A major goal of biomedical research has been the identification of molecular mechanisms that can enhance memory. Here we report a novel signaling pathway that regulates the conversion from short- to long-term memory. The mTOR complex 2 (mTORC2), which contains the key regulatory protein Rictor (Rapamycin-Insensitive Companion of mTOR), was discovered only recently, and little is known about its physiological role. We show that conditional deletion of rictor in the postnatal murine forebrain greatly reduces mTORC2 activity and selectively impairs both long-term memory (LTM) and the late (but not the early) phase of hippocampal long-term potentiation (LTP). Actin polymerization is reduced in the hippocampus of mTORC2-deficient mice and its restoration rescues both L-LTP and LTM. More importantly, a compound that selectively promotes mTORC2 activity converts early-LTP into late-LTP and enhances LTM. These findings indicate that mTORC2 could be a novel therapeutic target for the treatment of cognitive dysfunction.
mTOR complexes; actin assembly; spatial memory; fear memory; cognitive enhancer
Although cervical cardiac transplantation is a well recognized useful model in diverse experimental settings, its widespread use, however, has been significantly hampered by the technical challenges relevant to small vessel anastomosis. We herein introduced a simplified two-stitch sleeve technique into arterial anastomosis during the course of cervical cardiac transplantation in mice. Cervical transplantation of allogenic and syngeneic cardiac grafts was conducted to assess the feasibility of this two-stitch sleeve technique in arterial anastomosis. Venous anastomosis was completed by the one-suture end-to-end microsuture technique, while arterial anastomosis was conducted by invaginating the recipient right common carotid artery into the graft left common carotid artery along with two guiding stitches. The two-stitch sleeve technique significantly simplified the procedures for arterial anastomosis as compared with that of the traditional microsuture technique (5.5 ± 1.8 min vs. 15.7 ± 3.0 min). However, the survival time for allografts (8.0 ± 0.2 day vs. 8.0 ± 0.4 day) and the long-term patency for syngeneic grafts (> 120 days) were the same as the grafts implanted by the traditional microsuture technique. This simplified sleeve technique is easy to learn, particularly for beginners without microsuture experience, and therefore, it has the great potential for widespread use in transplant immunology.
Anastomosis; heart transplantation; microsuture; two-stitch sleeve technique
During aortic surgery, interruption of spinal cord blood flow might cause spinal cord ischemia-reperfusion injury (IRI). The incidence of spinal cord IRI after aortic surgery is up to 28%, and patients with spinal cord IRI might suffer from postoperative paraplegia or paraparesis. Spinal cord IRI includes two phases. The immediate spinal cord injury is related to acute ischemia. And the delayed spinal cord injury involves both ischemic cellular death and reperfusion injury. Inflammation is a subsequent event of spinal cord ischemia and possibly a major contributor to spinal cord IRI. However, the development of inflammatory mediators is incompletely demonstrated. And treatments available for inflammation in spinal cord IRI are insufficient. Improved understanding about spinal cord IRI and the development of inflammatory cells and cytokines in this process will provide novel therapeutic strategies for spinal cord IRI. Inflammatory cytokines (e.g., TNF-α and IL-1) may play an important role in spinal cord IRI. For treatment of several intractable autoimmune diseases (e.g., rheumatoid arthritis), where inflammatory cytokines are involved in disease progression, anti-inflammatory cytokine antagonist is now available. Hence, there is great potential of anti-inflammatory cytokine antagonist for therapeutic use of spinal cord IRI. We here review the mediators and several possibilities of treatment in spinal cord IRI.
Meiotic recombination creates genetic diversity and ensures segregation of homologous chromosomes. Previous population analyses yielded results averaged among individuals and impacted by evolutionary pressures. Here we sequenced 99 sperm from an Asian male using the newly developed amplification method—Multiple Annealing and Looping-Based Amplification Cycles (MALBAC)—to phase the personal genome and map at high resolution recombination events, which are non-uniformly distributed across the genome in the absence of selection pressure. The paucity of recombination near transcription start sites observed in individual sperm indicates such a phenomenon is intrinsic to the molecular mechanism of meiosis. Interestingly, a decreased crossover frequency in companion with an increase of autosomal aneuploidy is observable on a global per-sperm basis.
