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BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.
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Antituberculosos/química , Proteínas Bacterianas/química , Antituberculosos/aislamiento & purificación , Antituberculosos/farmacología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Estabilidad de Medicamentos , Endopeptidasa K/metabolismo , Calor , Humanos , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana , Mycobacterium bovis/efectos de los fármacos , Staphylococcus hominis/metabolismoRESUMEN
Introduction: Barleria prionitis is known for its medicinal properties from ancient times. Bioactive iridoid glycosides and phenolic compounds have been isolated from leaves of this plant. However, other parts of a medicinal plants are also important, especially roots. Therefore, it is important to screen all organs for complete chemical characterization. Method: All parts of B. prionitis, including leaf, root, stem and inflorescence in search of bioactive compounds, with a rapid and effective metabolomic method. X500R QTOF system with information dependent acquisition (IDA) method was used to collect high resolution accurate mass data (HRMS) on both the parent (MS signal) and their fragment ions (MS/MS signal). ESI spectra was obtained in positive ion mode from all parts of the plant. A comparative analysis of antioxidant and antibacterial activity was done and their correlation study with the identified compounds was demonstrated. Principal component analysis was performed. Result: Iridoid glycosides and phenolic compounds were identified from all parts of the showing variability in presence and abundance. Many of the compounds are reported first time in B. prionitis. Antioxidant and antibacterial activity was revealed in all organs, root being the most effective one. Some of the iridoid glycoside and phenolic compounds found to be positively correlated with the tested biological activity. Principal component analysis of the chemical profiles showed variability in distribution of the compounds. Conclusion: All parts of B. prionitis are rich source of bioactive iridoid glycosides and phenolic compounds.
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OBJECTIVE: To evaluate the capacity of the automatic detection system to accurately grade, from smartphones' selfie pictures, the severity of seven new facial signs added to the nine previously integrated. METHODS: A two-step approach was conducted: first, to check on 112 Korean women, how the AI-based automatic grading system may correlate with dermatological assessments, taken as reference; second, to confirm on 1140 women of three ancestries (African, Asian, and Caucasian) the relevance of the newly input facial signs. RESULTS: The sixteen specific Asian facial signs, detected automatically, were found significantly (P < .0001) highly correlated with the clinical evaluations made by two Korean dermatologists (wrinkles: r = .90; sagging: r = .75-.95; vascular: r = .85; pores: r = .60; pigmentation: r = .50-.80). When applied at a larger scale on women of different ethnicities, new signs were found of good accuracy and reproducibility, albeit depending on ethnicity. Due to contrast with the innate skin complexion, the facial signs dealing with skin pigmentation were found of a much higher relevance among Asian women than African or Caucasian women. The automatic gradings were even found of a slightly higher accuracy than the clinical gradings. CONCLUSION: The previously used automatic grading system is now completed by adding new facial signs apt at being detected. The continuous development is now integrating some limitations with regard to the constitutive skin complexion of the self-pictured subjects. Presenting reproducible assessments, highly correlated with medical grading, this system could change tremendously clinical researches, like in epidemiological studies, where it offers an easy, fast, affordable, and confidential approach in the objective quantification of facial signs.
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Etnicidad , Envejecimiento de la Piel , Anciano , Cara , Femenino , Humanos , Reproducibilidad de los Resultados , Población BlancaRESUMEN
S-adenosyl-L-homocysteine hydrolase of Plasmodium falciparum (PfSAHH) is a potential drug target against malaria, and selective inhibition of PfSAHH is the excellent strategy to prevent the growth of parasite inside the host. Therefore, a comparative analysis of human S-adenosyl-L-homocysteine hydrolase (HsSAHH) and PfSAHH has been performed to explore the structural differences. Structural superimposition of PfSAHH and HsSAHH has generated the RMSD of 0.749 Å over 394 alpha carbon pairs. Residues of PfSAHH from position Tyr152 to Lys193 aligned with insertion/deletion region in HsSAHH, and these extra residues results in an extent of variation in cavity region of PfSAHH. Nicotinamide adenine dinucleotide (NAD) was observed to form hydrogen bonding with Thr201, Thr202, Thr203, Asn235, Val268, Glu287, Asn322, Ile343, Asn391, Lys473, and Tyr477 and also forms hydrophobic interactions with Val268, Ile288, and Thr320 of PfSAHH. In comparison to HsSAHH, Asn322, Lys473, and Tyr477 residues of PfSAHH are unique in interaction with NAD. 2-Fluoroaristeromycin and other analogues of aristeromycin have shown the good binding affinity for both enzymes. Structural differences between PfSAHH and HsSAHH might be employed to design the potential inhibitor of PfSAHH. To find the target enzyme responsible for an anti-malarial effect, molecular docking and interaction analysis of curcumin were performed with 34 drug targets of P. falciparum. Curcumin shows high affinity for binding with HGPRT of PfHGPRT, and an anti-malarial effect of curcumin might be due to binding with PfHGPRT.
