N-ethyl-N-nitrosourea (ENU) is a potent mutagen in a mouse model by inducing point mutation in a random manner and, in particular, causing heritable base substitutions in spermatogonia. In this study, systematic development of phenotype-driven mutant mice with large scale was carried out by using ENU. Nine-week-old male mice of C57BL/6J received intraperitoneal injection at three times with 100 mg/kg of ENU at weekly intervals for three weeks. After injections with ENU, the changes of body weight, fatality, recovery of fertile period, and breeding record were measured in these mice. Body weight lost as a result of ENU treatments was reversed after the last ENU injection. Live fertile male mice recovered from infertility from 104 to 165 days after ENU treatments were mated with C57BL/6J female mice for generation of G1 offspring. An average birth rate was 5.9 mice from 1 pair of paternal and maternal mice. All of 231 G1 offspring mice were analyzed by modified-SHIRPA with standard procedure at nine weeks of age. Among G1 mice, 166 mice were identified as mutagenic phenotypes in 20 test items. The changes in mutagenic phenotypes after ENU treatments, for instance, pattern in the region with a different color, touch escape, changes in head morphology, pupil, and teeth, and negative geotaxis etc., were found in these mice. Taken together, these results indicate that ENU may be a trans-generational mutagen in C57BL/6J mice.

Apolipoprotein A1 (ApoA1) is the major protein component of high density lipoprotein (HDL) cholesterol in blood, and ApoA1 genetic polymorphisms modulate the blood lipid profiles. This study was conducted in order to investigate the association between three genetic polymorphisms (rs670, rs5069, and rs5070) of ApoA1 and blood lipid profiles in postmenopausal Korean women. A total of 130 post- menopausal women who visited a hospital in order to undergo screening tests were subjects of this study. Genetic polymporphisms and blood lipid profiles were determined using a direct sequencing and spectrophotometric assay, respectively. A significant linkage disequilibrium was observed between all tested single nucleotide polymorphisms. ApoA1 rs5070 genetic polymorphism showed a marginally significant association with HDL cholesterol levels (p=0.066). After adjusting for age, body mass index, smoking, alcohol drinking, medication, hypertension, and diabetes mellitus, we found that the ApoA1 rs5070 genetic polymorphism is a significant determinant of HDL cholesterol levels (β=4.421, p=0.037). According to the results of this study, ApoA1 rs5070 genetic polymorphism may be an important genetic marker associated with HDL cholesterol in postmenopausal Korean women.

Vitamin C (ascorbic acid) is an essential nutrient of most living tissues. We established a strain of Gulo-/- mice with known deficiency, in which vitamin C intake can be controlled by diet, like humans, and investigated the differen- tially expressed proteins following treatments with Helicobacter pylori and diethylnitrosamine (DENA) in the liver of Gulo-/- mice using a proteomic approach. Expression of p53, 14-3-3ε and 14-3-3δ in Gulo-/- mice liver tissue was analyzed by immunohistochemistry. 2-DE maps constructed from Gulo-/- mice liver and differentially expressed proteins in liver tissue were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/MS). In Gulo- /- mice after H. Pylori infection, followed by treatment with DENA, no differences in p53, 14-3-3ε and 14-3-3δ were observed by immunohistochemistry. Proteome analyses us- ing MALDI-TOF/MS resulted in successful identification of 12 proteins (nine proteins were up-regulated and three were down-regulated). Specifically, peroxiredoxin-6 and Alpha-1-antitrypsin 1-4 were up-regulated in liver after H. Pylori infection followed by treatment with DENA. These results indicated that oral supplementation with vitamin C led to rescue of Gulo-/- mice from vitamin deficiency, and protected the liver from H.pylori infection and/or DENA ef- fect, and vitamin C also protected the liver against oxidative stress.

