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1.
Nat Commun ; 14(1): 7238, 2023 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-37945559

RESUMEN

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range of the plasma proteome. Here we address these challenges with NUcleic acid Linked Immuno-Sandwich Assay (NULISA™), which improves the sensitivity of traditional proximity ligation assays by ~10,000-fold to attomolar level, by suppressing assay background via a dual capture and release mechanism built into oligonucleotide-conjugated antibodies. Highly multiplexed quantification of both low- and high-abundance proteins spanning a wide dynamic range is achieved by attenuating signals from abundant targets with unconjugated antibodies and next-generation sequencing of barcoded reporter DNA. A 200-plex NULISA containing 124 cytokines and chemokines and other proteins demonstrates superior sensitivity to a proximity extension assay in detecting biologically important low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA makes broad and in-depth proteomic analysis easily accessible for research and diagnostic applications.


Asunto(s)
Proteoma , Proteómica , Humanos , Proteínas Sanguíneas/genética , Anticuerpos , Citocinas
2.
Vaccines (Basel) ; 11(10)2023 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-37896937

RESUMEN

The spread of COVID-19 continues, expressed by periodic wave-like increases in morbidity and mortality. The reason for the periodic increases in morbidity is the emergence and spread of novel genetic variants of SARS-CoV-2. A decrease in the efficacy of monoclonal antibodies (mAbs) has been reported, especially against Omicron subvariants. There have been reports of a decrease in the efficacy of specific antiviral drugs as a result of mutations in the genes of non-structural proteins. This indicates the urgent need for practical healthcare to constantly monitor pathogen variability and its effect on the efficacy of preventive and therapeutic drugs. As part of this study, we report the results of the continuous monitoring of COVID-19 in Moscow using genetic and virological methods. As a result of this monitoring, we determined the dominant genetic variants and identified the variants that are most widespread, not only in Moscow, but also in other countries. A collection of viruses from more than 500 SARS-CoV-2 isolates has been obtained and characterized. The genetic lines XBB.1.9.1, XBB.1.9.3, XBB.1.5, XBB.1.16, XBB.2.4, BQ.1.1.45, CH.1.1, and CL.1, representing the greatest concern, were identified among the dominant variants. We studied the in vitro efficacy of mAbs Tixagevimab + Cilgavimab (Evusheld), Sotrovimab, Regdanvimab, Casirivimab + Imdevimab (Ronapreve), and Bebtelovimab, as well as the specific antiviral drugs Remdesivir, Molnupiravir, and Nirmatrelvir, against these genetic lines. At the current stage of the COVID-19 pandemic, the use of mAbs developed against early SARS-CoV-2 variants has little prospect. Specific antiviral drugs retain their activity, but further monitoring is needed to assess the risk of their efficacy being reduced and adjust recommendations for their use.

3.
bioRxiv ; 2023 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-37090549

RESUMEN

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA™) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout. A 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultrahigh sensitivity allowed the detection of previously difficult-to-detect, but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA addresses longstanding challenges in proteomic analysis of liquid biopsies and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

4.
Int J Mol Sci ; 23(23)2022 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-36498998

RESUMEN

Findings collected over two and a half years of the COVID-19 pandemic demonstrated that the level immunity resulting from vaccination and infection is insufficient to stop the circulation of new genetic variants. The short-term decline in morbidity was followed by a steady increase. The early identification of new genetic lineages that will require vaccine adaptation in the future is an important research target. In this study, we summarised data on the variability of genetic line composition throughout the COVID-19 pandemic in Moscow, Russia, and evaluated the virological and epidemiological features of dominant variants in the context of selected vaccine prophylaxes. The prevalence of the Omicron variant highlighted the low effectiveness of the existing immune layer in preventing infection, which points to the necessity of optimising the antigens used in vaccines in Moscow. Logistic growth curves showing the rate at which the new variant displaces the previously dominant variants may serve as early indicators for selecting candidates for updated vaccines, along with estimates of efficacy, reduced viral neutralising activity against the new strains, and viral load in previously vaccinated patients.


