RESUMO
The TRPM4 gene encodes a Ca2+-activated monovalent cation channel called transient receptor potential melastatin 4 (TRPM4) that is expressed in various tissues. Dysregulation or abnormal expression of TRPM4 has been linked to a range of diseases. We introduced the hemagglutinin (HA) tag into the extracellular S6 loop of TRPM4, resulting in an HA-tagged version called TRPM4-HA. This TRPM4-HA was developed to investigate the purification, localization, and function of TRPM4 in different physiological and pathological conditions. TRPM4-HA was successfully expressed in the intact cell membrane and exhibited similar electrophysiological properties, such as the current-voltage relationship, rapid desensitization, and current size, compared to the wild-type TRPM4. The presence of the TRPM4 inhibitor 9-phenanthrol did not affect these properties. Furthermore, a wound-healing assay showed that TRPM4-HA induced cell proliferation and migration, similar to the native TRPM4. Co-expression of protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP-1) with TRPM4-HA led to the translocation of TRPM4-HA to the cytosol. To investigate the interaction between PTPN6 and tyrosine residues of TRPM4 in enhancing channel activity, we generated four mutants in which tyrosine (Y) residues were substituted with phenylalanine (F) at the N-terminus of TRPM4. The YF mutants displayed properties and functions similar to TRPM4-HA, except for the Y256F mutant, which showed resistance to 9-phenanthrol, suggesting that Y256 may be involved in the binding site for 9-phenanthrol. Overall, the creation of HA-tagged TRPM4 provides researchers with a valuable tool to study the role of TRPM4 in different conditions and its potential interactions with other proteins, such as PTPN6.
RESUMO
Biogenic amines are cellular components produced by the decarboxylation of amino acids; however, excessive biogenic amine production causes adverse health problems. The relationship between hepatic damage and biogenic amine levels in nonalcoholic fatty liver disease (NAFLD) remains unclear. In this study, mice were fed a high-fat diet (HFD) for 10 weeks to induce obesity, presenting early-stage of NAFLD. We administered histamine (20 mg/kg) + tyramine (100 mg/kg) via oral gavage for 6 days to mice with HFD-induced early-stage NAFLD. The results showed that combined histamine and tyramine administration increased cleaved PARP-1 and IL-1ß in the liver, as well as MAO-A, total MAO, CRP, and AST/ALT levels. In contrast, the survival rate decreased in HFD-induced NAFLD mice. Treatment with manufactured or traditional fermented soybean paste decreased biogenically elevated hepatic cleaved PARP-1 and IL-1ß expression and blood plasma MAO-A, CRP, and AST/ALT levels in HFD-induced NAFLD mice. Additionally, the biogenic amine-induced reduction in survival rate was alleviated by fermented soybean paste in HFD-induced NAFLD mice. These results show that biogenic amine-induced liver damage can be exacerbated by obesity and may adversely affect life conservation. However, fermented soybean paste can reduce biogenic amine-induced liver damage in NAFLD mice. These results suggest a beneficial effect of fermented soybean paste on biogenic amine-induced liver damage and provide a new research perspective on the relationship between biogenic amines and obesity.
Assuntos
Alimentos Fermentados , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Histamina , Camundongos Obesos , Glycine max/química , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Aminas Biogênicas , Obesidade , Monoaminoxidase , Tiramina/uso terapêuticoRESUMO
Models created by the intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-GalN) have been widely used to study the pathogenesis of human acute liver failure (ALF) and drug development. Our previous study reported that oyster (Crassostrea gigas) hydrolysate (OH) had a hepatoprotective effect in LPS/D-GalN-injected mice. This study was performed to identify the hepatoprotective effect of the tyrosine-alanine (YA) peptide, the main component of OH, in a LPS/D-GalN-injected ALF mice model. We analyzed the effect of YA on previously known mechanisms of hepatocellular injury in the model. LPS/D-GalN-injected mice showed inflammatory, apoptotic, ferroptotic, and pyroptotic liver injury. The pre-administration of YA (10 mg/kg or 50 mg/kg) significantly reduced the liver damage factors. The hepatoprotective effect of YA was higher in the 50 mg/kg YA pre-administered group than in the 10 mg/kg YA pre-administered group. These results showed that YA had a hepatoprotective effect by reducing inflammation, apoptosis, ferroptosis, and pyroptosis in the LPS/D-GalN-injected ALF mouse model. We suggest that YA can be used as a functional peptide for the prevention of acute liver injury.
