RESUMEN
The formation of membrane-less organelles is driven by multivalent weak interactions while mediation of such interactions by small molecules remains an unparalleled challenge. Here, we uncovered a bivalent inhibitor that blocked the recruitment of TDRD3 by the two methylated arginines of G3BP1. Relative to the monovalent inhibitor, this bivalent inhibitor demonstrated an enhanced binding affinity to TDRD3 and capability to suppress the phase separation of methylated G3BP1, TDRD3, and RNAs, and in turn inhibit the stress granule growth in cells. Our result paves a new path to mediate multivalent interactions involved in SG assembly for potential combinational chemotherapy by bivalent inhibitors.
Asunto(s)
ADN Helicasas , ARN Helicasas , ADN Helicasas/metabolismo , ARN Helicasas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Separación de Fases , Gránulos Citoplasmáticos/metabolismoRESUMEN
<p><b>BACKGROUND</b>Occult stress urinary incontinence may lead to de novo stress urinary incontinence after pelvic floor repair surgery. A measurement of pudendal nerve terminal motor latency can reflect the integrity of the nerves. We aimed to explore the value of pudendal nerve terminal motor latency in the diagnosis of occult stress urinary incontinence in pelvic organ prolapse patients.</p><p><b>METHODS</b>Ten patients with stress urinary incontinence (SUI group), 10 with SUI and uterine or vaginal prolapse (POP + SUI group) and 10 with uncomplicated uterine or vaginal prolapse (POP group) were evaluated for their pudendal nerve terminal motor latency using a keypoint electromyogram.</p><p><b>RESULTS</b>The amplitude of positive waves was between 0.1 and 0.2 mV. The nerve terminal motor latency was between 1.44 and 2.38 ms. There was no significant difference in the wave amplitudes of pudendal nerve evoked action potential among the three different groups (P > 0.05). The pudendal nerve latency of the SUI group, POP + SUI group and POP group were (2.9 ± 0.7) seconds, (2.8 ± 0.7) seconds and (1.9 ± 0.5) seconds respectively. The difference between the SUI group and POP + SUI group was not statistically significant (P > 0.05), whereas the difference between the SUI and POP groups and between the POP + SUI and POP groups were statistically significant (P < 0.05). There was a positive correlation between pudendal nerve latency and the severity of SUI; the correlation coefficient was 0.720 (P < 0.01).</p><p><b>CONCLUSIONS</b>Patients with SUI may have some nerve demyelination injuries in the pudendal nerve but the damage might not involve the nerve axons. The measurement of pudendal nerve latency may be useful for the diagnosis of SUI in POP patients.</p>
Asunto(s)
Femenino , Humanos , Persona de Mediana Edad , Potenciales Evocados , Fisiología , Prolapso de Órgano Pélvico , Nervio Pudendo , Incontinencia Urinaria de Esfuerzo , Diagnóstico , Prolapso UterinoRESUMEN
Nek2A is a cell-cycle-regulated protein kinase that localizes to the centrosome and kinetochore. Our recent studies provide a link between Nek2A and spindle checkpoint signaling [J. Biol. Chem. 279 (2004) 20049]. Extracellular signal-regulated kinase 2 (Erk2) is an important kinase, which belongs to mitogen activating protein (MAP) kinase family. Here we demonstrated that Nek2A binds specifically to Erk2. Erk2 interacts with Nek2A via a conserved Erk2 docking site located to the C-terminus of Nek2A. Our studies indicate this docking site is essential and sufficient for a direct Nek2A-Erk2 interaction. In addition, our immunocytochemical studies show that Nek2A and Erk2 are co-localized to centrosome. Significantly, elimination of Nek2A by RNA interference delocalized Erk2 from its centrosomal location, while inhibition of Erk2 kinase activity did not affect the localization of Nek2A in centrosome. We propose that Erk2 links extracellular signaling to centrosome dynamics by Nek2A.
