The present study identified the proteins that are differentially expressed during ischemic brain injury. Adult male rats were performed a middle cerebral artery occlusion (MCAO) to induce cerebral ischemia, and brains were collected at 24 hr after MCAO. Protein analysis was performed on the cerebral cortex using two-dimensional gel electrophoresis. Protein spots with a greater than 3 fold change in intensity between the sham and MCAO groups were identified by mass spectrometry. Among these proteins, 60 kDa heat shock protein, dehydropyrimidinase-related protein 2, t-complex protein 1, and Rho GDP dissociation inhibitor levels were significantly increased in MCAO group compared to those of the sham group. In contrast, thioredoxin, peroxiredoxin-2, stathmin, ubiquitin carboxy-terminal hydrolase L1, guanine nucleotide-binding protein α, pyridoxal-5'-phosphate phosphatase, and apoplipoprotein A-I levels were significantly decreased in MCAO group. These results suggest that cerebral ischemia induces neuronal cells death by changing expression levels of several proteins.
A novel cataract model was identified in the ddY strain (outbred colony) reared at Osaka Prefecture University. Opacity appeared as a white pinpoint focus in the unpigmented eyes of cataract mice at 6 weeks of age. All mice, fully viable and fertile, were bilaterally affected by the time they were 10 weeks of age. There were no gender differences in the incidence of cataracts. Histologically, 5-month-old cataract mice showed vacuolation of epithelial cells, disruption of lens fibers, and dislocation of the lens nucleus to the posterior lens cortex. To elucidate the mode of inheritance, heterozygous mutant hybrids between cataract mice and wild-type ddY mice, as well as offspring between the heterozygous mutants, were analyzed. No affected mice were observed among the heterozygous mutants, and the ratio of affected to unaffected mice was 1:3 among offspring between heterozygous mutants. For linkage analysis, we produced backcross progeny [cataract mouse × (cataract mouse × MSM/Ms mouse)], and concluded that the cataracts are inherited by an autosomal recessive gene. Moreover, the locus of the cataract gene, mct, was mapped to the 3.91 cM region encompassed by D2Mit467 and D2Mit320 on mouse chromosome 2 by linkage analysis. Thus, the present cataract mice represent a novel cataract mouse model, and have been designated Morioka cataract (MCT) mice.
The tapetum lucidum is a light-reflective device improving visual sensitivity in mesopic vision. There have been few studies on tapetal distribution and its relationship with degree of pigmentation in the retinal pigment epithelium (RPE). In the present study, sheep's eyes were used for macroscopical observation of the tapetum, and then histological sections of the posterior eyecups were made to analyze the distribution of tapetal thickness and degree of pigmentation in the RPE. Macroscopically, with available light, the tapetum was located in the dorsal eye fundus to the optic disc and showed an L-shape with the horizontally elongated nasal part and the dorsally expanded temporal part. In photographs with a flash, the tapetal area expanded and showed a more triangular shape. The tapetum histologically consisted of layers of dense collagen fibers and was thicker in the temporal part than in the nasal part. The maximum tapetal thickness was approximately 70 μm. The histological tapetal area was similar to the tapetal shape with a flash light. The pigmentation of the RPE was divided into three types, nonpigmented, transitional, and pigmented areas. The nonpigmented area was similar to the tapetal shape with available light. It is suggested that approximately 55% of the histological tapetal area is covered with the nonpigmented area and is functional under a natural light condition. The functional tapetal area was similar to the L-shaped high-density area of retinal ganglion cells.
Early weaning induces villous atrophy in the small intestine of piglets. We evaluated an influence of early weaning at 16 days old in mice for the use of villous atrophy model observed in early-weaned piglets. Five pregnant BALB/c mice were obtained and half of pups were weaned at 16 days old (early-weaned), while the others were allowed to suckle. Their small intestine was collected at 17, 18 and 19 days old in each group. Villous was shorting at 17 and 18 days old, but obscured at 19 days old. The gene expressions of epidermal and platelet-derived growth factor were associated with the villous height. Early weaning induced villous atrophy in the mouse small intestine as well as the piglets.