Assuntos
Anti-Inflamatórios/farmacologia , Ostreidae , Peptídeos/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Organismos Aquáticos , Modelos Animais de Doenças , Galactosamina , Lipopolissacarídeos , Falência Hepática Aguda/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/uso terapêutico , Piroptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.
Assuntos
Agonistas de Receptores de GABA-A/farmacologia , Neurônios GABAérgicos/metabolismo , Potenciais da Membrana , Muscimol/farmacologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Receptores de GABA/química , Animais , Células Cultivadas , Neurônios GABAérgicos/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Canais de Potássio de Domínios Poros em Tandem/genética , RatosRESUMO
BACKGROUND: Obesity due to an excessive intake of nutrient disturbs the hypothalamus-mediated energy metabolism subsequently develops metabolic disorders. In this study, we investigated the effect of pine needle extract (PNE) on the hypothalamic proopiomelanocortin (POMC) neurons involved in the regulation of energy balance via melanocortin system and fat tissue metabolism. METHODS: We performed electrophysiological and immunohistochemical analyses to determine the effect of PNE on POMC neurons. Mice were fed a normal or high-fat diet for 12 weeks, then received PNE for the last 2 weeks to measure the following physiological indices: Body weight, food intake, fat/lean mass, glucose metabolism, and plasma leptin levels. In addition, changes of thermogenic, lipolytic, and lipogenetic markers were evaluated in brown adipose tissue (BAT) and white adipose tissue (WAT) by western blotting, respectively. RESULTS: PNE increased hypothalamic POMC neuronal activity, and the effect was abolished by blockade of melanocortin 3/4 receptors (MC3/4Rs). PNE decreased body weight, fat mass, plasma leptin levels, and improved glucose metabolism after high-fat-induced obesity. However, PNE did not change the expression of thermogenic markers of the BAT in HFD fed groups, but decreased only the lipogenetic markers of WAT. This study suggests that PNE has a potent anti-obesity effect, inhibiting lipogenesis in WAT, even though HFD-induced leptin resistance-mediated disruption of POMC neuronal activity.
Assuntos
Delftia , Humanos , beta-Lactamases/genética , Delftia/genética , RNA Ribossômico 16S/genéticaRESUMO
Accumulative alcohol hangovers cause liver damage through oxidative and inflammatory stress. Numerous antioxidant and anti-inflammatory reagents have been developed to reduce alcohol hangovers, but these reagents are still insignificant and have limitations in that they can cause liver toxicity. Oyster hydrolysate (OH), another reagent that has antioxidant and anti-inflammatory activity, is a product extracted through an enzymatic hydrolysis process from oysters (Crassostrea gigas), which can be easily eaten in meals. This study was aimed at determining the effects of OH on alcohol metabolism, using a single high dose of ethanol (EtOH) administered to rodents, by monitoring alcohol metabolic enzymes, oxidative stress signals, and inflammatory mediators. The effect of tyrosine-alanine (YA) peptide, a main component of OH, on EtOH metabolism was also identified. In vitro experiments showed that OH pretreatment inhibited EtOH-induced cell death, oxidative stress, and inflammation in liver cells and macrophages. In vivo experiments showed that OH and YA pre-administration increased alcohol dehydrogenase, aldehyde dehydrogenase, and catalase activity in EtOH binge treatment. In addition, OH pre-administration alleviated CYP2E1 activity, ROS production, apoptotic signals, and inflammatory mediators in liver tissues. These results showed that OH and YA enhanced EtOH metabolism and had a protective effect against acute alcohol liver damage. Our findings offer new insights into a single high dose of EtOH drinking and suggest that OH and YA could be used as potential marine functional foods to prevent acute alcohol-induced liver damage.
