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Biogenic polyamines are ubiquitous compounds. Dysregulation of their metabolism is associated with the development of various pathologies, including cancer, hyperproliferative diseases, and infections. The canonical pathway of polyamine catabolism includes acetylation of spermine and spermidine and subsequent acetylpolyamine oxidase (PAOX)-mediated oxidation of acetylpolyamines (back-conversion) or their direct efflux from the cell. PAOX is considered to catalyze a non-rate-limiting catabolic step. Here, we show that PAOX transcription levels are extremely low in various tumor- and non-tumor cell lines and, in most cases, do not change in response to altered polyamine metabolism. Its enzymatic activity is undetectable in the majority of cell lines except for neuroblastoma and low passage glioblastoma cell lines. Treatment of A549 cells with N1,N11-diethylnorspermine leads to PAOX induction, but its contribution to polyamine catabolism remains moderate. We also describe two alternative enzyme isoforms and show that isoform 4 has diminished oxidase activity and isoform 2 is inactive. PAOX overexpression correlates with the resistance of cancer cells to genotoxic antitumor drugs, indicating that PAOX may be a useful therapeutic target. Finally, PAOX is dispensable for the replication of various viruses. These data suggest that a decrease in polyamine levels is achieved predominantly by the secretion of acetylated spermine and spermidine rather than by back-conversion.
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Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , Poliaminas , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Poliaminas/metabolismo , Línea Celular Tumoral , Espermina/metabolismo , Espermina/análogos & derivados , Acetilación , Células A549RESUMEN
Hepatitis C virus (HCV) is an oncogenic virus that causes chronic liver disease in more than 80% of patients. During the last decade, efficient direct-acting antivirals were introduced into clinical practice. However, clearance of the virus does not reduce the risk of end-stage liver diseases to the level observed in patients who have never been infected. So, investigation of HCV pathogenesis is still warranted. Virus-induced changes in cell metabolism contribute to the development of HCV-associated liver pathologies. Here, we studied the impact of the virus on the metabolism of polyamines and proline as well as on the urea cycle, which plays a crucial role in liver function. It was found that HCV strongly suppresses the expression of arginase, a key enzyme of the urea cycle, leading to the accumulation of arginine, and up-regulates proline oxidase with a concomitant decrease in proline concentrations. The addition of exogenous proline moderately suppressed viral replication. HCV up-regulated transcription but suppressed protein levels of polyamine-metabolizing enzymes. This resulted in a decrease in polyamine content in infected cells. Finally, compounds targeting polyamine metabolism demonstrated pronounced antiviral activity, pointing to spermine and spermidine as compounds affecting HCV replication. These data expand our understanding of HCV's imprint on cell metabolism.
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Hepacivirus , Poliaminas , Prolina , Urea , Replicación Viral , Prolina/metabolismo , Humanos , Hepacivirus/fisiología , Hepacivirus/efectos de los fármacos , Poliaminas/metabolismo , Urea/metabolismo , Urea/farmacología , Replicación Viral/efectos de los fármacos , Arginasa/metabolismo , Antivirales/farmacología , Antivirales/metabolismo , Hepatitis C/metabolismo , Hepatitis C/virología , Línea Celular Tumoral , Prolina Oxidasa/metabolismoRESUMEN
Reactive oxygen species (ROS) play a major role in the regulation of various processes in the cell. The increase in their production is a factor contributing to the development of numerous pathologies, including inflammation, fibrosis, and cancer. Accordingly, the study of ROS production and neutralization, as well as redox-dependent processes and the post-translational modifications of proteins, is warranted. Here, we present a transcriptomic analysis of the gene expression of various redox systems and related metabolic processes, such as polyamine and proline metabolism and the urea cycle in Huh7.5 hepatoma cells and the HepaRG liver progenitor cell line, that are widely used in hepatitis research. In addition, changes in response to the activation of polyamine catabolism that contribute to oxidative stress were studied. In particular, differences in the gene expression of various ROS-producing and ROS-neutralizing proteins, the enzymes of polyamine metabolisms and proline and urea cycles, as well as calcium ion transporters between cell lines, are shown. The data obtained are important for understanding the redox biology of viral hepatitis and elucidating the influence of the laboratory models used.
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Carcinoma Hepatocelular , Hepatocitos , Neoplasias Hepáticas , Poliaminas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Redes y Vías Metabólicas , Oxidación-Reducción , Poliaminas/metabolismo , Prolina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , UreaRESUMEN
Hepatitis delta virus (HDV) is a viroid-like satellite that may co-infect individuals together with hepatitis B virus (HBV), as well as cause superinfection by infecting patients with chronic hepatitis B (CHB). Being a defective virus, HDV requires HBV structural proteins for virion production. Although the virus encodes just two forms of its single antigen, it enhances the progression of liver disease to cirrhosis in CHB patients and increases the incidence of hepatocellular carcinoma. HDV pathogenesis so far has been attributed to virus-induced humoral and cellular immune responses, while other factors have been neglected. Here, we evaluated the impact of the virus on the redox status of hepatocytes, as oxidative stress is believed to contribute to the pathogenesis of various viruses, including HBV and hepatitis C virus (HCV). We show that the overexpression of large HDV antigen (L-HDAg) or autonomous replication of the viral genome in cells leads to increased production of reactive oxygen species (ROS). It also leads to the upregulated expression of NADPH oxidases 1 and 4, cytochrome P450 2E1, and ER oxidoreductin 1α, which have previously been shown to mediate oxidative stress induced by HCV. Both HDV antigens also activated the Nrf2/ARE pathway, which controls the expression of a spectrum of antioxidant enzymes. Finally, HDV and its large antigen also induced endoplasmic reticulum (ER) stress and the concomitant unfolded protein response (UPR). In conclusion, HDV may enhance oxidative and ER stress induced by HBV, thus aggravating HBV-associated pathologies, including inflammation, liver fibrosis, and the development of cirrhosis and hepatocellular carcinoma.