The aim of this study was to determine the immunoexpression of mucins in jejunal and ileal villous epithelium using six antibodies against MUC1, MUC2, MUC4 MUC5AC, MUC5B and MUC6. The immunohistochemical score for MUC1 has significantly intense staining compared with MUC2 (P=0.008) and the immunohistochemical socre for MUC4 and MUC 6 has significantly intense staining compared with MUC2 (P=0.032) in ileal villous surface. The immunohistochemical score for MUC4 (P=0.008), MUC5AC (P=0.016) and MUC6 (P=0.016) in ileal villous surface has significantly intense staining compared with ileal cryptic surface. The results of this study demonstrated that six mucins gave distinctly different expression patterns throughout the 1 week-old porcine small intestinal tract.
Equine canker is a chronic pododermatitis of the hoof in horses. Although spirochetes are detectable histopathologically in the lesions, the precise etiology remains unclear. This study reports the 16S rRNA gene sequencing of randomly selected clones based on PCR with Treponema-specific primers, using the canker lesions from two horses and healthy frog and sole from a horse. A total of 114 clones were obtained from the lesions, but no clones were detected in the healthy hoof tissues. The clones from the canker lesions examined were grouped into 19 operational taxonomic units, such as treponemal phylotypes originating from papillomatous digital dermatitis lesions of dairy cattle and as-yet uncultured human oral treponemes, indicating the presence of multiple treponemes in the lesions.
Cell-mediated and humoral immune responses are attenuated with aging. Intracellular glutathione (GSH) levels also decrease with aging. Previously, we have reported that combined administration of L-cystine and L-theanine enhances antigen-specific IgG production, partly through augmentation of GSH levels and T helper 2-mediated responses in 12-week-old mice. These findings suggest that combined administration of L-cystine and L-theanine to aged mice improves immune responses via increase of GSH synthesis. Here, we examined the effects of combined administration of L-cystine and L-theanine on antigen-specific antibody production and influenza virus infection in aged mice. Combined administration of these amino acids for 14 days before primary immunization significantly enhanced the serum antigen-specific IgM and IgG levels in 24-month-old mice. Furthermore, 13-month-old mice co-treated with these amino acids orally for 10 days had significantly lower lung viral titers than controls at 6 days after influenza virus infection. In addition, this co-treatment also significantly prevented the weight loss associated with infection. Enhancement of anti-influenza-virus IgG antibodies by combined administration of L-cystine and L-theanine was seen 10 days after infection. The significantly elevated serum interleukin-10/interferon-γ ratio and γ-glutamylcysteine synthetase mRNA expression, which is the rate-limiting enzyme of GSH synthesis, in the spleen 3 days after infection may have contributed to the observed beneficial effects. These results suggest that combined administration of L-cystine and L-theanine enhances immune function and GSH synthesis which are compromised with advanced age, and may become a useful strategy in healthy aging.
In this study, cDNA of Toll-like receptors (TLR) 3, 7 and 9 were synthesized and completely sequenced. The coding regions of cDNA for bat TLR3, TLR7 and TLR9 were 2,718, 3,150 and 3,090 bp in length, respectively. The open reading frames encoded 905, 1,049 and 1,029 amino acids for TLR3, TLR7 and TLR9, respectively. The nucleotide sequences, predicted amino acid sequences and predicted domain structures of the three bat TLRs had high homology with those of other mammals. In addition, the expression profiles of each TLR in main organs were analyzed. Expression of TLR3 was highest in the liver, whereas the expressions of TLR7 and TLR9 were highest in the spleen.