Assuntos
Crassostrea/química , Dipeptídeos/farmacologia , Etanol/metabolismo , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Animais , Dipeptídeos/química , Etanol/administração & dosagem , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidrólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-DawleyRESUMO
Tandem pore domain weak inward rectifier potassium channel (TWIK)-related spinal cord K⺠(TRESK; K2P18.1) channel is the only member of the two-pore domain K⺠(K2P) channel family that is activated by an increase in intracellular Ca2+ concentration ([Ca2+]i) and linked to migraines. This study was performed to identify the effect of verapamil, which is an L-type Ca2+ channel blocker and a prophylaxis for migraines, on the TRESK channel in trigeminal ganglion (TG) neurons, as well as in a heterologous system. Single-channel and whole-cell currents were recorded in TG neurons and HEK-293 cells transfected with mTRESK using patch-clamping techniques. In TG neurons, changes in [Ca2+]i were measured using the fluo-3-AM Ca2+ indicator. Verapamil, nifedipine, and NiCl2 inhibited the whole-cell currents in HEK-293 cells overexpressing mTRESK with IC50 values of 5.2, 54.3, and >100 µM, respectively. The inhibitory effect of verapamil on TRESK channel was also observed in excised patches. In TG neurons, verapamil (10 µM) inhibited TRESK channel activity by approximately 76%. The TRESK channel activity was not dependent on the presence of extracellular Ca2+. In addition, the inhibitory effect of verapamil on the TRESK channel remained despite the absence of extracellular Ca2+. These findings show that verapamil inhibits the TRESK current independently of the blockade of Ca2+ influx in TG neurons. Verapamil will be able to exert its pharmacological effects by modulating TRESK, as well as Ca2+ influx, in TG neurons in vitro. We suggest that verapamil could be used as an inhibitor for identifying TRESK channel in TG neurons.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Neurônios/metabolismo , Canais de Potássio/metabolismo , Gânglio Trigeminal/metabolismo , Verapamil/farmacologia , Animais , Cálcio/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Camundongos , Níquel/farmacologia , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Transient receptor-potential, cation channel, subfamily M, member 4 (TRPM4) channels regulate a variety of physiological and pathological processes; however, their roles as functional channels under diverse conditions remain unclear. In this study, cytosolic protein tyrosine phosphatase non-receptor type 6 (PTPN6) interacted with TRPM4 channels. We confirmed their interaction by performing co-immunoprecipitation (Co-IP) assays following heterologous PTPN6 and TRPM4 channel expression in HEK293 cells. Furthermore, biomolecular fluorescence complementation (BiFC) image analysis confirmed TRPM4-PTPN6 binding. In addition, immunoblotting and Co-IP analyses revealed that TRPM4 expression significantly decreased in the membrane fraction of cells after PTPN6 was silenced with a specific short-hairpin RNA (shRNA-PTPN6). In agreement, TRPM4-induced changes in whole-cell currents were not detected in PTPN6-silenced HEK cells, in contrast to cells transfected with a scrambled RNA (scRNA) or in naïve HEK cells. These data suggest that PTPN6 inhibits TRPM4 channel activity by disrupting TRPM4 expression. Furthermore, TRPM4 channels were expressed in the membrane of naïve cells and scRNA transfectants, but not in those of PTPN6-silenced cells. These results indicated that PTPN6 is critically associated with TRPM4 trafficking. This role of PTPN6 in TRPM4 membrane localization was also demonstrated in HeLa cells. TRPM4 overexpression significantly enhanced cell proliferation in untreated HeLa cells, but not in HeLa cells with silenced PTPN6 expression. These findings indicate that PTPN6-dependent TRPM4 expression and trafficking to the plasma membrane is critical for cell proliferation in both HEK293 and HeLa cells. Therefore, PTPN6 is a novel therapeutic target for treating pathologic diseases involving TRPM4.