Canine parvovirus (CPV2) is one of the most virulent virus causing acute hemorrhagic enteritis and myocarditis in dogs. Infection mainly caused by the ingestion of virus through the mucosal route. Therefore, induction of mucosal immunity is essential in prevention of Canine Parvovirus (CPV2) infection. For safe and effective delivery of viral antigens to the mucosal immune system, a novel surface antigen display system for lactic acid bacteria using the poly-γ-glutamic acid synthetase A protein (pgsA) of Bacillus subtilis as an anchoring matrix was applied in order to display CPV2 antigen on the surface of the recombinant L. casei. Recombinant fusion proteins comprised of pgsA and the capsid protein (VP2-S1) showed stable expression in Lactobacillus casei. Surface localization of the fusion protein was verified by cellular fractionation analyses. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by ELISA using recombinant VP2-S1 proteins. Mice receiving intranasal immunization mounted higher antibody response than those receiving oral immunization. These results indicate that mucosal immunization with recombinant L. casei expressing CPV2 VP2-S1 protein on its surface provides an effective means for elicitation of strong antibody responses against CPV 2 VP2-S1.

The aim of the current study was to analyze the active ingredients and to screen the pharmacological properties of freshwater laver, Prasiola japonica, the only species grown in Korea. According to results of gas chromatography- mass spectrometry assay, components from P. japonica were more diverse than those from sea laver. Of particular interest, our results indicated that ethanol extract of P. japonica (PJE) contained loliolide, sorbitol, mannitol, and alverine, which were known to have an anti-oxidant, anti-oral microbial, osmotic diuresis, and smooth muscle relaxant, respectively. In addition, five solvent fractions of PJE (water, butanol, chloroform, ethyl acetate, and hexane) significantly inhibited the production of lipopolysaccharide-induced nitric oxide and a higher amount (>100 μg/mL) of chloroform, ethyl acetate, and hexane fraction were considered to play a specific role in cancer cell death. PJE and its solvent fractions found to be effective scavengers of free radicals, particularly, hydroxyl radicals. Glucose uptake in L6 myoblast cell line that stably expresses the glucose transporter type 4 (GLUT4) proteins was also remarkably enhanced upon treatment with solvent fractions, remarkably chloroform fraction. Taken together, we concluded that P. japonica may have potent pharmacological properties and thus contribute to development of novel natural candidates for various disease targets.

Onion (Allium cepa L.) contains high levels of dietary fibers and antioxidants, including vitamin C, D, and folates. Onion is also known as a quercetin-rich vegetable with high flavonoid content. Onion peel contains over 20 times more quercetin than onion flesh. The aim of this study was to examine the question of whether onion peel extract supplementation has an effect on maximal exercise performance in rat. Onion peel extracts were extracted with hot water. Thirty male Sprague Dawley rats were maintained on a pellet diet for one week, and then randomly divided into five groups: Normal control, Positive control (quercetin 20 mg/kg), Onion peel 4 mg/kg, Onion peel 20 mg/kg, and Onion peel 100 mg/kg. Oral administration was performed daily. The experimental period was four weeks. Thereafter, animals were then forced to swim in water and the maximal exercise performance period from the swimming start time to the exhausted time, in which they failed to rise to the surface of the water to breathe within a 7 second period, was measured. After necropsy, weights of gastrocnemius muscles were measured. Lactate dehydrogenase concentration in serum was measured using an enzymatic method, using a commercial kit. The maximal exercise performance period was significantly longer in the onion peel extracts fed groups, compared with the control group. The lactate dehydrogenase concentration of the onion peel extracts fed groups was significantly lower, compared with the control group. Based on these results, we suggest that onion peel water extract supplementation can enhance exercise capacity caused by the mechanism of decreasing lactate dehydrogenase concentration.

