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Two polyhedral silver-thiolate clusters, [S@Ag16(Tab)10(MeCN)8](PF6)14 (Ag16) and [Ag12(Tab)6(DMF)12](PF6)12 (Ag12), were synthesized by using electroneutral Tab species as protective ligands (Tab=4-(trimethylammonio)benzenethiolate, DMF=N,N-dimethylformamide, MeCN=acetonitrile). Ag16 has a decahedral shape composed of eight pentagon {Ag5} units and two square {Ag4} units. The structure of Ag12 is a cuboctahedron, a classical Archimedean structure composed of six triangular faces and eight square faces. The former configuration is discovered in silver-thiolate cluster for the first time, possibly benefited from the more flexible coordination between the Tab ligand and Ag+ facilitated by the electropositive -N(CH3)3 + substituent group. Third-order nonlinear optical studies show that both clusters in DMF exhibit reverse saturate absorption response under the irradiation of 532â nm laser.
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Quantum key distribution (QKD) allows two remote parties to share information-theoretic secret keys. Many QKD protocols assume the phase of encoding state can be continuous randomized from 0 to 2π, which, however, may be questionable in the experiment. This is particularly the case in the recently proposed twin-field (TF) QKD, which has received a lot of attention since it can increase the key rate significantly and even beat some theoretical rate-loss limits. As an intuitive solution, one may introduce discrete-phase randomization instead of continuous randomization. However, a security proof for a QKD protocol with discrete-phase randomization in the finite-key region is still missing. Here, we develop a technique based on conjugate measurement and quantum state distinguishment to analyze the security in this case. Our results show that TF-QKD with a reasonable number of discrete random phases, e.g., 8 phases from {0,π/4,π/2, ,7π/4}, can achieve satisfactory performance. On the other hand, we find the finite-size effects become more notable than before, which implies that more pulses should be emit in this case. More importantly, as a the first proof for TF-QKD with discrete-phase randomization in the finite-key region, our method is also applicable in other QKD protocols.
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Rolling bearing is an important part guaranteeing the normal operation of rotating machinery, which is also prone to various damages due to severe running conditions. However, it is usually difficult to extract the weak fault characteristic information from rolling bearing vibration signals and to realize a rolling bearing fault diagnosis. Hence, this paper offers a rolling bearing fault diagnosis method based on successive variational mode decomposition (SVMD) and the energy concentration and position accuracy (EP) index. Since SVMD decomposes a vibration signal of a rolling bearing into a number of modes, it is difficult to select the target mode with the ideal fault characteristic information. Comprehensively considering the energy concentration degree and frequency position accuracy of the fault characteristic component, the EP index is proposed to indicate the target mode. As the balancing parameter is crucial to the performance of SVMD and must be set properly, the line search method guided by the EP index is introduced to determine an optimal value for the balancing parameter of SVMD. The simulation and experiment results demonstrate that the proposed SVMD method is effective for rolling bearing fault diagnosis and superior to the variational mode decomposition (VMD) method.
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Gearboxes are widely used in drive systems of rotating machinery. The health status of gearboxes considerably influences the normal and reliable operation of rotating machinery. When a gearbox experiences tooth failure, a vibration signal with impulse features is excited. However, these impulse features tend to be relatively weak and difficult to extract. To solve this problem, a novel approach for gearbox fault feature extraction and fault diagnosis based on improved variational mode extraction (VME) is proposed. Since the initial value of the desired mode center frequency and the value of the penalty parameter in VME must be assigned, a short-time Fourier transform (STFT) was performed, and a new index, the standard deviation of differential values of envelope maxima positions (SDE), is proposed. The feasibility and effectiveness of the proposed approach was verified by a simulation signal and two datasets associated with a gearbox test bench. The results demonstrate that the VME-based approach outperforms the variational mode decomposition (VMD) approach.