Assuntos
Membrana Celular/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Canais de Cátion TRPM/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Transporte ProteicoRESUMO
Earlier studies have demonstrated that the tandem pore domain weak inward rectifying K⺠channel (TWIK)-related K⺠(TREK)-1 channel is inhibited by antidepressants and is associated with major depression. However, little is known about the effect of mood stabilizers that are commonly used for treatment of bipolar disorder on TREK channels, members of the two-pore domain K⺠(K2P) channel family. This study sought to investigate the effect of mood stabilizers on TREK-1 and TREK-2 channels. HEK-293A cells were transfected with human TREK-1 or TREK-2 DNA. The effect of mood stabilizers on TREK-1 and TREK-2 was studied using the patch clamp technique. Changes in TREK protein expression by mood stabilizers were studied in the HT-22 mouse hippocampal neuronal cells using western blot analysis. Lithium chloride (LiCl, 1 mM), gabapentin (100 µM), valproate (100 µM), and carbamazepine (100 µM) increased TREK-1 currents by 31 ± 14%, 25 ± 11%, 28 ± 12%, and 72 ± 12%, respectively, whereas they had no effect on TREK-2 channel activity. In addition, western blot analysis showed LiCl and carbamazepine slightly upregulated TREK-1 expression, but not TREK-2 in the HT-22 cells. These results suggest that TREK-1 could be a potential therapeutic target for treatment of bipolar disorders as well as depression, while TREK-2 is a target well suited for treatment of major depression.
Assuntos
Antimaníacos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Antidepressivos/farmacologia , Antipsicóticos/farmacologia , Células HEK293 , Hipocampo/metabolismo , Humanos , Cloreto de Lítio/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ácido Valproico/farmacologiaRESUMO
Trimethoprim-sulfamethoxazole is the first-line antimicrobial combination for Stenotrophomonas maltophilia infections. However, allergy or intolerance and increasing resistance limit the use of trimethoprim-sulfamethoxazole. Quinolones can be used as an alternative therapeutic option, but resistance can emerge rapidly during therapy. We analyzed the contribution of SmeABC and SmeDEF efflux pumps to levofloxacin resistance in clinical isolates of S. maltophilia. Nonduplicate clinical isolates of S. maltophilia were collected in 2010 from 11 university hospitals (n = 102). Fifty-five levofloxacin nonsusceptible (minimum inhibitory concentration [MIC] ≥4 µg/ml) and 47 susceptible (MIC ≤2 µg/ml) isolates were tested for efflux pump overexpression. Real-time reverse transcription-PCR was performed for amplification and quantification of smeB, smeC, smeD, and smeF mRNA. To determine which antimicrobials were affected by smeD overexpression, the growth rates of a levofloxacin-susceptible S. maltophilia isolate were compared by measuring absorbance of antimicrobial-supplemented Luria-Bertani broth (LB) cultures with or without triclosan. Significant relationships between sme gene overexpression and resistance were observed for smeD against levofloxacin, smeC and smeF against ceftazidime, and smeC against ticarcillin-clavulanate. The mean MICs of moxifloxacin and tigecycline did not significantly differ for isolates with or without overexpression of smeB, smeC, and smeF, but were significantly higher for isolates with smeD overexpression. The mean MICs of amikacin were significantly higher for smeC or smeF overexpressing isolates. Increased growth of a levofloxacin-susceptible isolate was observed in LB with 1/2 MIC levofloxacin in the presence of triclosan. These data suggest that the expression of smeD plays a role in levofloxacin resistance in S. maltophilia.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Levofloxacino/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Stenotrophomonas maltophilia/efeitos dos fármacos , Stenotrophomonas maltophilia/metabolismo , Ceftazidima/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana/métodos , Minociclina/análogos & derivados , Minociclina/farmacologia , Moxifloxacina , Ticarcilina/farmacologia , Tigeciclina , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
BACKGROUND: Extensively drug-resistant (XDR) Pseudomonas aeruginosa and Acinetobacter baumannii are a threat to hospitalized patients. We evaluated the effects of antimicrobial combinations on XDR P. aeruginosa and A. baumannii isolates. METHODS: P. aeruginosa and A. baumannii isolates, which were resistant to all antibiotics except colistin (CL), were collected from eight hospitals in Korea. Genes encoding metallo-ß-lactamases (MBLs) and OXA carbapenemases were detected by PCR in eight P. aeruginosa and 30 A. baumannii isolates. In vitro synergy of antimicrobial combinations was tested by using the checkerboard method. RESULTS: Minimum inhibitory concentrations of ß-lactams, aminoglycosides, and fluoroquinolones were very high, while that of CL was low