Asunto(s)
COVID-19 , Vacunas , Humanos , SARS-CoV-2/genética , COVID-19/epidemiología , COVID-19/prevención & control , Pandemias
5.
Protein Expr Purif ; 183: 105861, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33667651

RESUMEN

Sensitive and specific serology tests are essential for epidemiological and public health studies of COVID-19 and for vaccine efficacy testing. The presence of antibodies to SARS-CoV-2 surface glycoprotein (Spike) and, specifically, its receptor-binding domain (RBD) correlates with inhibition of SARS-CoV-2 binding to the cellular receptor and viral entry into the cells. Serology tests that detect antibodies targeting RBD have high potential to predict COVID-19 immunity and to accurately determine the extent of the vaccine-induced immune response. Cost-effective methods of expression and purification of Spike and its fragments that preserve antigenic properties are essential for development of such tests. Here we describe a method of production of His6-tagged S319-640 fragment containing RBD in E. coli. It includes expression of the fragment, solubilization of inclusion bodies, and on-the-column refolding. The antigenic properties of the resulting product are similar, but not identical to the RBD-containing fragment expressed in human cells.


Asunto(s)
COVID-19/virología , SARS-CoV-2/química , Glicoproteína de la Espiga del Coronavirus/química , Sitios de Unión , Clonación Molecular , Escherichia coli/química , Escherichia coli/genética , Expresión Génica , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Dominios Proteicos , Replegamiento Proteico , SARS-CoV-2/genética , Solubilidad , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificación
6.
Diagnostics (Basel) ; 11(1)2021 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-33440690

RESUMEN

Determining the presence of antibodies in serum is important for epidemiological studies, to be able to confirm whether a person has been infected, predicting risks of them getting sick and spreading the disease. During the ongoing pandemic of COVID-19, a positive serological test result can suggest if it is safe to return to work and re-engage in social activities. Despite a multitude of emerging tests, the quality of respective data often remains ambiguous, yielding a significant fraction of false positive results. The human organism produces polyclonal antibodies specific to multiple viral proteins, so testing simultaneously for multiple antibodies appeared a practical approach for increasing test specificity. We analyzed immune response and testing potential for a spectrum of antigens derived from the spike and nucleocapsid proteins of SARS-CoV-2, developed a dual-antigen testing system in the ELISA format and designed a robust algorithm for data processing. Combining nucleocapsid protein and receptor-binding domain for analysis allowed us to completely eliminate false positive results in the tested cohort (achieving specificity within a 95% confidence interval of 97.2-100%). We also tested samples collected from different households, and demonstrated differences in the immune response of COVID-19 patients and their family members; identifying, in particular, asymptomatic cases showing strong presence of studied antibodies, and cases showing none despite confirmed close contacts with the infected individuals.

7.
Oncotarget ; 7(48): 78281-78296, 2016 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-27835876

RESUMEN

Aggregation of proteins with the expansion of polyglutamine tracts in the brain underlies progressive genetic neurodegenerative diseases (NDs) like Huntington's disease and spinocerebellar ataxias (SCA). An insensitive cellular proteotoxic stress response to non-native protein oligomers is common in such conditions. Indeed, upregulation of heat shock factor 1 (HSF1) function and its target protein chaperone expression has shown promising results in animal models of NDs. Using an HSF1 sensitive cell based reporter screening, we have isolated azadiradione (AZD) from the methanolic extract of seeds of Azadirachta indica, a plant known for its multifarious medicinal properties. We show that AZD ameliorates toxicity due to protein aggregation in cell and fly models of polyglutamine expansion diseases to a great extent. All these effects are correlated with activation of HSF1 function and expression of its target protein chaperone genes. Notably, HSF1 activation by AZD is independent of cellular HSP90 or proteasome function. Furthermore, we show that AZD directly interacts with purified human HSF1 with high specificity, and facilitates binding of HSF1 to its recognition sequence with higher affinity. These unique findings qualify AZD as an ideal lead molecule for consideration for drug development against NDs that affect millions worldwide.