Asunto(s)
Centrosoma/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Mitosis , Datos de Secuencia Molecular , Quinasas Relacionadas con NIMA , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Conejos , Técnicas del Sistema de Dos HíbridosRESUMEN
OBJECTIVE: To observe the effect of Fufang Sishen Decoction (FFSSD) on arrhythmia after virus myocarditis. METHODS: One hundred and two cases of arrhythmia after virus myocarditis were randomly divided into two groups. The treatment group was treated with FFSSD, 6 g, b.i.d.; and the control group with propafenone, 150 mg, q 8 h. The therapeutic effects were observed in 4 weeks. RESULTS: The total anti-arrhythmia effects of FFSSD and propafenone were 71.9% and 78.9% respectively (P>0.05). FFSSD took effects relatively slowly with mild and lasting effect. CONCLUSION: The curative effect of FFSSD in treating arrhythmia after virus myocarditis is confirmed. FFSSD has no obvious side effects.
Asunto(s)
Arritmias Cardíacas/tratamiento farmacológico , Enterovirus Humano B , Infecciones por Enterovirus/complicaciones , Medicina Tradicional China , Miocarditis/complicaciones , Adulto , Arritmias Cardíacas/etiología , Femenino , Humanos , MasculinoRESUMEN
To study the effect of three positively charged arginine residues near the active site Cys(124) of the human dual-specific phosphatase on the catalytic function, six VHR mutants R(125)L, R(130)L, R(130)K, R(130)L/S(131)A, R(158)K and R(158)L were obtained using QuikChange site-directed mutagenesis method. The recombinant plasmids containing mutant genes were transformed into the Escherichia coli strain BL21(DE3), and the expressed proteins were found to be water soluble after the induction of IPTG. The proteins with purity greater than 90% were obtained using Ni(2+) chelating affinity chromatography. The measurement of the steady-state kinetic parameters and arsenate inhibition constants K(i) of the enzyme and their mutants showed that the k(cat)/K(m) values of Arg(130) and Arg(158) mutants decreased, and K(i) values increased obviously compared with those of the wild enzyme. These results indicated that Arg(130) and Arg(158) were necessary for the enzymatic activity, and were probably related to the binding with the negatively charged phosphate group of the substrate. In addition, the slight difference for the k(cat) values between the R(130)L and R(130)L/S(131)A mutants suggested that Arg(130) mutation disrupted the hydrogen bond between Ser(131) and Cys(124). Furthermore, the arsenate binding affinity for R(125)L, R(130)L and R(158)L mutants was decreased, suggesting that positive charges in the side chains of these three arginine residues may be helpful for the binding of the enzyme to the substrate.
Asunto(s)
Arginina/genética , Cisteína/genética , Proteínas Tirosina Fosfatasas/genética , Sitios de Unión/genética , Catálisis , Fosfatasa 3 de Especificidad Dual , Escherichia coli/genética , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
The production conditions of extracellular laccase from Armilliria mellea and the characteristics of the enzyme were studied. The experiment proved that initial pH5.5 of the culture medium and temperature at 25 degrees C were favorable for laccase synthesis. As carbon resources, cellobiose and raffinose were better in terms of productivity than maltose, sorbose and galactose. Organic nitrogen source was more suitable for Armilliria mellea to synthesize laccase than inorganic nitrogen source. Peat extract (PE) enhanced notably the yield of laccase; the maximal yield was 7 times as much as that of the control when PE concentration was 50%. Three isozymes were detected in culture supernatant named A, B and C respectively after their mobility on PAGE. After concentrated by (NH4)2SO4 precipitation, laccase A was further purified to homogeneity by preparative native PAGE and anion exchange column chromatography. The native enzyme was a single polypeptide with a molecular mass of approximately 59 kD estimated by SDS-PAGE, while 58 kD by gel filtration chromatography under non-denaturing conditions. Determined by IEF its isoelectric point was 4.0. The optimal pH value and temperature were 5.6 and 60 degrees C respectively in catalytic reaction of oxidizing guaiacol. At 60 degrees C and 65 degrees C, half-lives of laccase A were 45 min and 36.8 min, respectively. Enzyme activity was inhibited with 100 mmol/L Cl-, but was activated with 1 mmol/L SO4(2-). However, if the concentration of NaN3 was only 1 mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1 mmol/L Cu2+ could enhance its activity. Laccase A showed a good stability when the pH of the buffer varied from 5.2 to 7.2. Using guaiacol as the substrate, the Km was 1.026 mmol/L and the Vmax was 5 mumol/(min.mg); using ABTS instead, the Km was 0.22 mmol/L and Vmax was 69 mumol/(min.mg).