for majority of XDR P. aeruginosa and A. baumannii isolates. Antimicrobial combinations including Imipenem (IPM)-CL, ceftazidime (CAZ)-CL, and rifampin (RIF)-CL exerted only additive/indifferent effects on majority of XDR P. aeruginosa isolates. Proportions of XDR A. baumannii isolates that showed synergistic and additive/indifferent inhibition after treatment with antimicrobial combinations used are as follows: IPM-ampicillin-sulbactam (AMS), 17% and 80% isolates, respectively; IPM-rifampin (RIF), 13% and 81% isolates, respectively; IPM-CL, 13% and 87% isolates, respectively; and RIF-COL, 20% and 73% isolates, respectively. Significant proportion (19%) of XDR P. aeruginosa isolates produced MBLs, and majority (82%) of A. baumannii isolates produced either MBLs or OXA-23. CONCLUSIONS: Our results suggest that combinations of IPM-AMS, IPM-RIF, IPM-CL, and RIF-CL are more useful than individual drugs for treating 13-20% of XDR A. baumannii infections.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Aminoglicosídeos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sinergismo Farmacológico , Fluoroquinolonas/farmacologia , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Quality control (QC) processes are being performed in the majority of clinical microbiology laboratories to ensure the performance of microbial identification and antimicrobial susceptibility testing by using ATCC strains. To obtain these ATCC strains, some inconveniences are encountered concerning the purchase cost of the strains and the shipping time required. This study was focused on constructing a database of reference strains for QC processes using domestic bacterial strains, concentrating primarily on antimicrobial susceptibility testing. Three strains (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) that showed legible results in preliminary testing were selected. The minimal inhibitory concentrations (MICs) and zone diameters (ZDs) of eight antimicrobials for each strain were determined according to the CLSI M23. All resulting MIC and ZD ranges included at least 95% of the data. The ZD QC ranges obtained by using the CLSI method were less than 12 mm, and the MIC QC ranges extended no more than five dilutions. This study is a preliminary attempt to construct a bank of Korean QC strains. With further studies, a positive outcome toward cost and time reduction can be anticipated.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Povo Asiático , Humanos , Laboratórios , Controle de Qualidade , Valores de Referência , República da Coreia , Coloração e RotulagemRESUMO
BACKGROUND: We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-ß-lactamase (MBL)-producing Pseudomonas spp. METHODS: A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum ß-lactamase [GES]-5, 9 New Delhi metallo-ß-lactamase [NDM]-1, 5 Verona integron-encoded metallo-ß-lactamase [VIM]-2, 3 imipenem-hydrolyzing ß-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain. RESULTS: Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively. CONCLUSIONS: Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.
Assuntos
Proteínas de Bactérias/metabolismo , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão/métodos , Enterobacteriaceae/enzimologia , Pseudomonas/enzimologia , beta-Lactamases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Ácido Edético/química , Ácido Edético/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Infecções por Enterobacteriaceae/diagnóstico , Humanos , Pseudomonas/efeitos dos fármacos , Infecções por Pseudomonas/diagnóstico , Sensibilidade e Especificidade , beta-Lactamases/químicaRESUMO
Emerging resistance to trimethoprim/sulfamethoxazole (SXT) poses a serious threat to the treatment of Stenotrophomonas maltophilia infections. We determined the prevalence and molecular characteristics of acquired SXT resistance in recent clinical S. maltophilia isolates obtained from Korea. A total of 252 clinical isolates of S. maltophilia were collected from 10 university hospitals in Korea between 2009 and 2010. Antimicrobial susceptibility was determined by using the CLSI agar dilution method. The sul1, sul2, and sul3 genes, integrons, insertion sequence common region (ISCR) elements, and dfrA genes were detected using PCR. The presence of the sul1 gene and integrons was confirmed through sequence analysis. Among the 32 SXT-resistant isolates, sul1 was detected in 23 isolates (72%), all of which demonstrated high-level resistance (≥64 mg/L) to SXT. The sul1 gene (varying in size and structure) was linked to class 1 integrons in 15 of the 23 isolates (65%) harboring this gene. None of the SXT-susceptible isolates or the SXT-resistant isolates with a minimum inhibitory concentration of 4 and 8 mg/L were positive for sul1. Moreover, the sul2, sul3, and dfrA genes or the ISCR elements were not detected. The sul1 gene may play an important role in the high-level SXT resistance observed in S. maltophilia.