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VibraciónRESUMEN
Increased oxidative stress is well known to cause testicular dysfunction in aging males, but the detailed relationships between aging, oxidative stress, and testicular function remain to be elucidated. LIM and cysteine-rich domains 1 (LMCD1) regulates fundamentally cellular process by interacting with transcription factors. A recent study has identified Lmcd1 as one of the most upregulated nuclear proteins associated with Sertoli cell (SC) differentiation, raising the possibility that testicular actions of LMCD1 are likely to take place. Herein, we reported that LMCD1 was exclusively expressed in the nuclei of SCs. This expression was regulated by TNF-α signaling produced by apoptotic germ cells (GCs) and was suppressed by oxidative stress in a STAT3-dependent manner. Ablation of endogenous LMCD1 expression caused lipid accumulation and senescence in GC co-incubated SCs. Using a previously validated in vivo siRNA approach, we showed that LMCD1 depletion significantly impaired male fertility by inducing oligozoospermia and asthenospermia. Mechanistically, LMCD1 upregulation was associated with the nuclear enrichment of the nuclear factor of activated T cells 1 (NFAT1), a core component of Ca2+ /calmodulin-dependent pathway. LMCD1 facilitated the dephosphorylation and nuclear translocation of NFAT1, which consequently expedited the transactivation of Txlna, a binding partner of the syntaxin family essential for testicular phagocytosis, and thus promoted the removal of apoptotic GCs by phagocytic SCs. Collectively, LMCD1 may operate as a novel pretranscriptional integrator linking SC phagocytosis, lipid homeostasis, and cell senescence.
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Proteínas Co-Represoras/metabolismo , Proteínas con Dominio LIM/metabolismo , Factores de Transcripción NFATC/metabolismo , Células de Sertoli/metabolismo , Testículo/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Fagocitosis , Transducción de Señal , Espermatogénesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Neuropathic pain is a debilitating status that is insusceptible to the existing analgesics. It is important to explore the underlying pathophysiological changes and search for new pharmacological approaches. Transient receptor potential canonical 6 (TRPC6) is a mechanosensitive channel that is expressed by dorsal root ganglia and glial cells. It has been demonstrated that this channel in dorsal root ganglia plays essential roles in the formation of mechanical hyperalgesia in neuropathic pain. Recent pharmacological screening suggests that larixyl acetate (LA), a main constituent of larch resin, is able to selectively inhibit TRPC6 function. But whether LA is effective in treating neuropathic pain remains unknown. We investigated the efficacy of LA in rat neuropathic pain model, examined its effects on central neuroinflammation, and explored the possible molecular mechanisms by targeting the spinal dorsal horn. METHODS: Spared nerve injury (SNI) was conducted in Sprague-Dawley rats. Mechanical hypersensitivity and cold allodynia before and after single and multiple i.t. applications of LA at the dose of 3, 10, and 30 µM were evaluated by von Frey filament and acetone tests, respectively. Western blot, immunohistochemical, and immunocytochemical stainings were employed to examine the level and expression feature of ionized calcium-binding adaptor molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), TRPC6, and phosphorylated p38 kinase. The changes of cytokine concentrations, including that of TNF-α, IL-1ß, IL-6, and IL-10, were also assessed by multiplex analysis. TRPC6 antisense strategy was finally adopted to investigate the action mechanisms of LA. RESULTS: Single application of LA on day 5 post injury caused dose-dependent inhibition of mechanical allodynia with the ED50 value of 13.43 µM. Multiple applications of LA at 30 µM not only enhanced the analgesic efficacy but also elongated the effective duration without obvious influences on animal locomotor activities. Single and multiple administrations of LA at 30 µM played similar but weaker inhibitory effects on cold allodynia. In addition to behavioral improvements, multiple applications of LA for 6 days dose-dependently inhibited the upregulation of Iba-1, TNF-α, IL-1ß, and IL-6, whereas had no obvious effects on the levels of GFAP and IL-10. Combined Western blot and immunostaining assays revealed that the expression of TRPC6 was significantly increased in both spinal dorsal horn after nerve injury and the cultured microglia challenged by LPS, which was however suppressed by the addition of LA at 30 µM or 10 µM, respectively. Further knockdown of TRPC6 with antisense oligodeoxynucleotide produced prominent analgesic effects in rats with SNI, accompanied by the reduced phosphorylation level of p38 in the microglia. CONCLUSIONS: These data demonstrate that i.t. applied LA exhibits analgesic and anti-inflammatory action in neuropathic pain. The action of LA involves the suppression of TRPC6 and p38 signaling in the microglia. LA may be thus a promising pharmacological candidate for the treatment of intractable chronic pain.