Asunto(s)
ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Limoninas/farmacología , Enfermedades Neurodegenerativas/prevención & control , Fármacos Neuroprotectores/farmacología , Péptidos/metabolismo , Extractos Vegetales/farmacología , Agregación Patológica de Proteínas , Animales , Azadirachta/química , ADN/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células HCT116 , Células HEK293 , Factores de Transcripción del Choque Térmico/genética , Humanos , Limoninas/aislamiento & purificación , Limoninas/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Fármacos Neuroprotectores/aislamiento & purificación , Fármacos Neuroprotectores/metabolismo , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Unión Proteica , Semillas , Factores de Tiempo , Transfección
8.
Proc Natl Acad Sci U S A ; 111(4): E474-83, 2014 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-24474797

RESUMEN

Chronic inflammation is associated with increased cancer risk. Furthermore, the transcription factor NF-κB, a central regulator of inflammatory responses, is constitutively active in most tumors. To determine whether active NF-κB inherently contributes to malignant transformation, we isolated a set of NF-κB-activating genetic elements and tested their oncogenic potential in rodent cell transformation models. Genetic elements with desired properties were isolated using biologically active selectable peptide technology, which involves functional screening of lentiviral libraries encoding 20 or 50 amino acid-long polypeptides supplemented with endoplasmic reticulum-targeting and oligomerization domains. Twelve NF-κB-activating selectable peptides (NASPs) representing specific fragments of six proteins, none of which was previously associated with NF-κB activation, were isolated from libraries of 200,000 peptides derived from 500 human extracellular proteins. Using selective knockdown of distinct components of the NF-κB pathway, we showed that the isolated NASPs act either via or upstream of TNF receptor-associated factor 6. Transduction of NASPs into mouse and rat embryo fibroblasts did not, in itself, alter their growth. However, when coexpressed with oncogenic Ras (H-Ras(V12)), NASPs allowed rodent fibroblasts to overcome H-Ras(V12)-mediated p53-dependent senescence and acquire a transformed tumorigenic phenotype. Consistent with their ability to cooperate with oncogenic Ras in cell transformation, NASP expression reduced the transactivation activity of p53. This system provides an in vitro model of NF-κB-driven carcinogenesis and suggests that the known carcinogenic effects of inflammation may be at least partially due to NF-κB-mediated abrogation of oncogene-induced senescence.


Asunto(s)
Carcinogénesis , Genes ras , Inflamación/metabolismo , FN-kappa B/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Inflamación/genética , Ratones , Datos de Secuencia Molecular , Péptidos/química , Unión Proteica , Ratas
9.
Oncotarget ; 2(3): 209-21, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21444945

RESUMEN

Proteotoxic stress (PS) is generated in cells under a variety of conditions involving accumulation of misfolded proteins. To avoid the toxicity of unmitigated PS, cells activate the heat shock response (HSR). HSR involves upregulation of factors such as ubiquitin and the non-housekeeping chaperone Hsp70 which assist with metabolism of aberrant proteins. The PS-HSR axis is a potential anticancer treatment target since many tumor cells display constitutive PS and dependence on HSR due to their rapid rates of proliferation and translation. In fact, induction of PS via stimulation of protein misfolding (hyperthermia), inhibition of proteasomes (bortezomib) or inhibition of Hsp90 (geldanamycin) have all been considered or used for cancer treatment. We found that combination of bortezomib with an inducer of protein misfolding (hyperthermia or puromycin) resulted in enhanced PS. HSR was also induced, but could not mitigate the elevated PS and the cells died via largely p53-independent apoptosis. Thus, combination treatments were more cytotoxic in vitro than the component single treatments. Consistent with this, combination of non-toxic doses of puromycin with bortezomib significantly increased the antitumor activity of bortezomib in a mouse model of multiple myeloma. These results provide support for using combination treatments that disrupt the balance of PS and HSR to increase the therapeutic index of anticancer therapies.