Assuntos
Proteínas de Bactérias/genética , Integrons/genética , Stenotrophomonas maltophilia/efeitos dos fármacos , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/patologia , Humanos , Testes de Sensibilidade Microbiana , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/isolamento & purificaçãoRESUMO
Copine1 (CPNE1) has tandem C2 domains and an A domain and is known as a calcium-dependent membrane-binding protein that regulates signal transduction and membrane trafficking. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Akt phosphorylation in the hippocampal progenitor cell line, HiB5. To determine which region of CPNE1 is related to HiB5 cell neurite outgrowth, we constructed several mutants. Our results show that over-expression of each C2 domain of CPNE1 increased neurite outgrowth and expression of the neuronal marker protein neurofilament (NF). Even though protein localization of the calcium binding-deficient mutant of CPNE1 was not affected by ionomycin, this mutant increased neurite outgrowth and NF expression in HiB5 cells. Furthermore, Akt phosphorylation was increased by over-expression of the calcium binding-deficient CPNE1 mutant. These results suggest that neither cellular calcium levels nor the localization of CPNE1 affect its function in neuronal differentiation. Collectively, our findings indicating that the C2 domains of CPNE1 play a calcium-independent role in regulating the neuronal differentiation of HiB5 cells.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Células COS , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Chlorocebus aethiops , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Células HEK293 , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estrutura Terciária de Proteína , Ratos , Deleção de Sequência , Transdução de SinaisRESUMO
We analyzed the whole genome sequence and resistome of the outbreak Klebsiella pneumoniae strain MP14 and compared it with those of K. pneumoniae carbapenemase- (KPC-) producing isolates that showed high similarity in the NCBI genome database. A KPC-2-producing multidrug-resistant (MDR) K. pneumoniae clinical isolate was obtained from a patient admitted to a Korean hospital in 2011. The strain MP14 was resistant to all tested ß-lactams including monobactam, amikacin, levofloxacin, and cotrimoxazole, but susceptible to tigecycline and colistin. Resistome analysis showed the presence of ß-lactamase genes including bla KPC-2, bla SHV-11, bla TEM-169, and bla OXA-9. MP14 also possessed aac(6'-)Ib, aadA2, and aph(3'-)Ia as aminoglycoside resistance-encoding genes, mph(A) for macrolides, oqxA and oqxB for quinolone, catA1 for phenicol, sul1 for sulfonamide, and dfrA12 for trimethoprim. Both SNP tree and cgMLST analysis showed the close relatedness with the KPC producers (KPNIH strains) isolated from an outbreak in the USA and colistin-resistant strains isolated in Italy. The plasmid-scaffold genes in plasmids pKpQil, pKpQil-IT, pKPN3, or pKPN-IT were identified in MP14, KPNIH, and Italian strains. The KPC-2-producing MDR K. pneumoniae ST258 stain isolated in Korea was highly clonally related with MDR K. pneumoniae strains from the USA and Italy. Global spread of KPC-producing K. pneumoniae is a worrying phenomenon.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Klebsiella pneumoniae , Análise de Sequência de DNA , Proteínas de Bactérias/genética , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/genética , Surtos de Doenças , Estudo de Associação Genômica Ampla , Humanos , Itália , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , República da Coreia/epidemiologia , Estados Unidos , beta-Lactamases/genéticaRESUMO
BACKGROUND: TRPM4 channels are Ca2+-activated nonselective cation channels which are deeply involved in physiological and pathological conditions. However, their trafficking mechanism and binding partners are still elusive. RESULTS: We have found the 14-3-3γ as a binding partner for TRPM4b using its N-terminal fragment from the yeast-two hybrid screening. Ser88 at the N-terminus of TRPM4b is critical for 14-3-3γ binding by showing GST pull-down and co-immunoprecipitation. Heterologous overexpression of 14-3-3γ in HEK293T cells increased TRPM4b expression on the plasma membrane which was measured by whole-cell recordings and cell surface biotinylation experiment. Surface expression of TRPM4b was greatly reduced by short hairpin RNA (shRNA) against 14-3-3γ. Next, endogenous TRPM4b-mediated currents were electrophysiologically characterized by application of glutamate and 9-phenanthrol, a TRPM4b specific antagonist in HT-22 cells which originated from mouse hippocampal neurons. Glutamate-induced TRPM4b currents were significantly attenuated by shRNAs against 14-3-3γ or TRPM4b in these cells. Finally, glutamate-induced cell death was greatly prevented by treatment of 9-phenanthrol or 14-3-3γ shRNA. CONCLUSION: These results showed that the cell surface expression of TRPM4 channels is mediated by 14-3-3γ binding, and the specific inhibition of this trafficking process can be a potential therapeutic target for glutamate-induced neuronal cell death.