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Acetatos/uso terapéutico , Analgésicos/uso terapéutico , Naftalenos/uso terapéutico , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/metabolismo , Acetatos/farmacología , Analgésicos/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/patología , Masculino , Ratones , Naftalenos/farmacología , Neuralgia/patología , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Gorham disease, a rare disorder of unknown etiology, is characterized by the clinical and radiologic disappearance of bone. Because the etiology is unknown, diagnosis is difficult. Therefore, radiographic manifestations play a vital role in the diagnosis of this disease. Thus far, there has been no completely effective treatment. Most remedies are limited to symptom management. Despite the fact that any bone can be affected, one of the most prevalent sites is the maxillofacial region. In this paper, 2 cases of Gorham disease involving the maxillofacial region are reported, including preoperative and postoperative radiographic features.
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Enfermedades Maxilomandibulares , Osteólisis Esencial , Humanos , Enfermedades Maxilomandibulares/diagnóstico , Enfermedades Maxilomandibulares/terapia , Mandíbula , Osteólisis Esencial/diagnóstico , Osteólisis Esencial/terapia , Resultado del TratamientoRESUMEN
Chronic pain and depression frequently coexist in clinical setting, and current clinical treatments for this comorbidity have shown limited efficacy. Triptolide (T10), an active component of Tripterygium wilfordii Hook F., has been demonstrated to exert strong analgesic activities in experimental pain models, but whether it possesses anti-depressive actions remains unknown. Using a depression comorbidity of chronic pain rat model induced by spinal nerve ligation (SNL), we investigated the potency of T10 for the treatment of comorbid depression in comparison with a widely used antidepressant, fluoxetine (FLX). Concomitant neuroinflammation changes were also examined in the hippocampus. The results showed that prophylactic and reversal treatments with T10 dose-dependently (30, 100, 300µg/kg) inhibited the depression-like behaviors (DLB) assessed by the forced swim test, sucrose preference test and body weight measurement. The anti-depressive efficacy of T10 at 300µg/kg was significantly stronger than that of FLX at 18mg/kg. T10 at all three doses exhibited more efficient analgesic effects than FLX at 18mg/kg. The combined application of T10 with FLX markedly augmented the effects of T10 or FLX per se, with the facilitating effects of T10 at 30µg/kg being most prominent. In addition, nerve injury caused the activation of microglia and p38 MAPK, the upregulation of IL-1ß and TNF-α as well as the downregulation of IL-10 in the hippocampus at postoperative week (POW) 3. These neuroinflammatory responses were reversed by subchronic treatment with T10. Taken together, these results demonstrate that T10 possesses potent anti-depressive function, which is correlated with its immunoregulation in the hippocampus. The combination of a low dose of T10 with FLX may become a more effective medication strategy for the treatment of comorbid depression and chronic pain.
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Antidepresivos/administración & dosificación , Dolor Crónico/complicaciones , Depresión/tratamiento farmacológico , Diterpenos/administración & dosificación , Encefalitis/complicaciones , Hipocampo/efectos de los fármacos , Fenantrenos/administración & dosificación , Analgésicos/administración & dosificación , Animales , Conducta Animal/efectos de los fármacos , Dolor Crónico/tratamiento farmacológico , Dolor Crónico/metabolismo , Depresión/complicaciones , Depresión/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Encefalitis/metabolismo , Compuestos Epoxi/administración & dosificación , Fluoxetina/administración & dosificación , Hipocampo/metabolismo , Hipocampo/fisiopatología , Hiperalgesia/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Masculino , Microglía/efectos de los fármacos , Ratas Sprague-DawleyRESUMEN
Members of the Drosophila behavior/human splicing protein family, including splicing factor proline/glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and paraspeckle protein component 1 (PSPC1), are abundantly expressed in testicular Sertoli cells (SCs), but their roles remain obscure. Here, we show that treatment with mono-(2-ethylhexyl) phthalate (MEHP), a well-known SC toxicant, selectively stimulates the expression levels of NONO and PSPC1. Simultaneous inhibition of NONO and PSPC1 expression in SCs enhances MEHP-induced oxidative stress and potentiates SC death. Mechanistically, NONO and PSPC1 transcriptionally activate aldehyde dehydrogenase 1 (Aldh1a1), by synergistically binding to the distinct CCGGAGTC sequence in the Aldh1a1 promoter. Together, the NONO/PSPC1-ALDH1A1 cascade may serve as an indispensable defense mechanism against MEHP insult in SCs.
