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1.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33397703

RESUMEN

The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a coenzyme A (CoA)-activation-type catabolic pathway that is usually a common part of the microbial mineralization of chlorinated aromatic compounds. Previous studies have shown that the transport and dehalogenation genes for 4CBA are typically clustered as an fcbBAT1T2T3C operon and inducibly expressed in response to 4CBA. However, the associated molecular mechanism remains unknown. In this study, a gene (fcbR) adjacent to the fcb operon was predicted to encode a TetR-type transcriptional regulator in Comamonas sediminis strain CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host Escherichia coli, the expression of the Pfcb -fused green fluorescent protein (gfp) reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the ligand for FcbR may be an intermediate metabolite. Purified FcbR could bind to the Pfcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the P fcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 molar ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5'-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3') that consists of two sequence repeats containing four pseudopalindromic sequences (5'-TCNATNGA-3'). This binding motif overlaps with the -35 box of Pfcb and was proposed to prevent the binding of RNA polymerase. This study characterizes a transcriptional repressor of the fcb operon, together with its ligand, thus identifying halogenated benzoyl-CoA as belonging to the class of ligands of transcriptional regulators.IMPORTANCE The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation-type catabolic pathway that plays an important role in the biodegradation of polychlorinated biphenyls and some herbicides. With genetic and biochemical approaches, the present study identified the transcriptional repressor and its cognate effector of a 4CBA hydrolytic dehalogenation operon. This work extends halogenated benzoyl-CoA as a new member of CoA-derived effector compounds that mediate allosteric regulation of transcriptional regulators.


Asunto(s)
Acilcoenzima A/metabolismo , Proteínas Bacterianas/genética , Clorobenzoatos/metabolismo , Comamonas/genética , Factores de Transcripción/genética , Escherichia coli/genética , Hidrólisis , Operón
2.
Artículo en Inglés | MEDLINE | ID: mdl-36767955

RESUMEN

The bank slopes in the Three Gorges Reservoir area (TGRA) have experienced obvious deterioration under the action of the periodic fluctuations in the reservoir water level. Generally, laboratory tests have been used to reveal the evolution trend of the slope banks. However, this method has a certain degree of cross-scale problem, especially for the mechanical state in a complex environment. Therefore, in this study, we took the Yangjiaping bank slope in the TGRA as an example and proposed a comprehensive on-site detection method to further reveal the rock mass degradation phenomenon of this typical reverse sand-mudstone interbedded bank slope. Specifically, multi-scale laser scanning, cross-hole acoustic wave detection, and inclination measurements were performed to analyze the fractures, quality, and deformation of rocky banks. The results showed that the deterioration of the bank slope manifested as the expansion, deepening, and widening of the cracks, as well as the peeling off and loosening of rocky banks. Large-scale laser scanning revealed that the deterioration zone was deformed along large fracture zones and layers. Unlike limestone slopes, the intact sandstone underground might be degraded by changes in water. There are few inclinometers and no deformation or weak deformation, which requires long-term monitoring. The relevant research methods provide an important reference for determining the instability and failure trend of the reservoir bank slopes.


Asunto(s)
Arena , Suelo , Conservación de los Recursos Naturales , Carbonato de Calcio , Agua
3.
Vascul Pharmacol ; 145: 107017, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35680060

RESUMEN

Pulmonary hypertension (PH) is a progressive and life-threatening disease with poor prognosis despite many advances in medical therapy over the past 20 years. Novel therapies which target on the underlying pathology of PH are still urgent to be met. TPN171H is a recently found new compound that exhibits potent pharmacological effects in PH via inhibiting phosphodiesterase type 5 (PDE-5). However, as one icariin derivative, the anti-inflammatory effects of TPN171H for treating PH are not clear. The present study was designed to investigate the therapeutical effect of TPN171H against inflammation in PH and reveal the underlying mechanism. Hypoxia and monocrotaline (MCT)-induced PH rat models were established, which were treated by oral administration of TPN171H (5, 25 mg/kg/d) or sildenafil (25 mg/kg/d). The right ventricle systolic pressure (RVSP), right ventricle hypertrophy index (RVHI) and vascular remodeling were measured. The results suggested that TPN171H significantly reduced RVSP and RVHI, and reversed pulmonary vascular remodeling in rats with both models. Furthermore, in in vivo and in vitro research, our data suggested that TPN171H remarkably suppressed cathepsin B-mediated NLRP3 inflammasome activation, which may contribute to its therapeutical function for PH.