Bladder cancer is a common cancer in smoking men and may correlate with mechanosensitive potassium channels because the urinary bladder is a stretch sensing organ. Two-pore K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to play a critical role in both cell apoptosis and tumorigenesis. Of the channels, TREK1 can be activated by many physiological stimuli, including polyunsaturated fatty acids, and intracellular pH, hypoxia, and neurotransmitters. Here we attempted to determine whether TREK1 is functionally expressed in bladder cancer 253J cells. K2P channels, including TREK1, TREK2, TASK1, TASK3, and TWIK1, were quantified in cultured human bladder cancer 253J cells using real time quantitative RT-PCR (qRT-PCR) analysis. Among them, TREK1-like channel was recorded at a single channel level using the patch-clamp technique. The TREKl-like channel, with single-channel conductance of ~90 pS at −80 mV, was recorded in symmetrical 150 mM KCl using an excised inside-out patch configuration. The current- voltage relationships were linear and were insensitive to tetraethylammonium. The channel was activated by membrane stretch, free fatty acids, and intracellular acidosis. These results with electrophysiological properties resemble to those of K2P channel, for instance, TREK1. Therefore, we conclude that TREK1 channel is functionally present in bladder cancer 253J cells.

Uncovering enzyme (UCE), encoded by the human NAGPA, is a trans-Golgi enzyme that adds the mannose-6- phosphate recognition tag on lysosomal enzymes destined for the lysosome. Mutations in NAGPA are known to cause stuttering, a common speech disorder with unknown etiology. The human NAGPA gene is transcribed into two different forms, probably due to alternative splicing. One of them, known as a brain isoform, is lacking exon 8 (102-bp). We performed quantitative real-time PCR for the NAGPA brain and non-brain isoforms in a cDNA panel originating from 16 human tissues and 24 sub-brain regions. According to our findings, the relative quantity of the NAGPA brain isoform in the brain was 4.7 times more than that in the control cDNA, a pooled mixture of equal amounts of cDNAs from the 16 different tissues. Further analysis using the cDNA panel originating from 24 different sub-brain regions revealed that the cerebral cortex contained the largest amount of NAGPA brain isoform. Relative quantity in the cerebral cortex was 8.6 times more than that in the control cDNA (P=0.00004). The lowest quantity of this isoform was detected in cDNA from the pituitary gland. In conclusion, findings of the current study suggest that the cerebral cortex, expressing the highest quantity of the NAGPA brain isoform, might be the region associated with speech function.

Neuronal differentiation is a complex biological process accompanying cytoskeletal reorganization, including neurite outgrowth and growth cone formation. Therefore, neuronal differentiation is critically regulated by actin-related signaling proteins, such as small Rho GTPases, guanine nucleotide exchange factors (GEFs), and myosins. This study will demonstrate the change in activity of three small Rho GTPases, Rac, Cdc42, and Rho A, by treatment with blebbistatin (BBS), a specific inhibitor for myosin, during bFGF-induced neurite outgrowth in PC12 cells. Treatment with BBS induced morphological changes in growth cones and neurites during differentiation. A marked increase in protrusion and filopodia structures in growth cones, the shaft of neuritis, and cell membranes was observed in the cells treated with BBS. Activity of Rho GTPases showed the alterations in response to BBS. Activities of both Rac and Rho A were inhibited by BBS in a time-dependent manner. By contrast, Cdc42 activity was not changed by BBS. These results suggest that inactivation of myosin II by BBS induced morphological changes in neurites and growth cones and distinct regulation of three Rho GTPases during differentiation of PC12 cells.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the causative bacteria that can induce chronic enzootic pneumonia, resulting in low production in the swine industry. Potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia by M. hyopneumoniae has also been recognized. Although some available vaccines have been developed for prevention of M. hyopneumoniae infection, protective immunity is still poor. In this study, in order to provide valuable information on vaccine antigen, we investigated the immunogenicity of M. hyopneumoniae on mouse spleen cells. Concanavalin A (ConA) and lipopolysaccharide (LPS) were used for generation of activated T and B lymphocytes. M. hyopneumoniae made clusters of spleen cells and also affected the cellular activity and viability of spleen cells by alone or with mitogens. Of particular interest, it induced a significant increase in production of TNF-alpha in ConA- treated spleen cells, meaning T helper 1 response. In addition, cell size and mitochondrial membrane potential of M. hyopneumoniae–treated spleen cells were measured by flow cytometric analysis. M. hyopneumoniae did not affect the cell size by alone, whereas ConA or LPS profoundly increased the cell size. Taken together, M. hyopneumoniae significantly affect the cellular activity and cytokine production of spleen cells by alone or in a combination of ConA. This study provides valuable information for production of the vaccine against M. hyopneumoniae.