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Aldehído Deshidrogenasa/metabolismo , Proteínas de Unión al ADN/metabolismo , Dietilhexil Ftalato/análogos & derivados , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Células de Sertoli/metabolismo , Aldehído Deshidrogenasa/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Proteínas de Unión al ADN/genética , Dietilhexil Ftalato/farmacología , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Sertoli/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Regulación hacia ArribaRESUMEN
The main etiopathogenesis of rheumatoid arthritis (RA) is overexpressed inflammatory cytokines and tissue injury mediated by persistent NF-κB activation. MicroRNAs widely participate in the regulation of target gene expression and play important roles in various diseases. Here, we explored the mechanisms of microRNAs in RA. We found that microRNA (miR)-10a was downregulated in the fibroblast-like synoviocytes (FLSs) of RA patients compared with osteoarthritis (OA) controls, and this downregulation could be triggered by TNF-α and IL-1ß in an NF-κB-dependent manner through promoting the expression of the YingYang 1 (YY1) transcription factor. Downregulated miR-10a could accelerate IκB degradation and NF-κB activation by targeting IRAK4, TAK1 and BTRC. This miR-10a-mediated NF-κB activation then significantly promoted the production of various inflammatory cytokines, including TNF-α, IL-1ß, IL-6, IL-8, and MCP-1, and matrix metalloproteinase (MMP)-1 and MMP-13. In addition, transfection of a miR-10a inhibitor accelerated the proliferation and migration of FLSs. Collectively, our data demonstrates the existence of a novel NF-κB/YY1/miR-10a/NF-κB regulatory circuit that promotes the excessive secretion of NF-κB-mediated inflammatory cytokines and the proliferation and migration of RA FLSs. Thus, miR-10a acts as a switch to control this regulatory circuit and may serve as a diagnostic and therapeutic target for RA treatment.
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Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Glicoproteínas de Membrana/genética , FN-kappa B/metabolismo , Receptores Inmunológicos/genética , Sinoviocitos/metabolismo , Factor de Transcripción YY1/metabolismo , Artritis Reumatoide/patología , Secuencia de Bases , Sitios de Unión , Movimiento Celular/genética , Proliferación Celular/genética , Citocinas/biosíntesis , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/genética , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Persona de Mediana Edad , Modelos Biológicos , Interferencia de ARN , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
[This corrects the article DOI: 10.1007/s12264-010-0410-9.].
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OBJECTIVE: We aimed to investigate the expression characteristics of transient receptor potential canonical 7 (TRPC7) in normal and hypertrophic cardiac myocytes. METHODS: The 2-kidney 1-clip (2K1C) method was used to induce renovascular hypertension. Losartan, the potent inhibitor of angiotensin II (Ang II) receptor, was applied to the drinking water of 2K1C rats to inhibit Ang II-mediated responses. TRPC7 expression was examined by immunohisto/cytochemistry and Western blot analyses in normal and hypertrophic hearts. The expression level of protein kinase C (PKC), a negative regulator of TRPC7 channel in in vitro study, was also evaluated. RESULTS: In normal rat ventricles, strong TRPC7 immunoreactivity was distributed in the surface sarcolemma of cardiomyocytes, and a moderate but striated TRPC7 immunoreactivity was also detected in the subcellular regions. The 2K1C operation caused significant hypertension and cardiac hypertrophy as demonstrated by respective biophysical or biochemical assays. At this stage, expression of TRPC7 was significantly downregulated at both tissue and cell levels, whereas that of PKC was upregulated. Further analysis revealed a negative correlation between TRPC7 and PKC expression patterns. Oral application of losartan ameliorated the extent of experimentally induced hypertension and cardiac hypertrophy. Simultaneously, it effectively reversed the downregulation of TRPC7 and mildly antagonized the upregulation of PKC. CONCLUSIONS: Taken together, these results for the first time show that TRPC7 localizes in the surface and tubular sarcolemma of cardiomyocytes in normal adult rats and its expression significantly decreases in hypertrophied cardiomyocytes from renovascular hypertensive rats. TRPC7 may thus play a significant role in normal physiological settings in the heart.