Asunto(s)
Hipertensión Pulmonar , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Catepsina B/farmacología , Catepsina B/uso terapéutico , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/terapia , Hipertrofia Ventricular Derecha/tratamiento farmacológico , Hipertrofia Ventricular Derecha/prevención & control , Hipoxia/tratamiento farmacológico , Inflamasomas , Inflamación/patología , Monocrotalina , Proteína con Dominio Pirina 3 de la Familia NLR , Inhibidores de Fosfodiesterasa 5/farmacología , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Arteria Pulmonar , Ratas , Ratas Sprague-Dawley , Citrato de Sildenafil/farmacología , Citrato de Sildenafil/uso terapéutico , Remodelación Vascular
4.
ACS Synth Biol ; 10(3): 487-494, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33616380

RESUMEN

Bioaugmentation is a promising alternative in soil remediation. One challenge of bioaugmentation is that exogenous pollutant-degrading microbes added to soil cannot establish enough biomass to eliminate pollutants. Considering that methanotrophs have a growth advantage in the presence of methane, we hypothesize that genetically engineered methanotrophs could degrade contaminants efficiently in soil with methane. Here, methanotroph Methylomonas sp. LW13, herbicide bensulfuron-methyl (BSM), and two kinds of soil were chosen to confirm this hypothesis. The unmarked gene knock-in method was first developed for strain LW13. Then, BSM hydrolase encoding gene sulE was inserted into the chromosome of strain LW13, conferring it BSM-degrading ability. After inoculation, the cell amount of strain LW13-sulE in soil raised considerably (over 100 fold in 9 days) with methane provision; meanwhile, >90% of BSM in soil was degraded. This study provides a proof of the concept that genetically engineered methanotroph is a potential platform for soil remediation.


Asunto(s)
Biodegradación Ambiental , Metano/metabolismo , Plaguicidas/metabolismo , Contaminantes del Suelo/metabolismo , Técnicas de Sustitución del Gen , Hidrolasas/genética , Hidrolasas/metabolismo , Metano/química , Methylomonas/genética , Methylomonas/metabolismo , Plaguicidas/química , Microbiología del Suelo , Contaminantes del Suelo/química , Compuestos de Sulfonilurea/química , Compuestos de Sulfonilurea/metabolismo , Zea mays/metabolismo
5.
Front Pharmacol ; 12: 691405, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34658848

RESUMEN

Pulmonary hypertension (PH) is an extremely serious cardiopulmonary disease, finally leading to progressive right ventricular failure and death. Our previous studies have nominated HLQ2g, a pyrazolo[3,4-b] pyridine derivative stimulating soluble guanylate cyclase (sGC), as a new candidate for the treatment of PH, but the specific mechanism is still not clear. The PH model induced by hypoxia was established in rats. Right ventricular systolic pressure (RVSP) was assessed by jugular vein catheterization. RV weight was the index to evaluate RV hypertrophy. The protein levels of cGMP-dependent protein kinase type I (cGKI), bone morphogenetic protein receptor 2 (BMPR2), phosphorylated Smad1/5/8 (p-Smad1/5/8), and inhibitor of differention 1 (Id1) in pulmonary artery and human pulmonary artery smooth muscle cells (HPASMCs) were determined by western blotting. Cell proliferation and migration were evaluated. In the whole experiment, the first clinically available sGC stimulator Riociguat was used as the reference. In hypoxic PH rat model, elevated RVSP and RV hypertrophy were significantly reduced by HLQ2g treatment. Both Riociguat and HLQ2g attenuated vascular remodeling accompanied with up-regulated cGKI expression and BMP signaling pathway, which was characterized by elevated expression of BMPR2, p-Smad1/5/8, and Id1 in HPH rats. In addition, HLQ2g inhibited proliferation and migration of HPASMCs induced by hypoxia and platelet-derived growth factor (PDGF), restored BMPR2 signaling, which was recalled by Rp-8-Br-PET-cGMPS, the inhibitor of cGKI. In summary, the novel pyrazolo[3,4-b] pyridine derivative HLQ2g can alleviate HPH progression by up-regulating cGKI protein and BMP signaling pathway.