The molecular chaperone heat shock protein 90 (HSP90) facilitates the appropriate folding of various oncogenic proteins and is necessary for the survival of some cancer cells. HSP90 is therefore an attractive drug target, but the efficacy of HSP90 inhibitor may be limited by HSP90 inhibition induced feedback mechanisms. Through pooled RNA interference screens, we identified that heat shock factor 1(HSF1) is a sensitizer of HSP90 inhibitor. A striking combinational effect was observed when HSF1 knockdown plus with HSP90 inhibitors treatment in various cancer cell lines and tumor mouse models. Interestingly, HSF1 is highly expressed in hepatocellular carcinoma (HCC) patient samples and HCC is sensitive to combinational treatment, indicating a potential indication for the combinational treatment. To understand the mechanism of the combinational effect, we identified that a HSF1-target gene DEDD2 is involved in attenuating the effect of HSP90 inhibitors. Thus, the transcriptional activities of HSF1 induced by HSP90 inhibitors provide a feedback mechanism of limiting the HSP90 inhibitor's activity, and targeting HSF1 may provide a new avenue to enhance HSP90 inhibitors activity in human cancers.
HSF1; cancer cells; HSP90 inhibitor; Melanoma; HCC; DEDD2
Fixed vestibular appliances decrease the “self-cleansing” action of saliva and promote aggregation of dental plaque by disturbing the salivary flow field on tooth surfaces, leading to a higher prevalence of enamel demineralization and periodontal diseases. In the current study, we investigated the salivary dynamic characteristics of plaque retention and periodontal status around appliances during orthodontic treatment. By reconstructing lower central incisors and orthodontic appliances, we simulated saliva flow on the tooth surface and then characterized and quantified the salivary flow pattern surrounding the bracket and archwire. In parallel, we tested the total peri-bracket bacterial counts and periodontal status to assess interrelations. Our results demonstrate that orthodontic appliances disturb the salivary flow field on tooth surfaces and can lead to a decrease in salivary velocity and an increase in bacterial numbers. Local vortexes forming in the areas gingival to the bracket, together with the narrow space limitation, contributed to the periodontal inflammatory response. This study confirms that changes in salivary flow are an obvious predisposing factor for bacterial accumulation, and advances the ability to replicate, in
vitro, the salivary characteristics of plaque retention and periodontal status around appliances during orthodontic treatment.
AIM: To investigate whether hepatitis B virus (HBV) exacerbates hepatic cholesterol accumulation, and explore the underlying mechanisms.
METHODS: HepG2 cells were infected with adenovirus (Ad) containing 1.3-fold overlength HBV genome. Real-time polymerase chain reaction and Western blotting were used to measure mRNA and protein expression of target genes. Cholesterol accumulation was measured by fluorescence microscopy. Cell toxicity due to Ad-HBV treatment was determined by the mitochondrial tetrazolium assay. The protein levels of toll-like receptors (TLRs) were determined by Western blotting.
RESULTS: Ad-HBV increased hepatic cholesterol accumulation and enhanced the mRNA and protein levels of low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCoAr) mRNA and protein expression in HepG2 cells. In addition, these inductive effects were partly offset by suppressing TLR2 expression levels by small interfering RNA in HepG2 cells.
CONCLUSION: Ad-HBV increases LDLR and HMGCoAr expression, resulting in exacerbated cholesterol accumulation in HepG2 cells, which was mediated via the TLR2 pathway.
Hepatitis B virus; Toll-like receptors; Low-density lipoprotein receptor; 3-hydroxy-3-methylglutharyl-coenzyme A reductase
The double-stranded RNA-activated protein kinase (PKR) was originally identified as a sensor of virus infection, but its function in the brain remains unknown. Here, we report that the lack of PKR enhances learning and memory in several behavioral tasks while increasing network excitability. In addition, loss of PKR increases the late phase of long-lasting synaptic potentiation (L-LTP) in hippocampal slices. These effects are caused by an interferon-γ (IFN-γ)-mediated selective reduction in GABAergic synaptic action. Together, our results reveal that PKR finely tunes the network activity that must be maintained while storing a given episode during learning. Because PKR activity is altered in several neurological disorders, this kinase presents a promising new target for the treatment of cognitive dysfunction. As a first step in this direction, we show that a selective PKR inhibitor replicates the Pkr−/− phenotype in WT mice, enhancing long-term memory storage and L-LTP.