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Streptococcus pyogenes is one of the most important pathogens as it is involved in various infections affecting upper respiratory tract and skin. Due to the emergence of multidrug resistance and cross-resistance, S. Pyogenes is becoming more pathogenic and dangerous. In the present study, an in silico comparative analysis of total 65 metabolic pathways of the host (Homo sapiens) and the pathogen was performed. Initially, 486 paralogous enzymes were identified so that they can be removed from possible drug target list. The 105 enzymes of the biochemical pathways of S. pyogenes from the KEGG metabolic pathway database were compared with the proteins from the Homo sapiens by performing a BLASTP search against the non-redundant database restricted to the Homo sapiens subset. Out of these, 83 enzymes were identified as non-human homologous while 30 enzymes of inadequate amino acid length were removed for further processing. Essential enzymes were finally mined from remaining 53 enzymes. Finally, 28 essential enzymes were identified in S. pyogenes SF370 (serotype M1). In subcellular localization study, 18 enzymes were predicted with cytoplasmic localization and ten enzymes with the membrane localization. These ten enzymes with putative membrane localization should be of particular interest. Acyl-carrier-protein S-malonyltransferase, DNA polymerase III subunit beta and dihydropteroate synthase are novel drug targets and thus can be used to design potential inhibitors against S. pyogenes infection. 3D structure of dihydropteroate synthase was modeled and validated that can be used for virtual screening and interaction study of potential inhibitors with the target enzyme.
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Antibacterianos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Dihidropteroato Sintasa/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Redes y Vías Metabólicas/efectos de los fármacos , Streptococcus pyogenes/metabolismoRESUMEN
Determination of the native geometry of the enzymes and ligand complexes is a key step in the process of structure-based drug designing. Enzymes and ligands show flexibility in structural behavior as they come in contact with each other. When ligand binds with active site of the enzyme, in the presence of cofactor some structural changes are expected to occur in the active site. Motivation behind this study is to determine the nature of conformational changes as well as regions where such changes are more pronounced. To measure the structural changes due to cofactor and ligand complex, enzyme in apo, holo and ligand-bound forms is selected. Enzyme data set was retrieved from protein data bank. Fifteen triplet groups were selected for the analysis of structural changes based on selection criteria. Structural features for selected enzymes were compared at the global as well as local region. Accessible surface area for the enzymes in entire triplet set was calculated, which describes the change in accessible surface area upon binding of cofactor and ligand with the enzyme. It was observed that some structural changes take place during binding of ligand in the presence of cofactor. This study will helps in understanding the level of flexibility in protein-ligand interaction for computer-aided drug designing.
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Enzimas/química , Cristalografía por Rayos X , Bases de Datos de Proteínas , Ligandos , Unión Proteica , Conformación ProteicaRESUMEN
Determination of the native geometry of the enzymes and ligand complexe is a key step in the process of structure based drug designing. Enzymes and ligands show flexibility in structural behavior as they come in contact with each other. When ligand binds with active site of the enzyme, in presence of cofactor some structural changes are expected to occur in the active site. Motivation behind this study is to determine the nature of conformational changes as well as regions where such changes are more pronounced. To measure the structural changes due to cofactor and ligand complex, enzyme in Apo, holo and ligand bound form is selected. Enzyme data set was retrieved from protein data bank (PDB). 15 triplet groups were selected for the analysis of structural changes based on selection criteria. Structural features for selected enzymes were compared at the global as well as local region. Accessible surface area for the enzymes in entire triplet set was calculated, which describes the change in accessible surface area upon binding of cofactor and ligand with the enzyme. It was observed that some structural changes take place during binding of ligand in presence of cofactor. This study will helps in understanding the level of flexibility in protein-ligand interaction for computer aided drug designing.