6.
Front Microbiol ; 11: 441, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32296398

RESUMEN

Due to their fast growth rate and robustness, some haloalkalitolerant methanotrophs from the genus Methylotuvimicrobium have recently become not only promising biocatalysts for methane conversion but also favorable materials for obtaining fundamental knowledge on methanotrophs. Here, to realize unmarked genome modification in Methylotuvimicrobium bacteria, a counterselectable marker (CSM) was developed based on pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase. Two-point mutations (T252A and A306G) were introduced into PheS in Methylotuvimicrobium buryatense 5GB1C, generating PheS AG , which can recognize p-chloro-phenylalanine (p-Cl-Phe) as a substrate. Theoretically, the expression of PheS AG in a cell will result in the incorporation of p-Cl-Phe into proteins, leading to cell death. The P tac promoter and the ribosome-binding site region of mmoX were employed to control pheS AG , producing the pheS AG -3 CSM. M. buryatense 5GB1C harboring pheS AG -3 was extremely sensitive to 0.5 mM p-Cl-Phe. Then, a positive and counterselection cassette, PZ (only 1.5 kb in length), was constructed by combining pheS AG -3 and the zeocin resistance gene. A PZ- and PCR-based strategy was used to create the unmarked deletion of glgA1 or the whole smmo operon in M. buryatense 5GB1C and Methylotuvimicrobium alcaliphilum 20Z. The positive rates were over 92%, and the process could be accomplished in as few as eight days.

8.
J Clin Invest ; 111(12): 1933-43, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12813029

RESUMEN

TNF-alpha activates ASK1 in part by dissociating 14-3-3 from apoptosis signal-regulating kinase 1 (ASK1). In the present study, we identified a novel Ras GTPase-activating protein (Ras-GAP) as an ASK1-interacting protein (AIP1). AIP1 binds to the C-terminal domain of ASK1 via a lysine-rich cluster within the N-terminal C2 domain. AIP1 exists in a closed form through an intramolecular interaction between the N-terminus and the C-terminus, and TNF-alpha induces unfolding of AIP1 leading to association of AIP1 with ASK1. Thus, the N-terminus of AIP1 containing the C2 and GAP domains constitutively binds to ASK1 and facilitates the release of 14-3-3 from ASK1. In contrast to 14-3-3, AIP1 binds preferentially to dephosphorylated ASK1. Recruited AIP1 enhances ASK1-induced JNK activation, and the ASK1 binding and the GAP activity of AIP1 are critical for AIP1-enhanced ASK1 activation. Furthermore, TNF-induced ASK1/JNK activation is significantly blunted in cells where AIP1 is knocked down by RNA interference. These data suggest that AIP1 mediates TNF-alpha-induced ASK1 activation by facilitating dissociation of inhibitor 14-3-3 from ASK1, a novel mechanism by which TNF-alpha activates ASK1.


Asunto(s)
Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Tirosina 3-Monooxigenasa/metabolismo , Proteínas Activadoras de ras GTPasa/fisiología , Proteínas 14-3-3 , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Secuencia de Bases , Bovinos , Células Cultivadas , ADN Complementario/genética , Endotelio Vascular/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , MAP Quinasa Quinasa Quinasa 5 , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas , Datos de Secuencia Molecular , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Técnicas del Sistema de Dos Híbridos , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/genética
9.
Mol Cell Biol ; 22(21): 7512-23, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12370298