The development of a successful vaccine against human immunodeficiency virus type 1 (HIV-1) likely requires immunogens that elicit both broadly neutralizing antibodies against envelope spikes and T cell responses that recognize multiple viral proteins. HIV-1 virus-like particles (VLP), because they display authentic envelope spikes on the particle surface, may be developed into such immunogens. However, in one way or the other current systems for HIV-1 VLP production have many limitations. To overcome these, in the present study we developed a novel strategy to produce HIV-1 VLP using stably transfected Drosophila S2 cells. We cotransfected S2 cells with plasmids encoding HIV-1 envelope, Gag, and Rev proteins and a selection marker. After stably transfected S2 clones were established, HIV-1 VLP and their immunogenicity in mice were carefully evaluated. Here, we report that HIV-1 envelope proteins are properly cleaved, glycosylated, and incorporated into VLP with Gag. The amount of VLP released into culture supernatants is comparable to those produced by insect cells infected with recombinant baculoviruses. Moreover, cryo-electron microscopy tomography revealed average 17 spikes per purified VLP, and antigenic epitopes on the spikes were recognized by the broadly neutralizing antibodies 2G12, b12, VRC01, and 4E10 but not by PG16. Finally, mice primed with DNA and boosted with VLP in the presence of CpG exhibited anti-envelope antibody responses, including ELISA-binding, neutralizing, antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated viral inhibition, as well as envelope and Gag-specific CD8 T cell responses. Thus, we conclude that HIV-1 VLP produced by the S2 expression system has many desirable features to be developed into a vaccine component against HIV-1.
The gene AtCSR encodes peptidyl-prolyl cis/trans isomerases (PPIases) that accelerate energetically unfavorable cis/trans isomerization of the peptide bond preceding proline production. In our studies, we found that AtCSR was associated with cadmium (Cd)-sensitive response in Arabidopsis. Our results show that AtCSR expression was triggered by Cd-stress in wild type Arabidopsis. The expression of some genes responsible for Cd2+ transportation into vacuoles was induced, and the expression of the iron-regulated transporter 1 (IRT1) related to Cd2+ absorption from the environment was not induced in wild type with Cd2+ treatment. The expression of Cd-transportation related genes was not in response to Cd-stress, whereas IRT expression increased dramatically in atcsr-2 with Cd2+ treatment. The expression of glutathione 1 (GSH1) was consistent with GSH being much lower in atcsr-2 in comparison with the wild type with Cd2+ treatment. Additionally, malondialdehyde (MDA), hydrogen peroxide, and Cd2+ contents, and activities of some antioxidative enzymes, differed between the wild type and atcsr-2. Hydrogen sulfide (H2S) has been confirmed as the third gas-transmitter over recent years. The findings revealed that the expression pattern of H2S-releasing related genes and that of Cd-induced chelation and transportation genes matched well in the wild type and atcsr-2, and H2S could regulate the expression of the Cd-induced genes and alleviate Cd-triggered toxicity. Finally, one possible suggestion was given: down-regulation of atcsr-2, depending on H2S gas-transmitter not only weakened Cd2+ chelation, but also reduced Cd2+ transportation into vacuoles, as well as enhancing the Cd2+ assimilation, thus rendering atcsr-2 mutant sensitive to Cd-stress.
AtCSR; H2S gas-transmitter; Glutathione; Cadmium stress sensitivity
The single and combined effects of scavenger receptor-BI (SR-BI), ATP-binding cassette transporter (ABC) A1 and G1 on cholesterol efflux from Chinese Hamster Ovary (CHO) cells were investigated.
When apolipoproteinA-I (apoA-I) was used as an acceptor, ABCA1 overexpression led to an increase in total cholesterol (TC) in medium which is attributable to a 2-fold increase in free cholesterol (FC) content. When high-density lipoprotein 3 (HDL3) was used as an acceptor, SR-BI overexpression not only promoted FC efflux, but also promoted the uptake of cholesteryl ester (CE) into cells, resulting in no TC varieties in medium. Overexpression of ABCG1 increased both the FC and CE levels in medium. However, when apoA-I and HDL3 were both used as acceptors, coexpression of SR-BI has no effect on ABCA1-mediated increased FC and TC accumulation in medium. Interestingly, coexpression of SR-BI with ABCG1 blocked the ABCG1-mediated cholesterol efflux to HDL3, mostly by promoting the reuptake of CE from the medium. Furthermore, co-immunoprecipitation experiments revealed that SR-BI interacted with ABCG1 in BHK cells overexpressing ABCG1 and SR-BI.