A 10-year-old, castrated male, English cocker spaniel dog was presented for evaluation of a mass in the left forelimb. Physical examination revealed a solitary subcutaneous mass measuring 2.7 × 2.1 × 1 cm in size. Radiographs and ultrasonography showed a well-circumscribed, focally mineralized, non-invasive to muscle layer mass without signs of further bone invasion and periosteal reaction. Cytologic evaluation of the mass through fine needle aspiration revealed a mesenchymal cell type malignant tumor without distant metastasis. An excisional biopsy was performed for definitive diagnosis and the mass was diagnosed as cutaneous hemangiopericytoma. This case report presents disagreement between fine needle aspiration and histopathology during diagnostic procedures of cutaneous hemangiopericytoma in a dog.

A 14-year-old female South American sea lion (Otaria byronia) with persistent vaginal secretion and chronic hemorrhagic diarrhea was encountered. During postmortem examination, the uterus was found to resemble a balloon with mucosal congestion and was filled with grayish milky material. The ovaries also had abnormal features, including necrotic surface lesions and multiple whitish foci in the cut section. Hemorrhages and ulcerated changes due to toxemia were observed in other organs, including the liver, spleen, lung, intestines, and lymph nodes. Microscopically, the left ovary contained interlacing fascicles of fibroblast-like cells with blunt-end nuclei showing cytoplasmic positive immunoreactivity against alpha-smooth muscle actin and desmin. The right ovary contained cells with round to cigar-shaped nuclei showing cytoplasmic positive immunoreactivity against vimentin. In conclusion, based on classification of bilateral ovarian tumors as a leiomyoma in the left region and a fibroma in the right region, this sea lion was diagnosed with chronic closed pyometra.

The anti-diabetes mechanism of silkworm Bombyx mori L. powder and extracts was found to inhibit the activity of α-glycosidase. The major functional component of silkworm powder was 1-deoxynojirimycin (1-DNJ), which exerts a blood glucose-lowering effect. In this study, we aimed to compare the effects of the supplements, including red ginseng extract on the functional components of silkworm. Fifty silkworm larvae were divided into the control group (Con, N=50), group A (A, artificial diet 95% and mulberry leaf powder 5%), group B (B, artificial diet 95% and mulberry powder 5%), group C (C, artificial diet 95% and Rubus coreanus remainders 5%), group D (D, artificial diet 95% and red ginseng extract 5%), and group E (E, artificial diet 95% and yeast powder (Saccharomyces cerevisiae). Body weights and length of silkworm larvae showed significant improvement in group A, D. In particular, the growth rate in group D (artificial diet 95% and red ginseng extract 5%) was larger than that of Con. In addition, the results showed that 1-DNJ concentration was significantly largest in group D. From these results, it is concluded that the addition of red ginseng extract may be effective for larval growth and 1-DNJ accumulation in silkworm rearing with an artificial diet.

This study was conducted in order to examine the effects of alcohol-free cetylpyridinium chloride drinking water additive and oral gel on clinical parameters related to periodontal disease in beagle dogs. This study was conducted with healthy 15 beagle dogs. Following a professional teeth cleaning procedure, dogs were divided into three groups. Dogs in the control group received nothing, those in the drinking water additive (DWA) group received 800 ml water with 15 ml of alcohol-free cetylpyridinium chloride drinking water additive daily, and those in the Oral gel (OG) group were treated with oral gel containing alcohol-free cetylpyridinium chloride and 0.05% chlorhexidine gluconate daily. Clinical parameters, including plaque index (PI), calculus index (CI), and gingivitis index (GI) were evaluated at two and four weeks. Dogs in the DWA and OG groups had significantly less plaque than dogs in the control group at two and four weeks (P<0.01, P<0.05). And, at four weeks, CI was significantly lower in the OG group compared to the control group (P<0.05). On GI, similar scores were recorded for all groups during the experimental period. No significant difference was observed between the DWA group and the OG group. The effect of alcohol-free cetylpyridinium chloride drinking water additive was similar to the result for alcohol containing cetylpyridinium chloride mouthwash reported in a previous study. The effect in control of periodontal disease was better in the OG group because of additional chlorhexidine gluconate. However, use of drinking water additive will be more convenient for owners; thus, it will be more effective for achievement of long-term results.