Asunto(s)
Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/terapia , Inhibidores de Proteasoma , Deficiencias en la Proteostasis/metabolismo , Pirazinas/farmacología , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ácidos Borónicos/administración & dosificación , Bortezomib , Línea Celular Tumoral , Terapia Combinada , Sinergismo Farmacológico , Células HCT116 , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP90 de Choque Térmico/biosíntesis , Células HeLa , Respuesta al Choque Térmico/efectos de los fármacos , Humanos , Hipertermia Inducida , Ratones , Ratones Endogámicos BALB C , Mieloma Múltiple/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/metabolismo , Deficiencias en la Proteostasis/inducido químicamente , Puromicina/administración & dosificación , Puromicina/farmacología , Pirazinas/administración & dosificación
10.
Biometals ; 24(2): 279-89, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21229381

RESUMEN

Extracellular iron has been implicated in the pathogenesis of post-injury organ failure. However, the source(s) and biochemical species of this iron have not been identified. Based upon evidence that distant organ injury results from an increase in intestinal permeability, we looked for ferrous iron in mesenteric lymph in anesthetized rats undergoing hemorrhage and fluid resuscitation (H/R). Ferrous iron increased in lymph from 4.7 nmol/mg of protein prior to hemorrhage to 86.6 nmol/mg during resuscitation. Utilizing immuno-spin trapping in protein fractions that were rich in iron, we tentatively indentified protein carrier(s) of ferrous iron by MALDI-TOF MS. One of the identified proteins was the metalloproteinase (MMP) inhibitor, TIMP-2. Antibody to TIMP-2 immunoprecipitated 74% of the ferrozine detectable iron in its protein fraction. TIMP-2 binds iron in vitro at pH 6.3, which is typical of conditions in the mesentery during hemorrhage, but it retains the ability to inhibit the metalloproteases MMP-2 and MMP-9. In summary, there is a large increase in extracellular ferrous iron in the gut in H/R demonstrating dysregulation of iron homeostasis. We have identified, for the first time, the binding of extracellular iron to TIMP-2.


Asunto(s)
Hemorragia/metabolismo , Hierro/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Animales , Cromatografía en Gel , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Inmunoprecipitación , Masculino , Inhibidores de la Metaloproteinasa de la Matriz , Ratas , Ratas Sprague-Dawley , Resucitación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Inhibidor Tisular de Metaloproteinasa-2/farmacología
11.
Nutr J ; 9: 61, 2010 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-21092298

RESUMEN

Multivitamin/multimineral complexes are the most common dietary supplements. Unlike minerals in foods that are incorporated in bioorganic structures, minerals in dietary supplements are typically in an inorganic form. These minerals can catalyze the generation of free radicals, thereby oxidizing antioxidants during digestion. Here we examine the ability of a matrix consisting of an amino acid and non-digestible oligosaccharide (AAOS) to blunt metal-catalyzed oxidations. Monitoring of ascorbate radical generated by copper shows that ascorbate is oxidized more slowly with the AAOS matrix than with copper sulfate. Measurement of the rate of oxidation of ascorbic acid and Trolox® by catalytic metals confirmed the ability of AAOS to slow these oxidations. Similar results were observed with iron-catalyzed formation of hydroxyl radicals. When compared to traditional forms of minerals used in supplements, we conclude that the oxidative loss of antioxidants in solution at physiological pH is much slower when AAOS is present.


Asunto(s)
Suplementos Dietéticos/efectos adversos , Depuradores de Radicales Libres/química , Minerales/efectos adversos , Minerales/química , Estrés Oxidativo , Excipientes Farmacéuticos/química , Vitaminas , Aminoácidos/química , Antioxidantes/química , Ácido Ascórbico/química , Catálisis , Quelantes/química , Cromanos/química , Cobre/efectos adversos , Cobre/química , Radicales Libres/efectos adversos , Radicales Libres/química , Hierro/efectos adversos , Hierro/química , Cinética , Oligosacáridos/química , Oxidación-Reducción
12.
Magnes Res ; 23(4): S199-206, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20971697

RESUMEN

Hypomagnesemia continues to cause difficult clinical problems, such as significant cardiac arrhythmias where intravenous magnesium therapy can be lifesaving. Nutritional deficiency of magnesium may present with some subtle symptoms such as leg cramps and occasional palpitation. We have investigated dietary-induced magnesium deficiency in rodent models to assess the pathobiology associated with prolonged hypomagnesemia. We found that neuronal sources of the neuropeptide, substance P (SP), contributed to very early prooxidant/proinflammatory changes during Mg deficiency. This neurogenic inflammation is systemic in nature, affecting blood cells, cardiovascular, intestinal, and other tissues, leading to impaired cardiac contractility similar to that seen in patients with heart failure. We have used drugs that block the release of SP from neurons and SP-receptor blockers to prevent some of these pathobiological changes; whereas, blocking SP catabolism enhances inflammation. Our findings emphasize the essential role of this cation in preventing cardiomyopathic changes and intestinal inflammation in a well-studied animal model, and also implicate the need for more appreciation of the potential clinical relevance of optimal magnesium nutrition and therapy.