We found SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to HDL3.
The broadly neutralizing monoclonal antibodies (MAbs) 4E10, 2F5, and Z13e1 target membrane-proximal external region (MPER) epitopes of HIV-1 gp41 in a manner that remains controversial. The requirements for initial lipid bilayer binding and/or CD4 ligation have been proposed. To further investigate these issues, we probed for binding of these MAbs to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions with protein A-conjugated gold (PAG) nanoparticles using negative-stain electron microscopy. We found moderate levels of PAG associated with unliganded HIV-1 and SIV virions incubated with the three MAbs. Significantly higher levels of PAG were associated with CD4-liganded HIV-1 (epitope-positive) but not SIV (epitope-negative) virions. A chimeric SIV virion displaying the HIV-1 4E10 epitope also showed significantly higher PAG association after CD4 ligation and incubation with 4E10. MAbs accumulated rapidly on CD4-liganded virions and slowly on unliganded virions, although both reached similar levels in time. Anti-MPER epitope-specific binding was stable to washout. Virions incubated with an irrelevant MAb or CD4-only (no MAb) showed negligible PAG association, as did a vesicle-rich fraction devoid of virions. Preincubation with Fab 4E10 inhibited both specific and nonspecific 4E10 IgG binding. Our data provide evidence for moderate association of anti-MPER MAbs to viral surfaces but not lipid vesicles, even in the absence of cognate epitopes. Significantly greater MAb interaction occurs in epitope-positive virions following long incubation or CD4 ligation. These findings are consistent with a two-stage binding model where these anti-MPER MAbs bind first to the viral lipid bilayer and then to the MPER epitopes following spontaneous or induced exposure.
Crohn’s disease (CD) is characterized by the activation of Th1 and Th17 cells and deficiency of regulatory T cells (Tregs), leading to intestine tissue injury and destruction. As a novel cytokine of the interleukin (IL)-1 family, the role and underlying mechanisms of IL-33 in CD remain poorly understood. Here, we assess the effects and mechanisms of IL-33 on the trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis that mimics human CD. We found that IL-33 levels were increased in the TNBS-treated mice, whereas recombinant IL-33 (rIL-33) administration substantially ameliorated TNBS-mediated colonic tissue injury and clinical symptoms of colitis. The protective effect of rIL-33 was partly associated with the markedly increased induction of Th2-type cytokines. Importantly, rIL-33 treatment resulted in prominently upregulated Foxp3 expression in the TNBS-treated mice, and depletion of Tregs significantly abrogated the impact of IL-33 on reducing the development of colitis. Notably, the level of CD103+ dendritic cells (DCs), which promotes development of Tregs, is also increased in mesenteric lymph node and lamina propria of rIL-33–treated mice. The impact of rIL-33 on CD103+ DC induction was the result of indirectly upregulating intestine epithelial cells that produce thymic stromal lymphopoietin and retinoic acid but do not directly act on DCs. In conclusion, our data provide clear evidence that IL-33 plays a protective role in TNBS-induced colitis, which is closely related to a Th1-to-Th2/Treg switch. Thus, IL-33 is a promising candidate for the development of new treatments for CD.
Glutamate transporters regulate normal synaptic network interactions and prevent neurotoxicity by rapidly clearing extracellular glutamate. GLT-1, the dominant glutamate transporter in the cerebral cortex and hippocampus, is significantly reduced in Alzheimer's disease (AD). However, the role GLT-1 loss plays in the cognitive dysfunction and pathology of AD is unknown. To determine the significance of GLT-1 dysfunction on AD-related pathological processes, mice lacking one allele for GLT-1(+/−) were crossed with transgenic mice expressing mutations of the amyloid-β protein precursor and presenilin-1 (AβPPswe/PS1ΔE9) and investigated at 6 or 9 months of age. Partial loss of GLT-1 unmasked spatial memory deficits in 6-month-old mice expressing AβPPswe/PS1ΔE9, with these mice also exhibiting an increase in the ratio of detergent-insoluble Aβ42/Aβ40. At 9 months both behavioral performance and insoluble Aβ42/Aβ40 ratios among GLT-1(+/+)/AβPPswe/PS1 E9 and GLT-1(+/−)/AβPPswe/PS1ΔE9 mice were comparable. These results suggest that deficits in glutamate transporter function compound the effects of familial AD AβPP/PS1 mutant transgenes in younger animals and thus may contribute to early occurring pathogenic processes associated with AD.
