-positive sera were identified in 7/617 (1.1%) healthy donors, 5/150 port workers in the port of Hai Phong, and 2/185 residents of Ha Nam Province. In comparison, positive sera were detected in 5/220 (2.3%) fever patients in the provinces of Ha Nam (1/58) and Thanh Hoa (4/146). Antibody-positive Rattus norvegicus were found in the provinces of Ha Nam (7/52) and Thanh Hoa (1/67), in Haibatrung District (7/43) in Hanoi, and in Hai Phong Port (21/62), while antibody-positive R. rattus (2/17) were found in Hai Phong Port. Part of the Gc region from the viral genome was amplified by RT-PCR using lung tissue samples from R. norvegicus in Haibatrung (2/7) and Hai Phong Port (7/9), but not from R. rattus (0/2). Viral sequences were located in the SEOV clade and formed a single lineage with Indonesian SEOV, suggesting that Vietnamese SEOV is part of a distinct lineage among Asian SEOVs.