It is essential to develop a technique to culture purified skin-derived mast cells (SMCs) to facilitate immunological research on allergic diseases in dogs. This study was performed to develop an efficient culture system for canine SMCs and to characterize the cells in comparison to canine bone marrow-derived mast cells (BMMCs). Enzymatically digested skin biopsy samples were cultivated in serum-free AIM-V medium supplemented with recombinant canine stem cell factor. Three to five weeks after the initiation of culture, mast cells were collected by a magnetic activated cell separation system using anti-c-Kit antibody. The collected cells were composed of a uniform population showing morphological characteristics of mast cells with a round or oval nucleus and abundant toluidine blue-positive metachromatic granules in the cytoplasm. The results of flow cytometric analysis for the presence of cell membrane c-Kit and Fc epsilon receptor I (FcεRI) indicated that approximately 90% of the cells were mast cells. The cytoplasmic granules were positive for both tryptase and chymase. Apparent dose-dependent degranulation was induced by antibody-mediated cross-linking of immunoglobulin E (IgE) bound to the cells. These cytological and immunological characteristics observed in SMCs were mostly similar to those observed in BMMCs; however, IgE-mediated degranulation was significantly lower in SMCs than BMMCs. The culture system for canine SMCs developed in this study would be useful in understanding the pathophysiology and developing anti-allergic therapeutics in canine allergic dermatitis.
Polyethylene glycol (PEG) is attached to proteins in order to increase their half-life in circulation and reduce their immunogenicity in vivo. The present study was conducted to examine whether two different sizes of PEGylated bovine lactoferrin (40k- and 20k-PEG-bLf) would enhance the protective effect of native bLf on liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in rats. The treatment of PEGylated bLf more remarkably prevented the elevation of serum levels of hepatic enzyme markers and inhibited inflammatory and hemorrhagic changes and hepatic apoptosis induced by GalN/LPS than native bLf. The treatment of PEGylated bLf more significantly inhibited the increased concentration of proinflammatory cytokines (TNF-α and IL-6) in serum caused by GaIN/LPS, and enhanced anti-inflammatory cytokine (IL-10) production more than native bLf. PEGylated bLf decreased serum levels of nitric oxide (NO) more than native bLf. These results indicate that PEGylated bLf inhibits more significantly the induction of inflammatory mediators such as cytokines and NO than native bLf, resulting in the enhancement of its prevention of fulminant liver failure induced by GalN/LPS in rats. The present study provided evidence that PEGylated bLf may offer a novel alternative therapy for the prevention of acute hepatic failure through its anti-inflammatory and immunomodulatory properties.
Longicin, a defensin-like peptide, was recently identified in the hard tick Haemaphysalis longicornis. Longicin and one of its synthetic partial analogs (P4) displayed antimicrobial/fungicidal/parasiticidal activity. In the present study, we compared longicin-derived synthetic analogs in order to characterize the antimicrobial motif (P4) by analyzing some structural features using various bioinformatic tools and/or CD spectroscopy. According to the chemicophysical characteristics, P4 is suggested to be a cationic peptide with hydrophobic and amphipathic character. The predicted secondary structure indicated the existence of a β-sheet, which was also observed in the modeled tertiary structure. CD spectroscopic results also showed the existence of a β-sheet and transition to a helical conformation in the presence of membrane-mimicking conditions. These structural observations on P4 suggested that the antimicrobial activity could be due to the β-sheet as well as the α-helix. In addition, a sequence homology search showed that molecules identified in other ticks and organisms also have the P4 analogous domain at their C-terminal, which indicates P4 as a conserved domain. The peptide P4 also showed low cytolytic activity. Based on the present result and previously reported studies, the peptide P4 could be suggested as a novel antimicrobial domain indicating future therapeutic agent against bacteria.
Cryptosporidiosis is a diarrheal illness caused by apicomplexa parasite Cryptosporidium spp. In this study, to examine the overall infection status of Cryptosporidium spp. in individuals residing in southern parts of Korea, eight counties around Yeongsan, Seomjin and Nakdong River valleys was surveyed. The investigation was carried out from April to October 2005. A total of 9,498 stool samples were collected from individuals. Stool samples were analyzed for modified acid-fast stains, and DNA fragment extracted from positive samples was identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for 18S rRNA polymorphic region. Oocysts of Cryptosporidium spp. were detected in 239 specimens (2.5%) by a modified acid-fast stain. Infection rate was not significantly different between male (2.2%) and female (2.8%) individuals examined (P>0.05). In the infection rate by age, totally 1-9 (4.8%) and 80< (3.7%) age group were shown to the highest, and there was shown to significant differences (P<0.05). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 18S rRNA gene from 51 isolates showed that all the isolates were identified as C. parvum. Our data collectively suggested that C. parvum infection is prevalent in the studied areas of Korea and more comprehensive nation-wide epidemiological studies are needed to elucidate the infection status of Cryptosporidium infection in Korea.