Amyloid-β; dementia; excitatory; excitotoxicity; neurotransmission
The asymmetric unit of the title compound, C17H16O4, contains two crystallographically independent molecules with different absolute configurations.
There are two molecules in the asymmetric unit of the title compound, C19H22N2O. Both molecules have an E conformation about their C=N bonds and both piperdine rings adopt chair conformations with their N atoms adopting pyramidal geometries [bond angle sums = 329.8 (4) and 330.2 (4)°]. Both molecules feature an intramolecular O—H⋯N hydrogen bond, which generates an S(6) ring. The dihedral angles between the phenyl and benzene ring planes are 45.97 (18) and 66.0 (2)°. Short O—H⋯O contacts occur in the crystal.
In the title molecule, C10H7ClO3, the butyrolactone core, a furan-2(5H)-one, forms a dihedral angle of 59.21 (5)° with the benzene ring. In the crystal, two types of hydrogen bonds (O—H⋯O and C—H⋯Cl) link molecules into infinite chains along the b axis. π–π contacts [centroid–centroid distances = 3.6359 (10) and 3.8776 (11) Å] link the chains into a three-dimensional network.
In the title compound, C16H14O4·H2O, the dihedral angle betwen the benzene rings is 71.4 (6)°. The pyran ring is in a sofa conformation. In the crystal, O—H⋯O hydrogen bonds connect the components into a two-dimensional network parallel to (010), incorporating C
2(4) and C
2(11) chains. In addition, weak C—H⋯O, C—H⋯π and π–π stacking interactions [centroid–centroid distance = 3.768 (2) Å] are present.
Mimotopes are peptides with affinities to given targets. They are readily obtained through biopanning against combinatorial peptide libraries constructed by phage display and other display technologies such as mRNA display, ribosome display, bacterial display and yeast display. Mimotopes have been used to infer the protein interaction sites and networks; they are also ideal candidates for developing new diagnostics, therapeutics and vaccines. However, such valuable peptides are not collected in the central data resources such as UniProt and NCBI GenPept due to their ‘unnatural’ short sequences. The MimoDB database is an information portal to biopanning results of random libraries. In version 2.0, it has 15 633 peptides collected from 849 papers and grouped into 1818 sets. Besides the core data on panning experiments and their results, broad background information on target, template, library and structure is included. An accompanied benchmark has also been compiled for bioinformaticians to develop and evaluate their new models, algorithms and programs. In addition, the MimoDB database provides tools for simple and advanced searches, structure visualization, BLAST and alignment view on the fly. The experimental biologists can easily use the database as a virtual control to exclude possible target-unrelated peptides. The MimoDB database is freely available at http://immunet.cn/mimodb.
In the title compound, C16H12BrN3O4, the furan-2(5H)-one ring forms a dihedral angle of 33.19 (9)° with the 4-bromobenzene unit and is nearly perpendicular to the 4-nitrobenzene segment, making a dihedral angle of 89.93 (10)°. In the crystal, N—H⋯O hydrogen bonds link the molecules, generating an infinite chain along . The chains are linked into a three-dimensional network by C—H⋯O, C—H⋯π and π–π contacts [centroid–centroid separation = 3.805 (2) Å].
The title compound, C16H12ClNO2, featuring a furan-2(5H)-one (γ-butyrolactone) core, contains two molecules (A and B) in the asymmetric unit, with different dihedral angles between the central ring and the pendant phenyl and chlorobenzene rings [43.33 (8) and 20.16 (8)°, respectively, for A, and 47.79 (8) and 13.87 (8)°, respectively, for B]. In the crystal, the A molecules are linked into  chains by single C—H⋯O interactions. The B molecules also form  chains, but their relative orientations in the chains are quite different to those of the A molecules so that adjacent B molecules are linked by two C—H⋯O hydrogen bonds. Finally, C—H⋯O interactions and aromatic π–π stacking contacts [centroid–centroid separations = 3.754 (1) and 3.817 (1) Å] link the chains into a two-dimensional array parallel to (010).