Asunto(s)
Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Deficiencia de Magnesio/metabolismo , Deficiencia de Magnesio/fisiopatología , Animales , Humanos , Deficiencia de Magnesio/inmunología , Receptores de Neuroquinina-1/metabolismo , Sustancia P/metabolismo
13.
Cell Cycle ; 8(23): 3960-70, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19901558

RESUMEN

The number of physical conditions and chemical agents induce accumulation of misfolded proteins creating proteotoxic stress. This leads to activation of adaptive pro-survival pathway, known as heat shock response (HSR), resulting in expression of additional chaperones. Several cancer treatment approaches, such as proteasome inhibitor Bortezomib and hsp90 inhibitor geldanamycin, involve activation of proteotoxic stress. Low efficacy of these therapies is likely due to the protective effects of HSR induced in treated cells, making this pathway an attractive target for pharmacological suppression. We found that the anti-malaria drugs quinacrine (QC) and emetine prevented HSR in cancer cells, as judged by induction of hsp70 expression. As opposed to emetine, which inhibited general translation, QC did not affect protein synthesis, but rather suppressed inducible HSF1-dependent transcription of the hsp70 gene in a relatively selective manner. The treatment of tumor cells in vitro with a combination of non-toxic concentrations of QC and proteotoxic stress inducers resulted in rapid induction of apoptosis. The effect was similar if QC was substituted by siRNA against hsp70, suggesting that the HSR inhibitory activity of QC was responsible for cell sensitization to proteotoxic stress inducers. QC was also found to enhance the antitumor efficacy of proteotoxic stress inducers in vivo: combinatorial treatment with 17-DMAG + QC resulted in suppression of tumor growth in two mouse syngeneic models. These results reveal that QC is an inhibitor of HSF1-mediated HSR. As such, this compound has significant clinical potential as an adjuvant in therapeutic strategies aimed at exploiting the cytotoxic potential of proteotoxic stress.


Asunto(s)
Antimaláricos/farmacología , Antineoplásicos/farmacología , Respuesta al Choque Térmico/efectos de los fármacos , Quinacrina/farmacología , Apoptosis , Benzoquinonas/farmacología , Ácidos Borónicos/farmacología , Bortezomib , Proteínas de Unión al ADN/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Factores de Transcripción del Choque Térmico , Humanos , Lactamas Macrocíclicas/farmacología , Neoplasias/tratamiento farmacológico , Pirazinas/farmacología , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/metabolismo
14.
Am J Med Sci ; 338(1): 22-7, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19593099

RESUMEN

Hypomagnesemia continues to be a significant clinical disorder that is present in patients with diabetes mellitus, alcoholism, and treatment with magnesuric drugs (diuretics, cancer chemotherapy agents, etc.). To determine the role of magnesium in cardiovascular pathophysiology, we have used dietary restriction of this cation in animal models. This review highlights some key observations that helped formulate the hypothesis that release of substance P (SP) during experimental dietary Mg deficiency (MgD) may initiate a cascade of deleterious inflammatory, oxidative, and nitrosative events, which ultimately promote cardiomyopathy, in situ cardiac dysfunction, and myocardial intolerance to secondary stresses. SP acts primarily through neurokinin-1 receptors of inflammatory and endothelial cells, and may induce production of reactive oxygen and nitrogen species (superoxide anion, NO*, peroxynitrite, hydroxyl radical), leading to enhanced consumption of tissue antioxidants; stimulate release of inflammatory mediators; promote tissue adhesion molecule expression; and enhance inflammatory cell tissue infiltration and cardiovascular lesion formation. These SP-mediated events may predispose the heart to injury if faced with subsequent oxidative stressors (ischemia/reperfusion, certain drugs) or facilitate development of in situ cardiac dysfunction, especially with prolonged dietary Mg restriction. Significant protection against most of these MgD-mediated events has been observed with interventions that modulate neuronal SP release or its bioactivity, and with several antioxidants (vitamin E, probucol, epicaptopril, d-propranolol). In view of the clinical prevalence of hypomagnesemia, new treatments, beyond magnesium repletion, may be needed to diminish deleterious neurogenic and prooxidative components described in this article.