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OBJECTIVE: Sleep disturbance, which is characterized by excessive daytime sleepiness and sleep attacks, is frequently observed in patients with Parkinson's disease (PD). Loss of orexin neurons in hypothalamus and the resultant decreased level of orexin in cerebrospinal fluid (CSF) found in narcolepsy patients may also play an essential role in the pathogenesis of sleep disturbance. The present study aimed to investigate the possible changes in the orexin system during PD progression. METHODS: After the establishment of a rat PD model by injecting 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, the numbers of orexin-A- and tyrosine hydroxylase (TH)-positive neurons, and the levels of orexin-A fibers and orexin-A in CSF were examined by immunohistochemistry and ELISA assay, respectively. RESULTS: Compared to the TH-containing neurons that exhibited fast degeneration in response to 6-OHDA, orexin-A-containing neurons were less sensitive to 6-OHDA. The number of orexin-A-positive neurons began to decrease at day 21 after operation, and at day 49, it decreased by 30% of the initial level. The orexin-A level in CSF of PD rats did not show any obvious fluctuations compared to the control, and there was no obvious reduction in the density of orexin-A-positive fibers in brain areas such as tuberomammillary nucleus. CONCLUSION: These results reveal for the first time the dynamic changes of orexin system during the progression of PD. This may provide valuable information for drug development to reverse the loss of orexin neurons and sleep disturbance in PD patients.
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Péptidos y Proteínas de Señalización Intracelular/líquido cefalorraquídeo , Neuropéptidos/líquido cefalorraquídeo , Oxidopamina/toxicidad , Trastornos Parkinsonianos/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Péptidos y Proteínas de Señalización Intracelular/fisiología , Masculino , Haz Prosencefálico Medial/metabolismo , Haz Prosencefálico Medial/fisiopatología , Neuropéptidos/fisiología , Orexinas , Trastornos Parkinsonianos/etiología , Trastornos Parkinsonianos/fisiopatología , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Edwardsiella tarda is the pathogen responsible for edwardsiellosis, a serious infectious disease of freshwater and marine fish species, and currently recognized to be the species pathogenic for human. An anti-idiotypic monoclonal antibody (mAb), 1E11, has been developed. It mimics the protective epitope of E. tarda and can prevent fish from infection of E. tarda. In this study, the correct variable heavy (VH) and variable light (VL) genes were obtained from 1E11 by using bioinformatics methods, and a 15 amino acid (Gly4Ser)3 linker was used to hold the two V domains together for the construction of VL-linker-VH form of single chain variable fragment (scFv) gene. Then, the scFv was subcloned into the vector pET-28a, expressed in the Escherichia coli BL21 cells, and identified by SDS-PAGE and western blotting. Red drum (Sciaenops ocellatus L.) weighing about 50 g was subjected to challenge with different E. tarda strains after 4 weeks followed by vaccination, the mortality rates and relative percentage survival were recorded and calculated, and the survival rate of fish in the scFv subgroups was obviously higher than that of control subgroups (P<0.01). Enzyme-linked immunosorbent assay results show that after 4 weeks of post-vaccination, the level of specific antibody in fish sera of scFv groups was significantly higher than control groups. This study indicates that the recombinant antibody scFv was successfully developed, and it may serve as an effective vaccine candidate against E. tarda.
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Edwardsiella tarda/efectos de los fármacos , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/veterinaria , Enfermedades de los Peces/tratamiento farmacológico , Enfermedades de los Peces/inmunología , Anticuerpos de Cadena Única/uso terapéutico , Animales , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/uso terapéutico , Edwardsiella tarda/inmunología , Edwardsiella tarda/patogenicidad , Infecciones por Enterobacteriaceae/inmunología , Peces , Anticuerpos de Cadena Única/inmunología , Resultado del TratamientoRESUMEN
AIM: The aim of the present study was to explore the mechanism for the Ca2+- dependent inactivation of the canonical transient receptor potential (TRPC) 7 channel expressed in human embryonic kidney 293 cells. METHOD: The whole-cell patch-clamp technique was used in the study. RESULTS: With Ca2+-free external solution, the perfusion of 100 micromol/L carbachol to, or dialysis of the cell with 100 micromol/L guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), induced large inward currents, respectively. These currents were rapidly inhibited by the addition of 1 mmol/L Ca2+ into the bath, and recovery from this inhibition was only partial after the Ca2+ removal, unless vigorous intracellular Ca2+ buffering with 10 mmol/L 1,2 bis(2- aminophenoxy)ethane-N,N,No,No-tetraacetic acid (BAPTA) (plus 4 mmol/L Ca2+) was employed. In contrast, the current induced by a membrane-permeable analog of diacylglycerol (DAG), 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 micromol/L) did not undergo the inhibition persisting after Ca2+ removal. Interestingly, the inclusion of inositol 1,4,5 trisphosphate (IP3; 100 micromol/L) in the patch pipette rendered the OAG-induced current susceptible to the persistent Ca2+-mediated inhibition independent of the IP3 receptor in the majority of the tested cells, as evidenced by the inability of heparin and thapsigargin in reversing the effect of IP3. CONCLUSION: The present results suggest that Ca2+ entry via the activated TRPC7 channel plays a critical role in inactivating the channel where the cooperative actions of DAG and IP3 are essentially involved.