The aims of the present study are to determine the miRNA expression signature in rat hearts after ischaemic preconditioning (IP) and to identify an IP-regulated miRNA, miR-21, in IP-mediated cardiac protection, and the potential cellular and molecular mechanisms involved.
Methods and results
The miRNA expression signature was investigated in rat hearts. Among the 341 arrayed miRNAs, 40 miRNAs were differentially expressed (21 up and 19 down) in rat hearts with IP, compared with their controls. Some of these differentially expressed miRNAs were further verified by quantitative reverse transcriptase–polymerase chain reaction. Remarkably, miR-21 was one of most upregulated miRNAs in hearts after IP. In vivo, IP-mediated cardiac protection against ischaemia/reperfusion injury was inhibited by knockdown of cardiac miR-21. In cultured cardiac myocytes, we identified that miR-21 also had a protective effect on hypoxia/reoxygenation-induced cell apoptosis that was associated with its target gene, programmed cell death 4. The protective effect of miR-21 on cardiac cell apoptosis was further confirmed in rat hearts after ischaemia/reperfusion injury in vivo.
The results suggest that miRNAs are involved in IP-mediated cardiac protection. Identifying the roles of IP-regulated miRNAs in cardiac protection may provide novel therapeutic and preventive targets for ischaemic heart disease.
Ischaemic preconditioning; MicroRNA; Gene expression; Ischaemia/reperfusion injury; Ischaemic heart disease
Although “chest distress” is the most frequent complication in the aged with chronic atrial frbrillation (AF) in clinical practice, there are few data on the association between chronic AF and coronary artery disease (CAD) in the aged in terms of medical treatment and long-term outcome. We assessed coronary artery lesions in such patients and evaluated the efficacy of medical treatment in long-term follow-ups. Of 315 elderly patients (mean age: 77.39 ± 6.33 years) who had undergone coronary angiography for chest distress, 297 exhibited sinus rhythm (SR), whereas 18 patients exhibited chronic AF. Patients with AF were followed for 4.22 ± 2.21 years. Average diastolic blood pressure (DBP) of AF patients was observed to be markedly less than that of patients with SR (57.33 ± 6.87 mmHg vs 71.08 ± 10.54 mmHg, t-test: P < 0.01). Compared with SR patients, severe stenosis of the coronary artery in AF patients was reduced (73.06% vs 44.44%, Chi-square test: P < 0.01). AF patients with chest distress had high CHADS2 score (3.72 ± 1.27), but only 33.3% patients received oral anticoagulants, and such patients had a significantly lower rate of revascularization (21.43% vs 55.63%, Chi-square test: P < 0.01), and higher rate of all-cause death (22.22% vs 4.38%, Chi-square test: P < 0.01) and thromboembolism (16.67% vs 1.68%, Chi-square test: P < 0.01) in the long-term follow-ups compared with SR patients. Chest distress in the aged with AF was related to insufficient coronary blood supply that was primarily due to a reduced DBP rather than to occult CAD. Adequate and safe medical therapy was difficult to achieve in these patients. Such patients typically have a poor prognosis, and optimal therapeutic strategies to treat them are urgently needed.
aging; atrial fibrillation; coronary artery disease; antithrombotic therapy; revascularization; outcome
In the title compound, C12H10Br2O3, the dihedral angle between the furan-5(2H)-one ring and the benzene ring is 1.2 (3)°. Two intramolecular C—H⋯O interactions occur in the molecule, both of which generate S(6) rings. The bromoethyl fragment is disordered over two sets of sites in a 0.773 (8):0.227 (8) ratio. In the crystal, inversion dimers linked by pairs of C—H⋯π interactions occur.
The asymmetric unit of the title compound, C10H13NO3, contains two crystallographically independent molecules with different conformations of the ethoxycarbonyl groups; the terminal C—C—O—C torsion angles in the two molecules are 83.6 (6) and −171.1 (3)°, resulting in twisted and straight chain conformations, respectively. The crystal structure is stabilized by intermolecular N—H⋯O, O—H⋯N and C—H⋯O hydrogen bonds. Intramolecular hydrogen bonds occur between the amino N and phenolic O atoms.