Asunto(s)
Cardiomiopatías , Deficiencia de Magnesio/complicaciones , Inflamación Neurogénica , Animales , Cardiomiopatías/etiología , Cardiomiopatías/fisiopatología , Dieta , Endotoxemia/metabolismo , Humanos , Daño por Reperfusión Miocárdica/metabolismo , Inflamación Neurogénica/etiología , Inflamación Neurogénica/fisiopatología , Neuropéptidos/metabolismo , Estrés Oxidativo , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Neuroquinina-1/metabolismo
15.
Proc Natl Acad Sci U S A ; 105(38): 14453-8, 2008 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-18796602

RESUMEN

Functional selection of genetic suppressor elements (GSEs), engineered gene fragments that interfere with the function of a particular gene product, was used to identify regulators of FAS-induced apoptosis. Chicken DF-1 cells expressing human FAS receptor and susceptible to FAS-induced apoptosis were infected with a GSE library consisting of randomly fragmented normalized chicken cDNAs in a replication-competent avian retroviral vector. Virus-producing cells were subjected to several rounds of selection using FAS agonistic antibodies, resulting in isolation of a set of GSEs conferring resistance to FAS-induced apoptosis. Surprisingly, one of the isolated GSEs encoded a 42 amino acid-long polypeptide derived from the C-terminal half of cytochrome b (Cyt b) encoded by the mitochondrial genome. Subsequent experiments showed that caspase 8-dependent cleavage of mitochondrial Cyt b and translocation of its C-terminal half into the cytoplasm occurred during FAS-induced apoptosis in both chicken and human cells. Ectopic cytoplasmic expression of either full-length Cyt b or its C-terminal half in several human cell lines induced apoptosis, which could be suppressed by the isolated GSE, but not by Bcl2 over-expression or Apaf-1 or cytochrome c knock-down. These results reveal a cytochrome c-independent branch of FAS-induced apoptosis involving cleavage and cytoplasmic release of mitochondrial Cyt b.


Asunto(s)
Apoptosis , Citocromos b/metabolismo , Mitocondrias/metabolismo , Receptor fas/metabolismo , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasa 8/metabolismo , Línea Celular , Pollos , Citocromos c/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Supresión Genética , Receptor fas/genética
16.
Heart Fail Rev ; 11(1): 35-44, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16819576

RESUMEN

Magnesium is a micronutrient essential for the normal functioning of the cardiovascular system, and Mg deficiency (MgD) is frequently associated in the clinical setting with chronic pathologies such as CHF, diabetes, hypertension, and other pathologies. Animal models of MgD have demonstrated a systemic pro-inflammatory/pro-oxidant state, involving multiple tissues/organs including neuronal, hematopoietic, cardiovascular, and gastrointestinal systems; during later stages of MgD, a cardiomyopathy develops which may result from a cascade of inflammatory events. In rodent models of dietary MgD, a significant rise in circulating levels of proinflammatory neuropeptides such as substance P (SP) and calcitonin gene-related peptide among others, was observed within days (1-7) of initiating the Mg-restricted diet, and implicated a neurogenic trigger for the subsequent inflammatory events; this early "neurogenic inflammation" phase may be mediated in part, by the Mg-gated N: -methyl-D-aspartate (NMDA) receptor/channel complex. Deregulation of the NMDA receptor may trigger the abrupt release of neuronal SP from the sensory-motor C-fibers to promote the subsequent pro-inflammatory changes: elevations in circulating inflammatory cells, inflammatory cytokines, histamine, and PGE(2) levels, as well as formation of nitric oxide, reactive oxygen species, lipid peroxidation products, and depletion of key endogenous antioxidants. Concurrent elevations of tissue CD14, a high affinity receptor for lipopolyssacharide, suggest that intestinal permeability may be compromised leading to endotoxemia. If exposure to these early (1-3 weeks MgD) inflammatory/pro-oxidant events becomes prolonged, this might lead to impaired cardiac function, and when co-existing with other pathologies, may enhance the risk of developing chronic heart failure.