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Calcio/fisiología , Diglicéridos/farmacología , Inosina Trifosfato/farmacología , Canales Catiónicos TRPC/antagonistas & inhibidores , Calcio/metabolismo , Línea Celular , Sinergismo Farmacológico , Electrofisiología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Técnicas de Placa-ClampRESUMEN
Cytokine-induced antiapoptosis inhibitor 1 (CIAPIN1) is a newly identified antiapoptosis molecule and a mediator of gastric MDR. We cloned cDNA of human CIAPIN1 by RT-PCR and constructed prokaryotic expression vectors of human CIAPIN1 by inserting human CIAPIN1 coding region into pET28-a(+) and pGEX- 4T-1, respectively. The fusion proteins were expressed in Escherichia coli and purified by affinity chromotography. Monoclonal antibody (MAb) against CIAPIN1 was obtained with standard cell fusion technique and ELISA screening. Immunohistochemistry and Western blot showed that the anti-CIAPIN1 MAb recognizes human and mouse CIAPIN1 protein in both native and denatured form. Western blotting confirmed that the expression of CIAPIN1 was upregulated in MDR gastric cancer cell lines. This MAb will be a useful tool for the detection of CIAPIN1 protein in future studies.
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Anticuerpos Monoclonales/inmunología , Apoptosis , Inmunización , Interleucina-3/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Western Blotting , Fusión Celular , Línea Celular Tumoral , Clonación Molecular , Citocinas , ADN Complementario , Resistencia a Múltiples Medicamentos/inmunología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Vectores Genéticos , Humanos , Hibridomas , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Regulación hacia ArribaRESUMEN
Ribosomal proteins (RP) L6 was previously identified as an up-regulated gene in multidrug-resistant gastric cancer cells SGC7901/ADR comparing to its parental cells SGC7901 by subtractive hybridization. The aim of this study was to explore the roles of RPL6 in multidrug resistance (MDR) in gastric cancer cells. Northern and Western blot analysis confirmed that RPL6 was overexpressed in SGC7901/ADR cells. By gene transfection, RPL6 was genetically upregulated in SGC7901 or down-regulated in SGC7901/ ADR cells. Upregulation of RPL6 was associated with enhanced resistance to multiple anticancer drugs (adriamycin, vincristine, etoposide, 5-fluorouracil and cisplatin) and to adriamycin-induced apoptosis. Downregulation of RPL6 reversed MDR and sensitized cells to adriamycin-induced apoptosis. Alteration of RPL6 showed no obvious influence on intracellular adriamycin accumulation, glutathione content and expression of glutathione S-transferase. RPL6 could upregulate Bcl-2 and downregulate Bax in cells. Together, this work demonstrates that RPL6 could regulate MDR in gastric cancer cells by suppressing drug-induced apoptosis.