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Previously, we have shown that 4-hydroxynonenal (4-HNE) induces Fas-mediated apoptosis in HLE B-3 cells through a pathway which is independent of FasL, FADD, procaspase 8, and DISC (Li, J., et al. (2006) Biochemistry 45, 12253-12264). The involvement of Daxx has also been suggested in this pathway, but its role is not clear. Here, we report that Daxx plays an important regulatory role during 4-HNE-induced, Fas-mediated apoptosis in Jurkat cells. 4-HNE induces Fas-dependent apoptosis in procaspase 8-deficient Jurkat cells via the activation of ASK1, JNK, and caspase 3, and the apoptosis can be inhibited by masking Fas with the antagonistic anti-Fas antibodies. We demonstrate that 4-HNE exposure to Jurkat cells leads to the induction of both Fas and Daxx. 4-HNE binds to both Fas and Daxx and promotes the export of Daxx from the nucleus to the cytosol, where it binds to Fas and inhibits apoptosis. Depletion of Daxx results in an increase in the activation of ASK1, JNK, and caspase 3 along with exacerbation of 4-HNE-induced apoptosis, suggesting that Daxx inhibits apoptosis by binding to Fas. 4-HNE-induced translocation of Daxx is also accompanied by the activation of the transcription factor HSF1. The results of these studies are consistent with a model in which, by interacting with Fas, 4-HNE promotes proapoptotic signaling via ASK1, JNK, and caspase 3. In parallel, 4-HNE induces Daxx and promotes its export from the nucleus to the cytosol, where it interacts with Fas to self-limit the extent of apoptosis by inhibiting the downstream proapoptotic signaling. Cytoplasmic translocation of Daxx also results in up-regulation of HSF1-associated stress-responsive genes.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aldehídos/farmacología , Apoptosis/efectos de los fármacos , Núcleo Celular/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Proteína Ligando Fas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Antracenos/farmacología , Western Blotting , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Proteínas Co-Represoras , Citosol/efectos de los fármacos , Citosol/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Humanos , Inmunoprecipitación , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Células Jurkat , MAP Quinasa Quinasa Quinasa 5/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Chaperonas Moleculares , Proteínas Nucleares/genética , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/genética , Factores de Transcripción/metabolismoRESUMEN
RLIP76 or Ral binding protein (RalBP-1) was initially cloned as a Ral-effector that was proposed as a link between Ral and Ras pathways. This protein is encoded in humans on chromosome 18p11.3 by a gene with 11 exons and 9 introns and is found ubiquitously from drosophila to humans. RLIP76 displays inhibitory GTPase activity toward Rho/Rac class G-protein cdc42 which is involved in regulation of cytoskeletal organization, lamellipodia, cell migration and apoptosis via Ras. We have recently shown that RLIP76 is also a multispecific transporter of chemotherapeutic agents and glutathione conjugates (GS-E). In human cells RLIP76 accounts for more than two third of the transport activity for GS-E and drugs as opposed to the ABC-transporters including MRP1, which account for less than one third of this activity. Evidence is mounting that RLIP76 is a stress-responsive multi-specific, non-ABC transporter which represents an entirely novel link between stress-inducible G-protein signaling, receptor tyrosine-kinase signaling, endocytosis, heat-shock and stress defense pathways, and transport mediated drug-resistance. The expression of RLIP76 is significantly greater in human cancer cells of diverse origin as compared to the non-malignant cells. Inhibition of RLIP76, using antibodies towards a cell surface epitope, or depletion of RLIP76 using either siRNA or anti-sense phosphorothioate oligonucleotides preferentially causes apoptosis in malignant cells. Administration of RLIP76 antibodies, siRNA, or anti-sense oligonucleotides to mice bearing syngeneic B16 mouse melanoma tumors causes rapid and complete regression of tumors. Studies summarized in this review strongly suggest that RLIP76 is a logical target for clinical intervention of not only multi-drug resistance but also for diseases resulting from oxidative stress.