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.
Acidosis ; Anoxia ; Apoptosis ; Cell Transformation, Neoplastic ; Fatty Acids, Nonesterified ; Fatty Acids, Unsaturated ; Humans* ; Hydrogen-Ion Concentration ; Male ; Membranes ; Neurotransmitter Agents ; Patch-Clamp Techniques ; Potassium Channels* ; Potassium* ; Smoke ; Smoking ; Tetraethylammonium ; Urinary Bladder Neoplasms ; Urinary Bladder*

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.
Alternative Splicing ; Brain ; Cerebral Cortex ; DNA, Complementary* ; Exons ; Humans* ; Lysosomes ; Mannosephosphates ; Phosphoric Diester Hydrolases ; Pituitary Gland ; Protein Isoforms* ; Real-Time Polymerase Chain Reaction ; Stuttering

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.
Animals ; Biological Processes ; Cell Membrane ; Growth Cones ; Guanine Nucleotide Exchange Factors ; Heterocyclic Compounds with 4 or More Rings ; Myosin Type II ; Myosins ; Neurites ; Neuritis ; Neurons ; PC12 Cells* ; Proteins ; Pseudopodia ; rho GTP-Binding Proteins*

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.
Animals ; B-Lymphocytes ; Bacteria ; Cell Size ; Concanavalin A ; Membrane Potential, Mitochondrial ; Mice* ; Mitogens ; Mycoplasma hyopneumoniae* ; Mycoplasma* ; Pneumonia ; Porcine Reproductive and Respiratory Syndrome ; Spleen* ; Swine ; Tumor Necrosis Factor-alpha ; Vaccines

Sucrose and alcohol are rewarding and appetitive. They are occasionally over-consumed and cause addiction. The parabrachial nuclei (PbN) are the second taste relay in the central taste pathway. The nucleus accumbens (NAcc) is an important neural substrate in the reward system. Intake of sucrose or alcohol induces dopamine release in the NAcc. Although alcohol is not classified as a taste stimulus, a substantial number of sucrose-responsive neurons in the PbN respond to stimulation by alcohol on the tongue. In the present study, we investigated whether or not application of 0.5 M sucrose, 10% ethanol (EtOH), mixture of sucrose and EtOH, and double-distilled water (DDW) to the tongue induces c-Fos-like immunoreactivity (cFLI) in the PbN and NAcc. We also examined whether or not the number of cFLI following sucrose/EtOH is comparable to the number of cFLIs following sucrose and EtOH, respectively. Male Sprague-Dwaley rat was anesthetized with a mixture of Zoletil and Rompun while stimulation solution was applied to the anterior tongue. The rat was sacrificed by perfusion, and the fixed brain was sectioned and immunostained. Data from a total of 18 animals were analyzed. The number of cFLI following stimulation with sucrose and/or EtOH was greater than that of DDW in the PbN. Numbers of cFLI following sucrose, EtOH, and sucrose/EtOH were not significantly different from each other in the PbN. The number of cFLI in response to stimulation solution was not different from that of DDW in the NAcc. The result of the present study suggests that not only sucrose but also EtOH activates some neurons in the PbN, and that some pontine neurons possibly respond to both sucrose and EtOH.
Animals ; Brain ; Dopamine ; Ethanol ; Humans ; Male ; Neurons ; Nucleus Accumbens ; Perfusion ; Rats ; Reward ; Sucrose* ; Tongue ; Water ; Xylazine

Insulin detemir is a long-acting basal insulin analogue recently introduced in veterinary medicine for treatment of canine diabetes mellitus. As there are only limited studies in dogs, long-term evaluation of insulin detemir in veterinary medicine is required. In this study, we investigated trends in12-hour blood glucose concentration during hospitalization and evaluated initial and following doses of insulin detemir for several months in six diabetic dogs. The mean levels of blood glucose over 12-hour periods were between 113.5 to 327.2 mg/dL, and the average glucose nadir was 103 mg/dL in the six dogs. The dogs were treated with a mean dosage of 0.24 U/kg of insulin detemir, but hypoglycemia was observed in four of the dogs at the first monthly follow-up. Thus, insulin doses were adjusted according to the nadir levels of glucose observed during the follow-up periods (range, 1 to 16 months). The total range of insulin doses throughout the study period was between 0.1 and 0.4 U/kg. Changes in insulin doses in each dog during the follow-up period were not variable. We suggest that insulin detemir might be not only an alternative choice against traditional insulin for patients with insulin resistance or concurrent disease but also an effective home therapy medication in canine patients with DM. This study could help inform veterinary practitioners regarding the use of insulin detemir for canine insulin-dependent DM.
Adrenocortical Hyperfunction ; Animals ; Blood Glucose ; Diabetes Mellitus* ; Diabetic Ketoacidosis ; Dogs* ; Follow-Up Studies ; Glucose ; Hospitalization ; Humans ; Hypoglycemia ; Insulin Resistance ; Insulin* ; Insulin Detemir ; Veterinary Medicine