To determine whether developmental hypothyroidism causes permanent disruption of neuronal development, we first performed a global gene expression profiling study targeting hippocampal CA1 neurons in male rats at the end of maternal exposure to anti-thyroid agents on weaning (postnatal day 20). As a result, genes associated with nervous system development, zinc ion binding, apoptosis and cell adhesion were commonly up- or down-regulated. Genes related to calcium ion binding were up-regulated and those for myelination were often down-regulated. We, then, examined immunohistochemical cellular distribution of Ephrin type A receptor 5 (EphA5) and Tachykinin receptor (Tacr)-3, those selected based on the gene expression profiles, in the hippocampal formation at the adult stage (11-week-old) as well as at the end of exposure. At weaning, both EphA5- and Tacr3-immunoreactive cells with strong intensities appeared in the pyramidal cell layer or stratum oriens of the hippocampal CA1 region. Although the magnitude of the change was decreased at the adult stage, Tacr3 in the CA1 region showed a sustained increase in expressing cells until the adult stage after developmental hypothyroidism. On the other hand, EphA5-expressing cells did not show sustained increase at the adult stage. The results suggest that developmental hypothyroidism caused sustained neuronal expression of Tacr3 in the hippocampal CA1 region, probably reflecting a neuroprotective mechanism for mismigration.
We report a rare case of benign sex cord-stromal tumor consisted largely of luteoma with minor portion of Sertoli cell tumor located at the position of the left ovary excision in an 11-year-old ovariectomized bitch. Granulosa cell component was lacking, and both luteal and Sertoli cell portions were entirely positive for inhibin α and neuron-specific enolase, whereas luteoma portion alone was positive for Wilms' tumor-1 (WT1), immunohistochemically. The results suggest that this tumor is a possible complication of incomplete ovarian excision at the time of ovariectomy and consisted of uncommon hybrid of luteal and Sertoli cells to be diagnosed as an unclassified sex cord-stromal tumor if applied in human cases. WT1-expression pattern suggested the signature of the difference in the phenotype of these cell types.
This case describes a subcutaneous soft tissue tumour in a German Shepherd dog. Histologically, the lesion was characterized by proliferating ovoid cells, loosely arranged in a collagenous to myxoid stroma, and by numerous pseudoglandular structures lined by neoplastic cells. Immunohistochemically, neoplastic cells were labelled with vimentin, glial fibrillary acidic protein and S100 antibodies, but not with cytokeratin, desmin and smooth muscle actin antibodies. Ultrastructurally, neoplastic cells were characterized by numerous mitochondria surrounded by endoplasmic reticulum and contained few secondary lysosomes. This tumour was diagnosed as a subcutaneous peripheral nerve sheath tumour (PNST) with pseudoglandular architecture. This case illustrates the morphological diversity of PNST and provides new insight into the differential diagnosis of cutaneous tumours of similar morphology in the dog.
A 10-year-old miniature sow was died, showing inappetence and weight loss. Grossly, neoplastic enlargement of the uterus was found. Histopathologically, the lesions consisted of acinar, ductular and cystic proliferations of mono- and multilayered epithelial cells; these cells reacted immunohistochemically strongly with three different cytokeratin antibodies, and occasionally to vimentin. Myofibroblastic desmoplastic cells, positive to α-smooth muscle actin, were present among neoplastic cells. Metastatic lesions were seen in the lungs and liver. Based on these findings, a diagnosis of uterine adenocarcinoma with marked desmoplasia was made. This case is the second report of uterine adenocarcinoma in the miniature pig.