Asunto(s)
Insuficiencia Cardíaca/fisiopatología , Deficiencia de Magnesio/fisiopatología , Animales , Antioxidantes/farmacología , Insuficiencia Cardíaca/etiología , Humanos , Inflamación/metabolismo , Deficiencia de Magnesio/complicaciones , Deficiencia de Magnesio/metabolismo , Miocardio/metabolismo , Antagonistas del Receptor de Neuroquinina-1 , Neuronas/fisiología , Óxido Nítrico/fisiología , Estrés Oxidativo/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Sustancia P/fisiología
17.
Mol Cell Biochem ; 288(1-2): 213-7, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16718379

RESUMEN

In this study we have tested the effects of d-propranolol (D-Pro) on the iron uptake, iron release and oxidative response of iron-loaded cells in a cellular model of iron-overload using isolated rat peritoneal macrophages incubated with iron-dextran (Fe-D). Pretreatment of macrophages with D-Pro (5-200 microM) prior to Fe-D exposure decreased the cellular iron content and partially prevented iron release from latex-activated macrophages. Release of reactive oxygen species from activated cells was detected by dichlorodihydrofluorescein (DCDHF, 5 microM) oxidation. We found that loading cells with Fe-D increased their response to latex, which was prevented by the lysosomotropic antioxidant agent D-Pro (10 microM).


Asunto(s)
Hierro/metabolismo , Macrófagos Peritoneales/metabolismo , Propranolol/farmacología , Animales , Relación Dosis-Respuesta a Droga , Complejo Hierro-Dextran/metabolismo , Activación de Macrófagos , Macrófagos Peritoneales/efectos de los fármacos , Masculino , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo
18.
Biochem Pharmacol ; 70(12): 1814-22, 2005 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-16242673

RESUMEN

The metabolic reduction of hexavalent chromium [Cr(VI)] in the presence of DNA generates several lesions which impede DNA replication and gene transcription. However, the relative contribution of molecular oxygen to Cr-induced genetic damage is unclear. To elucidate the role of dioxygen in Cr genotoxicity, we studied the formation of Cr-induced lesions in DNA treated with either Cr(VI) and the physiological reductant, ascorbic acid (Asc), or Cr(III), under ambient and hypoxic (<1% oxygen) conditions. We found that hypoxia did not impede the reduction of Cr(VI) by Asc throughout a 2 h treatment. In contrast, Cr-DNA binding under these conditions was reduced up to 70% by hypoxia, and a 50-90% decrease in the frequency of Cr-induced Taq polymerase-arresting DNA adducts was also observed. In the presence of Cr(VI)/Asc, formation of Cr-DNA interstrand crosslinks (ICLs) under hypoxia was 50% or less of that under ambient conditions. Kinetic studies found that hypoxia reduced the rate at which Cr interacted with DNA, but not the ultimate steady state level of Cr-DNA binding. The inhibitory effect of hypoxia on Cr(VI)/Asc genotoxicity could not be explained solely by alterations in the reactivity of intermediate Cr(V) species because Cr(III)-DNA binding and Cr(III)-induced ICL formation were also impaired by hypoxia. Moreover, Cr(V) was generated to similar levels in ambient and hypoxic reactions. Hypoxia did not affect ICL formation by the inorganic chemotherapeutic agent cisplatin, suggesting that these effects were specific for Cr(III). Taken together, these results support a role for dioxygen in facilitating the formation of Cr-DNA coordination complexes.