Assuntos
Proteínas 14-3-3/deficiência , Membrana Celular/metabolismo , Glutamatos/toxicidade , Neurônios/citologia , Canais de Cátion TRPM/metabolismo , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Animais , Morte Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Hipocampo/citologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Serina/metabolismo , Relação Estrutura-Atividade , Canais de Cátion TRPM/químicaRESUMO
BACKGROUND: Mitochondrial trans-2-enoyl-CoA reductase (MECR) is involved in mitochondrial synthesis of fatty acids and is highly expressed in mitochondria. MECR is also known as nuclear receptor binding factor-1, which was originally reported with yeast two-hybrid screening as a binding protein of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). However, MECR and PPARα are localized at different compartment, mitochondria, and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for functional interaction between MECR and PPARα. METHODS: To identify the expression pattern of MECR and the cytosolic form of MECR (cMECR), we performed reverse transcription polymerase chain reaction (RT-PCR) with various tissue samples from Sprague-Dawley rats. To confirm the interaction between cMECR and PPARα, we performed several binding assays such as yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation. To observe subcellular localization of these proteins, immunocytochemistry was performed. A luciferase assay was used to measure PPARα activity. RESULTS: We provide evidence of an alternatively spliced variant of the rat MECR gene that yields cMECR. The cMECR lacks the N-terminal 76 amino acids of MECR and shows uniform distribution in the cytoplasm and nucleus of HeLa cells. cMECR directly bound PPARα in the nucleus and increased PPARα-dependent luciferase activity in HeLa cells. CONCLUSION: We found the cytosolic form of MECR (cMECR) was expressed in the cytosolic and/or nuclear region, directly binds with PPARα, and enhances PPARα activity.
RESUMO
Neural epidermal growth factor-like protein-like 2 (NELL2) is a secreted glycoprotein that is predominantly expressed in the nervous system, but little is known about the intracellular movement and secretion mechanism of this protein. By monitoring the localization and movements of enhanced green fluorescent protein (EGFP)-labeled NELL2 in living cultured hippocampal neuroprogenitor HiB5 cells, we determined the subcellular localization of NELL2 and its intracellular movement and secretion mechanism. Cterminal EGFP-fused NELL2 showed a typical expression pattern of secreted proteins, especially with respect to its localization in the endoplasmic reticulum, Golgi apparatus, and punctate structures. Vesicles containing NELL2 exhibited bidirectional movement in HiB5 cells. The majority of the vesicles (70.1%) moved in an anterograde direction with an average velocity of 0.454 µm/s, whereas some vesicles (28.7%) showed retrograde movement with an average velocity of 0.302 µm/s. The movement patterns of NELL2 vesicles were dependent upon the presence of microtubules in HiB5 cells. Anterograde movement of NELL2 did not lead to a detectable accumulation of NELL2 in the peripheral region of the cell, indicating that it was secreted into the culture medium. We also showed that the N-terminal 29 amino acids of NELL2 were important for secretion of this protein. Taken together, these results strongly suggest that the N-terminal region of NELL2 determines both the pattern of its intracellular expression and transport of NELL2 vesicles by high-velocity movement. Therefore, NELL2 may affect the cellular activity of cells in a paracrine or autocrine manner.