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Resistencia a Medicamentos , Proteínas Activadoras de GTPasa/metabolismo , Glutatión/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfatasas/química , Animales , Antineoplásicos/metabolismo , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Proteínas Activadoras de GTPasa/química , Proteínas de Choque Térmico/metabolismo , Humanos , Inflamación/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Conformación Proteica , Proteína Quinasa C/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Ral-binding protein 1 (RALBP1) is a stress-responsive and stress-protective multispecific transporter of glutathione conjugates (GS-E) and xenobiotic toxins. It is frequently overexpressed in malignant cells and plays a prominent antiapoptotic role selectively in cancer cells through its ability to control cellular concentration of proapoptotic oxidized lipid byproducts. In the absence of chemotherapy, depletion or inhibition of RALBP1 causes regression of syngeneic mouse B16 melanoma. Because RALBP1 transports anthracycline and Vinca alkaloid drugs, as well as GS-E, and because it confers resistance to these drugs, we proposed that depletion or inhibition of RALBP1 should cause regression of human solid tumors that overexpress RALBP1 and augment chemotherapy efficacy. Non-small-cell lung cancer (NSCLC) H358 and H520 and colon SW480 cell lines were used. Cytotoxic synergy between anti-RALBP1 immunoglobulin G (IgG), cis-diammine-dichloroplatinum (II) [CDDP], and vinorelbine was examined in cell culture and xenografts of NSCLC cells. Effects of RALBP1 depletion by antisense were examined in xenografts of NSCLC H358, NSCLC H520, and colon SW480 cells. RALBP1 depletion by phosphorothioate antisense was confirmed and was associated with rapid, complete, and sustained remissions in established s.c. human lung and colon xenografts. RALBP1 inhibition by anti-RALBP1 IgG was equally as effective as antisense and enhanced CDDP-vinorelbine in lung cancer xenografts. These studies show that RALBP1 is a transporter that serves as a key effector function in cancer cell survival and is a valid target for cancer therapy, and confirm that inhibitory modulation of RALBP1 transport activity at the cell surface is sufficient for antitumor effects.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Cisplatino/administración & dosificación , Neoplasias del Colon/genética , Neoplasias del Colon/patología , ADN sin Sentido/genética , Proteínas Activadoras de GTPasa/deficiencia , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Transfección , Vinblastina/administración & dosificación , Vinblastina/análogos & derivados , Vinorelbina , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Available evidence from a multitude of studies on the effects of 4-hydroxynonenal (HNE) on cellular processes seem to converge on some common themes: (i) concentration-dependent opposing effects of HNE on key signaling components (e.g. protein kinase C, adenylate cyclase) predict that certain constitutive levels of HNE may be needed for normal cell functions - lowering of this constitutive HNE level in cells promotes proliferative machinery while an increase in this level promotes apoptotic signaling; (ii) HNE is a common denominator in stress-induced apoptosis caused by H(2)O(2), superoxide, UV, heat or oxidant chemicals such as doxorubicin; and (iii) HNE can modulate ligand-independent signaling by membrane receptors such as EGFR or Fas (CD95) and may act as a sensor of external stimuli for eliciting stress-response. Against a backdrop of various reported effects of HNE, in vitro and in vivo, we have critically evaluated the above mentioned hypotheses suggesting a key role of HNE in signaling.
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Aldehídos/farmacología , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Receptores de Superficie Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Rayos UltravioletaRESUMEN
The Fas (apo/CD95) receptor which belongs to the TNF-alpha family is a transmembrane protein involved in the signaling for apoptosis through the extrinsic pathway. During this study, we have examined a correlation between intracellular levels of 4-HNE and expression of Fas in human lens epithelial (HLE B-3) cells. Our results show that in HLE B-3 cells, Fas is induced by 4-HNE in a concentration- and time-dependent manner, and it is accompanied by the activation of JNK, caspase 3, and the onset of apoptosis. Fas induction and activation of JNK are also observed in various tissues of mGsta4 null mice which have elevated levels of 4-HNE. Conversely, when 4-HNE is depleted in HLE B-3 cells by a transient transfection with hGSTA4, Fas expression is suppressed. However, upon the cessation of hGSTA4 expression in these transiently transfected cells, Fas and 4-HNE return to their basal levels. Fas-deficient transformed HLE B-3 cells stably transfected with hGSTA4 show remarkable resistance to apoptosis. Also, the wild-type HLE B-3 cells in which Fas is partially depleted by siRNA acquire resistance to 4-HNE-induced apoptosis, suggesting an at least partial role of Fas in 4-HNE-induced apoptosis in HLE B-3 cells. We also demonstrate that during 4-HNE-induced apoptosis of HLE B-3 cells, Daxx is induced and it binds to Fas. Together, these results show an important role of 4-HNE in regulation of the expression and functions of Fas.