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Apoptosis/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Regulación Neoplásica de la Expresión Génica , Proteínas Ribosómicas/metabolismo , Neoplasias Gástricas/metabolismo , Antibióticos Antineoplásicos/farmacología , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Northern Blotting , Western Blotting , Línea Celular Tumoral , Cisplatino/farmacología , Doxorrubicina/farmacología , Etopósido/farmacología , Fluorouracilo/farmacología , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Ribosómicas/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Transfección , Vincristina/farmacología , Proteína X Asociada a bcl-2RESUMEN
In our previous work, cellular prion protein (PrPc) was identified as an upregulated gene in adriamycin-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901. Here we investigate the expression of PrPc in gastric cancer and whether it was involved in multidrug resistance (MDR) of gastric cancer. We demonstrated that PrPc was ubiquitously expressed in gastric cancer cell lines and tissues. PrPc conferred resistance of both P-glycoprotein (P-gp)-related and P-gp-nonrelated drugs on SGC7901, which was accompanied by decreased accumulation and increased releasing amount of adriamycin in PrPc-overexpressing cell line. Inhibition of PrPc expression by antisense or RNAi technology could partially reverse multidrug-resistant phenotype of SGC7901/ADR. PrPc significantly upregulated the expression of the classical MDR-related molecule P-gp but not multidrug resistance associated protein and glutathione S-transferase pi. The PrPc-induced MDR could be partially reversed by P-gp inhibitor verapamil. PrPc could also suppress adriamycin-induced apoptosis and alter the expression of Bcl-2 and Bax, which might be another pathway contributing to PrPc-related MDR. The further study of the biological functions of PrPc may be helpful for understanding the mechanisms of occurrence and development of clinical gastric carcinoma and PrPc-related MDR and developing possible strategies to treat gastric cancer.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Resistencia a Múltiples Medicamentos , Proteínas PrPC/biosíntesis , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Adenocarcinoma/patología , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacología , Apoptosis , Bloqueadores de los Canales de Calcio/farmacocinética , Bloqueadores de los Canales de Calcio/farmacología , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Humanos , Fenotipo , Neoplasias Gástricas/patología , Células Tumorales Cultivadas , Regulación hacia Arriba , Verapamilo/farmacocinética , Verapamilo/farmacologíaRESUMEN
OBJECTIVE: To investigate the effect of gene Af116609 on gastric cancer multi-drug resistance (MDR) by introducing it into gastric cancer multi-drug resistant (MDR) cell line SGC7901/VCR. METHODS: Gene Af116609 was cloned from SGC7901/VCR by RT-PCR and its differential expression between gastric cancer MDR cells and its parental cells was displayed by Northern blot. The gene was introduced to gastric cancer cells by transfection of recombinant eukaryotic expression vector by electroporation. MTT assay in vitro was applied to investigate its effect on multi-drug resistance phenotype of gastric cancer cells. RESULTS: The full length CDS of gene Af116609, as long as 327 bp, was cloned from gastric cancer MDR cell line SGC7901/VCR and its sequence was coincident with the hypothetical gene Af116609 in GenBank. It was overexpressed in MDR cells than its parental cells at mRNA level. In the MTT assay in vitro, the drug sensitive cells transfected with sense eukaryotic expression vector showed upregulated targeted gene, with increased resistance to vincristine, 5-fliorouracil and arabinoside, and decreased resistance to adriamycin, but no influence on resistance to methotrexate. However, the drug resistant cells transfected with anti-sense eukaryotic expression vector, showed down regulated targeted gene, with less resistance to all the five anticancer drugs to different degrees. CONCLUSION: Gene Af116609 is related to MDR phenotype of gastric cancer cells and may become a candidate molecular target to reverse the MDR of gastric cancer.
Asunto(s)
Autoantígenos/genética , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , ARN Citoplasmático Pequeño/genética , Ribonucleoproteínas/genética , Neoplasias Gástricas/genética , Vincristina/farmacología , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Humanos , Neoplasias Gástricas/patología , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
ZNRD1, a new zinc ribbon gene, has been previously identified as an upregulated gene in a multidrug-resistant gastric cancer cell line SGC7901/VCR comparing to its parental cell SGC7901 by subtractive hybridization and RT-PCR. The antisense nucleic acid for ZNRD1 could enhance adriamycin accumulation in SGC7901/VCR cells and sensitize SGC7901/VCR cells to vincristine. The present study aims to explore the role of ZNRD1 in multidrug resistance in gastric cancer cells. Upregulation of ZNRD1 protein in SGC7901/VCR cells was confirmed by Western blot and immunocytochmical staining. ZNRD1 was genetically overexpressed in SGC7901 cells by gene transfection. It was found that overexpression of ZNRD1 could sensitize SGC7901 cells to P-glycoprotein (P-gp)-related anticancer drugs (vincristine, adriamycin, etoposide) but not to P-gp-nonrelated drugs (5-fluorouracil and cisplatin), which was accompanied with significantly decreased adriamycin accumulation and retention and increased adriamycin releasing in SGC7901 cells. Verapamil, an inhibitor for P-gp, could reverse the effects of ZNRD1 on drug sensitivity and drug accumulation in SGC7901 cells to a great extent. Western blot and Northern blot revealed that overexpression of ZNRD1 could upregulate P-gp at both protein and mRNA levels. Together, these results suggest that overexpression of ZNRD1 could promote multidrug-resistant phenotype of gastric cancer cells through upregulation of P-gp.