Leptospirosis is a zoonotic disease of global importance, and has a worldwide distribution. The present study aimed to determine leptospiral seroprevalence in clinically healthy racing horses from all three racecourses in Korea. Serum samples from 1,226 racing horses were examined using a microscopic agglutination test to detect the presence of antibodies against 18 Leptospira serovars. Of the tested samples, 307 (25.0%) were found to be positive. The distribution of seroprevalence differed significantly by racecourse (P=0.004); the Jeju course had the highest incidence (31.1%), followed by the Seoul (25.2%) and Busan (19.5%) racecourses. Seasonal variation in seropositivity was also apparent (P=0.000), being lower in spring (13.0%) and winter (12.5%), and higher in summer (36.7%) and autumn (34.7%). No significant age- or gender-related difference in seroprevalence was noted in this study (P>0.05). Seroprevalence was higher (P=0.006) among ponies than among thoroughbreds. Sejroe was the most frequently detected serovar (n=236), followed by Bratislava (n=35), Ballum (n=16), Autumnalis (n=10), and Canicola (n=10). The majority of serum titers were relatively low; most values ranged from 1:100 (n=217) to 1:200 (n=69). These results suggest that the Sejroe serovar may be maintained in the racing horse population in Korea.
Lidocaine hydrochloride (Lido) is widely used for analgesia in veterinary medicine; however, in humans, it has been suggested that Lido attenuates granulocyte functions, such as adhesion and reactive oxygen species (ROS) production. Thus, Lido may affect canine granulocyte function; however, there have been no reports on the effects of Lido on canine granulocyte function. Thus, we studied the effects of Lido on canine granulocyte CD11b expression and ROS production. We further studied the effects of Lido on the priming of canine granulocyte CD11b expression and ROS production by recombinant canine granulocyte macrophage colony stimulating factor (rcGM-CSF). Isolated granulocytes were incubated with 3, 30 or 300 μg/ml Lido, or with Lido followed by priming with 5 ng/ml rcGM-CSF. CD11b was detected by the immune fluorescent antibody method, and the mean fluorescence intensity (MFI) was assayed by flow cytometry. ROS production was assessed by the peak time (PT) of ROS production and area under the luminol reaction curve (AUC), which represents total ROS production quantity against opsonized zymosan stimuli. Only 300 μg/ml Lido (tissue level observed by regional block) significantly attenuated both the MFI of CD11b and its enhancement by rcGM-CSF. Moreover, at this concentration, the AUC and its enhancement by rcGM-CSF were significantly attenuated by Lido; in contrast, Lido did not affect PT. In conclusion, Lido suppressed granulocyte adhesion to the endothelium and antiseptic capability by suppressing CD11b expression and/or ROS production. Particular care should thus be exercised when performing regional anesthesia block using Lido.
In this study, we determined a partial sequence of CYP1A1 in the camel and its phylogenetic position. The deduced amino acid sequence of camel CYP1A1 showed the highest identity 94% with those of sheep and cattle CYP1A1. In a phylogenetic analysis, the camel CYP1A1 isoform was located beside sheep and cattle CYP1A1. When we studied the distribution of camel CYP1A1 mRNA in different tissues, we found that this isoform was expressed in all tissues except the hump. Interestingly, the lungs of all the camels and tongues of two of the three animals showed high expressions of CYP1A1 mRNA, and this may indicate exposure to ligands of aryl hydrocarbon receptor such as environmental pollutants or flavonoids.
The macaque is widely used for investigation of drug metabolism due to its evolutionary closeness to the human. However, the genetic backgrounds of drug-metabolizing enzymes have not been fully investigated; therefore, identification and characterization of drug-metabolizing enzyme genes are important for understanding drug metabolism in this species. In this study, we isolated and characterized a novel cytochrome P450 2C18 (CYP2C18) cDNA in cynomolgus macaques. This cDNA was highly homologous (96%) to human CYP2C18 cDNA. Cynomolgus CYP2C18 was preferentially expressed in the liver and kidney. Moreover, a metabolic assay using cynomolgus CYP2C18 protein heterologously expressed in Escherichia coli revealed its activity toward S-mephenytoin 4'-hydroxylation. These results suggest that cynomolgus CYP2C18 could function as a drug-metabolizing enzyme in the liver.