RESUMEN

Tumor necrosis factor (TNF) is a cytokine that mediates many pathophysiologial processes, including angiogenesis. However, the molecular signaling involved in TNF-induced angiogenesis has not been determined. In this study, we examined the role of Etk/Bmx, an endothelial/epithelial tyrosine kinase involved in cell adhesion, migration, and survival in TNF-induced angiogenesis. We show that TNF activates Etk specifically through TNF receptor type 2 (TNFR2) as demonstrated by studies using a specific agonist to TNFR2 and TNFR2-deficient cells. Etk forms a preexisting complex with TNFR2 in a ligand-independent manner, and the association is through multiple domains (pleckstrin homology domain, TEC homology domain, and SH2 domain) of Etk and the C-terminal domain of TNFR2. The C-terminal 16-amino-acid residues of TNFR2 are critical for Etk association and activation, and this Etk-binding and activating motif in TNFR2 is not overlapped with the TNFR-associated factor type 2 (TRAF2)-binding sequence. Thus, TRAF2 is not involved in TNF-induced Etk activation, suggesting a novel mechanism for Etk activation by cytokine receptors. Moreover, a constitutively active form of Etk enhanced, whereas a dominant-negative Etk blocked, TNF-induced endothelial cell migration and tube formation. While most TNF actions have been attributed to TNFR1, our studies demonstrate that Etk is a TNFR2-specific kinase involved in TNF-induced angiogenic events.


Asunto(s)
Antígenos CD/metabolismo , Endotelio Vascular/patología , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Tirosina Quinasas/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Western Blotting , Bovinos , Movimiento Celular , Células Cultivadas , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Humanos , Immunoblotting , Ligandos , MAP Quinasa Quinasa 4 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Neovascularización Patológica , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Receptores Tipo II del Factor de Necrosis Tumoral , Factores de Tiempo , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Venas Umbilicales/citología , Cicatrización de Heridas
10.
Riv Biol ; 103(1): 18-21, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-21110460
12.
Am J Pathol ; 163(1): 243-51, 2003 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12819028

RESUMEN

There is increasing support for the idea that excessive production of proinflammatory mediators such as tumor necrosis factor (TNF) and reactive oxygen species (ROS) contribute to the pathogenesis of cardiac dysfunction. However, the mechanisms by which cytokine/ROS production mediates cardiac dysfunction have not been established. Given that apoptosis signal-regulating kinase 1 (ASK1) is highly expressed in cardiac muscle and that ASK1 is an important mediator in the signaling pathways induced by tumor necrosis factor, interleukin-1, and ROS, we used the yeast two-hybrid system with ASK1 as bait to identify ASK1 substrates from a human heart cDNA library. The cDNA encoding the cardiac troponin T (cTnT) was isolated. ASK1 specifically interacted with cTnT, but not cTnI, in vitro and in vivo via the C-terminal ASK1 domain. ASK1 specifically phosphorylated cTnT in vitro and in vivo. Mutations in cTnT (T194/S198) at an ASK1-phosphorylation consensus sequence significantly reduced phosphorylation by ASK1. ROS-induced ASK1 activation, cTnT phosphorylation, and contractile dysfunction in cardiomyocytes showed similar kinetics. Moreover, overexpression of constitutively active ASK1 induces cTnT phosphorylation and inhibits shortening and calcium transient in adult cardiomyocytes. We conclude that ASK1 plays an important role in regulation of cardiac contractile function by phosphorylating cTnT and may participate in cytokine/ROS-induced pathogenesis of cardiomyopathy and heart failure.


Asunto(s)
Quinasas Quinasa Quinasa PAM/metabolismo , Contracción Muscular/fisiología , Miocitos Cardíacos/metabolismo , Troponina T/metabolismo , Animales , Células Cultivadas , Activación Enzimática , Biblioteca de Genes , Humanos , Peróxido de Hidrógeno/metabolismo , MAP Quinasa Quinasa Quinasa 5 , Quinasas Quinasa Quinasa PAM/genética , Miocitos Cardíacos/citología , Oxidantes/metabolismo , Fosforilación , Unión Proteica , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Troponina T/genética , Técnicas del Sistema de Dos Híbridos
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