Asunto(s)
Cromo/metabolismo , Aductos de ADN/metabolismo , ADN/metabolismo , Oxígeno/fisiología , Sistema Libre de Células , Células Cultivadas , Cromo/toxicidad , Cisplatino/metabolismo , Humanos , Plásmidos
19.
Biochim Biophys Acta ; 1688(3): 257-64, 2004 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-15062877

RESUMEN

Azidothymidine (AZT) and AZT monophosphate (AZT-MP) in concentrations as low as 10 and 50 microM, respectively, promote oxidation of chemically deacetylated 2',7'-dichlorodihydrofluorescein (DCDHF) to 2',7'-dichlorofluorescein (DCF) by rat peritoneal macrophages activated with latex. Cells were incubated with AZT and AZT-MP for 18 h, washed out from residual AZT or AZT-MP and activated with latex for 30 or 60 min in the presence of DCDHF. Latex-activated cells oxidize DCDHF extracellularly due to release of hydrogen peroxide and low-molecular iron complexes, which is verified using catalase, desferal and the peroxidase inhibitor sodium azide. AZT and AZT-MP increase DCDHF oxidation due to additional release of hydrogen peroxide as demonstrated by catalase inhibition of DCDHF oxidation and direct H(2)O(2) measurement. Thymidine and thymidine phosphates did not show any effect on macrophage activation. In separate experiments we evaluated the in vitro prooxidant activity of AZT, AZT-MP, AZT triphosphate (AZT-TP), AZT glucuronide (GAZT) and 3'-amino-3'-deoxythymidine (AMT) in a cell-free system using the hydrogen peroxide-iron-mediated oxidation of DCDHF. Under these conditions, AZT and AZT phosphates exhibit a prooxidant effect in concentrations as low as 100 microM. Furthermore, GAZT is a less effective prooxidant and AMT acts like an antioxidant. Thymidine did not show any effect.


Asunto(s)
Radicales Libres/metabolismo , Peróxido de Hidrógeno/farmacología , Hierro/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/fisiología , Estallido Respiratorio/fisiología , Zidovudina/análogos & derivados , Zidovudina/farmacología , Animales , Fármacos Anti-VIH/farmacología , Sistema Libre de Células , Didesoxinucleótidos , Peróxido de Hidrógeno/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Masculino , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Estallido Respiratorio/efectos de los fármacos , Nucleótidos de Timina/farmacología
20.
Mol Cell Biochem ; 245(1-2): 141-8, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12708753

RESUMEN

Cardioprotection by Mg Sulfate (MgSO4) during ischemia/reperfusion (I/R) is attributed largely to the Mg2+ cation. However, Mg-gluconate (MgGl2) may provide added benefit, possibly through its anion's antioxidant properties. Protective effects of both Mg-salts and their anions during 30 min global I and 50 min R were assessed in Langendorff-perfused (Krebs-Henseleit buffer) rat hearts. Recovery of function was compared between untreated hearts and those receiving supplement (2.4 mM MgGl2, MgSO4, or Na2SO4, or 4.8 mM NaGI) for 5 min prior to I and during the initial 30 min R. The final 20 min R was conducted without supplement. End diastolic pressure (EDP, mmHg) of the 50 min reperfused MgGl2 group (2.6) was lower than MgSO4 (16.2) and untreated (35.6) groups, and the NaGI group (25.2) was considerably lower than Na2SO4 (38.8). Recovery of developed pressure (% preischemic DP) at the onset of R for MgGl2 (74.9) was greater than MgSO4 (37.9) and untreated (33.2). After 50 min, MgGl2 (77.9) and MgSO4 (66.9) provided protection compared to untreated (51.8). In separate studies, ESR spin trapping with alpha-phenyl-N-tert-butylnitrone (3 mM PBN) showed that I/R alkoxyl radical production was reduced with MgGl2 (0.0 vs. 2.4 vs. 3.6 mM: 184 vs. 97 vs. 54.8 nM/g tissue x min) to a greater extent than seen with MgSO4 (3.6 mM: 108). Additional studies suggest that Gl(1-), unlike SO4(2-), may scavenge hydroxyl radicals, accounting for the added protection. MgGl2 treated hearts exhibited less postischemic dysfunction and oxidative injury compared to MgSO4, suggesting the contribution of Gl(1-) to cardioprotection.


Asunto(s)
Ácido Glucurónico/metabolismo , Magnesio/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Estrés Oxidativo , Sulfatos/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Gasto Cardíaco/efectos de los fármacos , Radicales Libres/metabolismo , Corazón/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo
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