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Aldehídos/metabolismo , Apoptosis/fisiología , Transducción de Señal/fisiología , Receptor fas/genética , Aldehídos/farmacología , Animales , Caspasa 3 , Caspasas/metabolismo , Transformación Celular Viral , Células Cultivadas , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cristalino , MAP Quinasa Quinasa 4/metabolismo , Ratones , Receptor fas/biosíntesisRESUMEN
Tolerance to clinically important organic nitrates such as nitroglycerin (NTG) has been experimentally related to endothelial dysfunction and vascular oxidative stress. Anti-oxidant enzymes such as the glutathione-S-transferases GSTs) could potentially play a protective role in NTG tolerance. Our previous work showed that an alpha-class glutathione-S-transferase (GSTA4-4) defends against oxidative damage in the vascular wall; therefore, we asked whether overexpression of GSTA4-4 in endothelial cells and smooth muscle cells might alter the development of tolerance to NTG. Stable transfections of mouse pancreatic islet endothelial cells (MS1) with cDNA of mGSTA4-4, and human fetal aortic vascular smooth muscle cells (FLTR) with cDNA of hGSTA4-4 were established. MTT cytotoxicity, apoptosis, nitric oxide (NO) synthases, both endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) and cyclic guanosine mono-phosphate (cGMP) were measured. Endothelial cells overexpressing mGSTA4-4, and smooth muscle cells overexpressing hGSTA4-4 were more resistant to cytotoxic injury by NTG, assessed at 24 h (p < 0.05). In both endothelial and smooth muscle cells, NTG-induced apoptosis was inhibited by GST overexpression. Following dosing in a relevant tolerance-inducing NTG protocol, we found that GSTA4-4-overexpressing cells demonstrated significant downregulation of NOS enzymes; NO release, unchanged by the tolerance protocol in both wild-type and vector-transfected cells, was augmented in GST-overexpressing cells (p < 0.01); cGMP levels in control cells fell, whereas it rose in GSTA4-4-overexpressing cells (p < 0.05). Our results demonstrate that overexpression of GST isozymes can protect endothelial cells and smooth muscle cells against oxidative stress associated with NTG, and markedly alter cellular responses to repeated doses, or tolerance. By manipulating GSTs, physiological tolerance to NTG may be diminished or eliminated.
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Glutatión Transferasa/fisiología , Nitroglicerina/farmacología , Vasodilatadores/farmacología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular , GMP Cíclico/fisiología , Tolerancia a Medicamentos , Células Endoteliales/efectos de los fármacos , Glutatión Transferasa/efectos de los fármacos , Glutatión Transferasa/genética , Ratones , Microscopía Confocal , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ácido Peroxinitroso/metabolismo , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Glutathione S-transferases (GSTs) play a key role in cellular detoxification of environmental toxicants through their conjugation to glutathione (GSH). Recent studies have shown that the alpha-class GSTs also provide protection against oxidative stress and lipid peroxidation (LPO). GSTA4-4 is a member of a sub group of the alpha-class GSTs. It has been shown to metabolize 4-hydroxynonenal (4-HNE) with high catalytic efficiency through its conjugation to glutathione (GSH) and has been suggested to be a major component of cellular defense against toxic electrophiles such as 4-HNE generated during LPO. Since the hepatotoxicity of carbon tetrachloride (CCl(4)) has been suggested to be due to the generation of free radicals leading to membrane LPO, the present studies were designed to compare hepatotoxicity of CCl(4) in GSTA4-4 null (-/-) and wild type (+/+) mice. The results show that administration of a single dose of CCl(4) (1 ml/kg i.p.) resulted in time dependent hepatotoxicity in both -/- and +/+ mice; the extent of cellular damage by serum enzymes suggests that progression was more rapid in -/- mice, although injury was similar by 24 h. Histopathologic examination showed similar degrees of centrilobular necrosis by 24 h but much greater surrounding degenerative change, including cellular swelling, disarray, and vacuolization, in the liver of -/- mice. As expected -/- mice did not show any expression of mGSTA4-4; after CCl(4) a compensatory increase in the activities of total GST activity was noted at 24 h. Major alterations in other antioxidant enzymes was not observed. 4-HNE levels in the liver of -/- mice were about four-fold higher than in +/+ mice, suggesting a positive correlation between 4-HNE levels and the altered course of CCl(4) hepatotoxicity. These studies suggest that GSTA4-4 is an important component during the early stages (1-6 h) of cellular defense against oxidative stress and LPO although, it is not effective in protecting against the ultimate degree of overall cell injury.