In this study, we observed anti-diabetic effects of acid hydrolyzed silk peptides, where the amount of peptides in the total amino acid mixture was strictly regulated. Using in vitro diabetes models, silk peptide-containing amino acid mixtures of 5.60% (G5), 11.30% (G10), 14.50% (G15), and 20.50% (G20) were examined separately in order to determine whether they have biological activities. According to our results, a cytoprotective effect was observed following treatment of interleukin-1β in RINm5f pancreas β-cells. As a consequence, Bax, a pro-apoptotic gene, was down-regulated, while Bcl-2, a pro-survival gene, was retained at normal level. Results of the 4’,6-diamidino-2-phentylindole (DAPI) staining assay confirmed that G20 has a better cytoprotective effect. Insulin release from RINm5f cells showed a significant increase following treatment with G5-G20, suggesting that silk peptide effectively regulated and induced insulin production. Single treatment with G5-G20 resulted in enhanced glucose uptake in L6 skeletal muscle cells. In addition, a higher amount of each group inhibited the activity of α-glucosidase. In summary, these data suggest that silk peptide may have an anti-diabetic effect through protection of pancreas β-cells and enhancement of insulin release, which showed a close association with Type 1 diabetes mellitus (DM), and can improve glucose uptake, which was the major target for therapy of Type 2 diabetes. Taken together, we concluded that acid hydrolyzed silk peptides can be used effectively for control of blood sugar metabolism via improvement of the problematic indices of Type 1 and Type 2 DM.

The aim of this study was to evaluate immunopotentiating activities of β-glucan derived from Saccharomyces (S.) cerevisiae and to select new strains having possibility as an immune-enhancing substance. We examined SB20 strains derived from commercial product as a control, and extracted β-glucans from the four strains of S. cerevisiae. RAW264.7 macrophages were treated with heat-killed yeasts, β-glucans, and lipopolysaccharide (LPS). The production of nitric oxide (NO) and cytokines such as TNF-α and IL-1β were then quantified. When macrophages were induced directly by in vitro addition of β-glucan, little production of NO and IL-1β was observed. When pretreated with strong stimulants, i.e., LPS, most yeasts showed down-modulation of NO and IL-1β production. However, TNF-α secretion was triggered by β-glucans and even more increased by the mixture effect of LPS and β-glucans. In particular, S6 strain induced TNF-α secretion more than other strains. Therefore, we can conclude that the S6 strain has possibility as an immune-enhancing substance.

The objective of this experiment was to assess the relationship between electrical resistance of the vaginal mucosa and plasma progesterone for optimal mating time in the bitch. Eight mature beagle bitches were examined, and we observed eight times of estrus. Vaginal electric resistance was recorded weekly using a Draminski ovulation detector in anestrus, and daily in estrus. Plasma progesterone concentration was estimated by radioimmunoassay. In the bitch, incline in vaginal electric resistance (376.20 ± 105.63 units) showed a closely association with the onset of proestrus. Ovulation day was determined as the first day when plasma progesterone concentration increased above 5.0 ng/ml (Day 0). On Day 0, vaginal mucous electric resistance was 438 ± 132 units. Vaginal mucous electric resistance showed a slight decrease or was maintained until Day 0. However, it showed an explosive increase, and peaked on Day 1~3, which was above 600 units. Two of eight cases peaked on Day 1, three of eight cases were revealed on Day 2, and others were revealed on Day 3. After Day 4, resistance showed a rapid drop to below 600 units and reached 200 units on Day 8. The optimal mating time was determined when vaginal mucous electric resistance was above 600 units.