Canine respiratory coronavirus (CRCoV) is commonly associated with canine kennel cough worldwide. Clinically infected dogs present coughing, sneezing, and nasal discharge. Severe infections may progress to pneumonia. Through serological surveys, CRCoV has been identified as a worldwide pathogen found in the respiratory tracts of dogs suffering from mild or severe respiratory disease. In this study, three dogs were obtained from a dog kennel. Over the previous 5 days, the dogs showed coughing, sneezing, and nasal discharge. To detect the etiologic pathogen, we performed multiplex RT-PCR (mRT-PCR) to amplify the genes encoding canine influenza virus matrix protein, canine distemper virus nucleocapsid protein, and CRCoV spike protein. Dot blotting was achieved with a CRCoV-specific probe. Nasal-secreting CRCoV was detected by the 442 bp CRCoV-positive PCR reaction in the nasal swabbing samples from dogs. Further, CRCoV-positive reactions by dot blot hybridization were detected in the nasal swabbing samples from dogs. In conclusion, we detected CRCoV in kenneled dogs with respiratory disease in Korea. Multiplex RT-PCR was able to detect successfully CRCoV infection in dogs. We suggest that mRT-PCR would be useful and effective for monitoring CRCoV infection in various kinds of dogs.
Animals ; Coronavirus* ; Cough ; Distemper Virus, Canine ; Dogs* ; Korea ; Nucleocapsid Proteins ; Orthomyxoviridae ; Pneumonia ; Polymerase Chain Reaction ; Respiratory System ; Sneezing

Neural crest and placodes share a number of important features, pointing to a possible common evolutionary origin. They both arise from the neural plate border, which is the boundary between the non-neural ectoderm and neural plate. The transcription factor Sox9 has been implicated in neural crest and otic placode induction in several species. To investigate the differential regulation of neural crest and otic placode induction by Sox9, a gain of function assay was performed using a hormone-inducible version of the Sox9 construct at different doses and time periods. Sox9 was expressed in both neural crest and otic placode cell populations in the same stage embryos by in situ hybridization. Using a gain of function approach, increased expression of neural crest marker (Snail2) and otic placode marker (Pax8) in Sox9-overexpressed embryos was observed. Higher dose of Sox9 reduced or eliminated both neural crest and placode cells in the embryos. Interestingly, otic placodes cells were more strongly affected as compared to neural crest cells. So, optimal dosage and timing of Sox9 expression are important for the development of the neural crest and otic placode. The development of the neural crest and otic placode are affected by Sox9 in a time- and dose-dependent manner.
Ectoderm ; Embryonic Structures ; In Situ Hybridization ; Neural Crest* ; Neural Plate ; Transcription Factors ; Xenopus

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin-Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.
Cell Line* ; Collagenases ; Distemper Virus, Canine* ; Embryonic Structures ; Epithelial Cells ; Family Characteristics ; Fibroblasts ; Kidney* ; Madin Darby Canine Kidney Cells ; Retroviridae ; Telomerase

Antibiotics have been used to prevent disease, promote growth rate, and improve feed efficiency. However, the use of antibiotics in livestock has been restricted worldwide due to problems such as bacterial resistance. Therefore, probiotics among alternatives to antibiotics have gained attention in the livestock feed industry these days. This study was conducted to investigate the effects of dietary supplementation with probiotic 379D on safety, growth rate, and feed efficiency. In this study, bacterial strain 379D was isolated from soil and identified as a Bacillus sp. according to 16S rRNA sequence analysis. In an in vitro test, in-gel activity assay and antimicrobial susceptibility test were conducted to evaluate 379D. In an in vivo study, 379D was administered at concentrations of 0.1% and 1% to broiler chickens for 28 days. The results of in-gel activity assay and antimicrobial susceptibility test showed that strain 379D had broad spectrum antimicrobial activity. Furthermore, no adverse 379D-related effects were observed in 0.1% and 1% groups. Feed efficiency was higher in the 379D-treated groups than in the control group. In conclusion, 379D is expected to be used as a safe alternative to antibiotics in a feed supplement and will improve feed efficiency in broiler chickens.
Anti-Bacterial Agents* ; Bacillus ; Chickens* ; Dietary Supplements ; Livestock ; Probiotics* ; Sequence Analysis ; Soil