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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The current study was conducted in order to investigate bone formation using matrigel and angiogenic factors with HA and poly ε-caprolactone (HA/PCL) in a rat calvarial defect model. Calvarial defect formation was surgically created in Sprague Dawley rats (n=36). Rats in the control group (CD group, n=6) did not receive a graft. The HA/ PCL scaffold was grafted with matrigel (M-HA/PCL group, n=6) or without matrigel (HA/PCL group, n=6); and 100 ng of vascular endothelial growth factor with HA/ PCL scaffold containing matrigel (VEGF100 group, n=6), 100 ng (PDGF100 group, n=6) and 300 ng (PDGF300 group, n=6) of PDGF with HA/PCL scaffold containing matrigel were grafted in calvarial defects, respectively. Four weeks after surgery, bone formation was evaluated with micro computed tomography (micro CT) scanning, and histologically. According to the results, bone mineral density was significantly increased in the VEGF100, PDGF100, and PDGF300 groups compared to the HA/PCL group, in which angiogenic factors were not applied. In histological evaluation, more new bone formation around scaffolds was observed in the PDGF100 and the PDGF300 groups, compared with the VEGF100 group. Thus, the results indicate that HA/PCL containing matrigel with VEGF and PDGF is an effective grafting material for enhancement of bone formation in critical-sized bone defects. Especially, due to its price and capacity for bone formation, PDGF may be more effective than VEGF.
A two-year-old, castrated male Jindo dog presented with a three-week history of non-pruritic alopecia and scales on dorsum. The dermatologic lesions were progressive and did not respond to antibiotics and treatment for ectoparasite. Silver-white scales were adhered to the hair shafts and a follicular cast was marked on a trichogram. Findings on histopathologic examination of the skin lesions were consistent with sebaceous adenitis. Treatment with systemic cyclosporine and topical shampoo treatment was recommended for long term management. This case report is the first to describe a sebaceous adenitis in our country and suggests combination treatment approaches to long term management of this disease.
Cholangiocarcinoma is a relatively rare neoplasm in animals and humans. A four-year-old, neutered male ferret presented with depression, anorexia, cachexia diarrhea, and icterus. Necropsy findings included ascites multiple white nodules on the surface of the liver, stomach and duodenum, gross enlargement of the bile duct and right atrium, hemorrhage of the gastric and duodena mucosa, and icterus of the mesenteric fat. Infiltrative well differentiated neoplastic biliary epithelial cells forming ducts and acini with a prominent collagenous stroma were observed on microscopic examination of neoplastic lesions within the liver, mesentery, and the serosa of the stomach and duodenum. This is a report on a rare case of obstructive jaundice due to cholangiocarcinoma in a ferret.