Taste receptors of the anterior tongue are innervated by the chorda tympani (CT) branch of the facial (VIIth) nerve. The CT nerve transmits information on taste to the ipsilateral nucleus of the solitary tract (NST), which is the first taste central nucleus in the medulla. Taste information is known to be transferred ipsilaterally along the taste pathway in the central nervous system. Some patients with unilateral CT damage often retain their ability to sense taste. This phenomenon is not explained by the unilateral taste pathway. We examined whether neurons in the NST receive information on taste from the contralateral side of the tongue by measuring c-Fos-like Immunoreactivity (cFLI) following taste stimulation of the contralateral side of the tongue in the anesthetized rats. We used four basic taste stimuli, 1.0 M sucrose, 0.3 M NaCl, 0.01 M citric acid, 0.03 M QHCl, and distilled water. Stimulation of one side of the tongue with taste stimuli induced cFLI in the NST bilaterally. The mean number of cFLI ranged from 23.28 ± 2.46 by contralateral QHCl to 30.28 ± 2.26 by ipsilateral NaCl stimulation. The difference between the number of cFLI in the ipsilaterl and contralateral NST was not significant. The result of the current study suggests that neurons in the NST receive information on taste not only from the ipsilateral but also the contralateral side of the tongue.

Amoxicillin, a well-known antibiotic, has a broad spectrum against gram-negative and gram-positive bacteria. This experiment was conducted in order to investigate the effect of micronized and non-micronized amoxicillin prepared using different comminution techniques on change in blood concentration of rats. Forty adult male Sprague Dawley rats (6~7 weeks of age, body weight 128.3 ± 10.7 g) were randomly allocated to two treatment groups: micronized amoxicillin (MA) group treated with micronized amoxicillin trihydrate powder (particle size, over 90% of 10 μm), non-micronized amoxicillin (NMA) group treated with non-micronized amoxicillin trihydrate powder (particle size, over 70% of 100 μm), given 480 mg/kg body weight once daily for four days. The results showed a significant increase in serum concentration in the MA group on days 3 and 4, compared to the NMA group (P<0.05). In particular, serum concentration of the MA group on day 4 was increased almost two times that of the NMA group. The results indicate that due to the increase of the drug’s oral bioavailability, higher serum concentration would be achieved with the micronized amoxicillin trihydrate than with the non-micronized drug.

Cancer is the result of damage to the genetic system, i.e., dysfunction of the DNA repair system, resulting in dysregulated expression of various molecules, leading to cancer formation, migration, and invasion. In cancer progression, several proteases play a critical role in metastasis; however, their biological mechanism in cancer metastasis is not clearly understood. Among these proteases, cathepsins are a family of lysosomal proteases found in most animal cells. Cathepsins have an important role in protein turnover of mammalian, and are classified into 15 types based on their structure as serine (cathepsin A and G), aspartic (cathepsin D and E), and cysteine cathepsins (cathepsin B, C, F, H, K, L, O, S, V, X, and W). Cysteine cathepsins appear to accelerate the progression of human and rodent cancers, which can be a biomarker of the potency of malignancy or metastasis in mammalian. Overexpression of cyteine cathepsins causes the activation of angiogenesis promoting factor, whereas their downregulation reduces the angiogenesis of cancer progression. Under physiological conditions, cysteine cathepsins are essential in inflammation, infection, and cancer development. Activity of cysteine proteases, i.e., cathepsin B, is required for cancer progression or metastasis. Elevation of cysteine cathepsin is associated with cancer metastasis, angiogenesis, and immunity. Therefore, in this review, we suggest that cysteine cathepsin may be an anticancer target of strong clinical interest, although the exact mechanism of cathepsins in cancer metastasis is under investigation.