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Tetracloruro de Carbono/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Glutatión Transferasa/biosíntesis , Hígado/efectos de los fármacos , Aldehídos/metabolismo , Animales , Western Blotting , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Glutatión Transferasa/genética , Peroxidación de Lípido/efectos de los fármacos , Hígado/enzimología , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Bazo/enzimología , Testículo/enzimologíaRESUMEN
RLIP76 (RALBP1) is a glutathione-conjugate transporter that is a critical component of clathrin-coated pit-mediated endocytosis, as well as in stress responses. In cultured cells, it provides protection from stressors including heat, oxidant chemicals, chemotherapeutic agents, UV irradiation, and X-irradiation. Here, we show marked reduction in glutathione conjugate transport capacity and stepwise increase in radiation sensitivity associated with heterozygous or homozygous loss of the RLIP76 gene in mice. Survival after radiation in homozygous knockout animals was significantly shorter than either the heterozygous knockouts or the wild type. Delivery of recombinant RLIP76 to mice lacking RLIP76 via a liposomal delivery system rescued radiation sensitivity. Furthermore, treatment of wild-type mice with RLIP76-containing liposomes conferred resistance to radiation. These findings suggest that inhibiting RLIP76 could be used for sensitization to radiation during cancer therapy and that RLIP76 liposomes could be radioprotective agents useful for treatment of iatrogenic or catastrophic radiation poisoning.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Proteínas Activadoras de GTPasa/fisiología , Tolerancia a Radiación/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Femenino , Proteínas Activadoras de GTPasa/deficiencia , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Glutatión/metabolismo , Humanos , Inmunohistoquímica , Liposomas/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tolerancia a Radiación/genéticaRESUMEN
PURPOSE: To study the mechanism and physiological significance of the transport of the glutathione (GSH) conjugate of 4-hydroxynonenal (4-HNE) from lens epithelial cells. METHODS: HLE B-3 cells were treated with [(3)H] 4-HNE and efflux of its GSH conjugate, [(3)H] GS-HNE, into the medium was quantitated and characterized by HPLC and mass spectrometry. Inside-out vesicles (IOVs) were prepared from HLE B-3 cell membranes. The kinetics of adenosine triphosphate (ATP)-dependent uptake of GS-HNE and dinitrophenyl S-glutathione (DNP-SG) by these IOVs and inhibition of GS-HNE uptake by anti-RLIP76 IgG was studied. Localization of RLIP76 was studied by immunogold electron microscopy and kinetics of the adenosine triphosphatase (ATPase) activity of purified RLIP76 was determined. 4-HNE-induced apoptosis was compared in HLE-B3 cells coated with anti-RLIP76 IgG or preimmune IgG, by caspase activation assay. RESULTS: The results showed the presence of RLIP76 in plasma membranes of HLE B-3 cells and that it mediated ATP-dependent transport of GS-HNE as well as of DNP-SG. The transport was saturable with respect to GS-HNE (K(m) = 8.4 micro M), DNP-SG (100 micro M) as well as to ATP (K(m) 0.45 mM) and was sensitive to temperature and osmolarity of the medium. Anti-RLIP76 IgG inhibited approximately 65% of the transport of GS-HNE and DNP-SG, indicating that most of the transport was mediated by RLIP76. Compatible with its transport function, the EGTA- and ouabain-insensitive ATPase activity of purified RLIP76 was stimulated by DNP-SG and GS-HNE. 4-HNE-induced caspase activation in HLE-B3 cells was exacerbated when the transport of GS-HNE from these cells was blocked by anti-RLIP76 IgG. CONCLUSIONS: RLIP76 provides a defense against the deleterious effects of 4-HNE by transporting GS-HNE and can modulate apoptotic signaling by regulating the intracellular concentrations of 4-HNE.