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The fast spread of bacteria that are resistant to many classes of antibiotics (multidrug resistant) is a global threat to human and animal health with a worrisome scenario ahead. Novel therapeutical strategies are of crucial importance to combat this phenomenon. For this purpose, we investigated the antimicrobial properties of the naturally occurring tripeptide Bialaphos and a dipeptide L-leucyl-L-phosphinoithricin, the synthesis and diastereomers separation of which are herein described. We demonstrate that these compounds are effective on clinical isolates of the human pathogen Klebsiella pneumoniae, causing hospital-acquired and community-acquired infections. The tested isolates were remarkable for their resistance to more than 20 commercial antibiotics of different classes. Based on previous literature data and our experiments consisting of glutamine supplementation, we suggest that both compounds release phosphinothricin-a well-known nanomolar inhibitor of glutamine synthetase-after their penetration in the bacterial cells; and, in this way, exert their antibacterial effect by negatively affecting nitrogen assimilation in this pathogen.
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Antiinfecciosos , Infecciones por Klebsiella , Humanos , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Farmacorresistencia Bacteriana Múltiple , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Péptidos/farmacologíaRESUMEN
Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and rapidly caused a pandemic that led to the death of >6 million people due to hypercoagulation and cytokine storm. In addition, SARS-CoV-2 triggers a wide array of pathologies, including liver dysfunction and neurological disorders. It remains unclear if these events are due to direct infection of the respective tissues or result from systemic inflammation. Here, we explored the possible infection of hepatic and CNS cell lines by SARS-CoV-2. We show that even moderate expression levels of the angiotensin-converting enzyme 2 (ACE2) are sufficient for productive infection. SARS-CoV-2 infects hepatoma Huh7.5 and HepG2 cells but not non-transformed liver progenitor or hepatocyte/cholangiocyte-like HepaRG cells. However, exposure to the virus causes partial dedifferentiation of HepaRG cells. SARS-CoV-2 can also establish efficient replication in some low-passage, high-grade glioblastoma cell lines. In contrast, embryonal primary astrocytes or neuroblastoma cells did not support replication of the virus. Glioblastoma cell permissiveness is associated with defects in interferon production. Overall, these results suggest that liver dysfunction during COVID-19 is not due to infection of these tissues by SARS-CoV-2. Furthermore, tumors may potentially serve as reservoirs for the virus during infection.
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Phosphorylation of beta-amyloid peptide (Aß) at the Ser8 residue affects its neurotoxicity, metal-dependent oligomerisation, amyloidogenicity, and other pathogenic properties. Phosphorylated Aß (pS8-Aß) was detected in vivo in AD model mice and in the brains of patients with AD. However, the pS8-Aß production and the regulation of its levels have not been previously studied in detail. In this paper, immunochemical methods together with radioactive labelling were used to study the Aß phosphorylation by intracellular and surface protein kinases of HEK293 cells and brain endothelial cells (bEnd.3). It was found that HEK293 robustly phosphorylated Aß, likely with contribution from casein kinase 2 (CK2), whereas in bEnd.3, the activity of Aß phosphorylation was relatively low. Further, the study showed that both HEK293 and bEnd.3 could dephosphorylate pS8-Aß, mainly due to the activity of protein phosphatases PP1 and PP2A. The Aß dephosphorylation efficiency in bEnd.3 was three times higher than in HEK293, which correlated with the reduced abundance of pS8-Aß in vascular amyloid deposits of patients with AD compared to senile plaques. These data suggest an important role of CK2, PP1, and PP2A as regulators of Aß phosphorylation, and point to the involvement of the blood-brain barrier in the control of Aß modification levels.
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Anti-SARS-CoV-2 antibody testing is an efficient tool to assess the proportion of seropositive population due to infection and/or vaccination. Numerous test systems utilizing various antigen composition(s) are routinely used for detection and quantitation of anti-SARS-CoV-2 antibodies. We determined their diagnostic specificity using archived true-negative samples collected before the onset of the COVID-19 pandemic. Using test systems demonstrating 98.5-100% specificity, we assessed the dynamics of SARS-CoV-2 seroconversion and durability of anti-spike (S) antibodies in healthcare professionals (n = 100) working in Moscow during the first two cycles of the pandemic (May 2020 to June 2021) outside of the "red zone". Analysis revealed a rapid increase in anti-SARS-CoV-2 seropositivity from 19 to 80% (19/100 and 80/100, respectively) due to virus exposition/infection; only 16.3% of seroconversion cases (13/80) were due to vaccination, but not the virus exposure, although massive COVID-19 vaccination of healthcare workers was performed beginning in December 2020. In total, 12.7% (8/63) remained positive for anti-SARS-CoV-2 IgM for >6 months, indicating unsuitability of IgM for identification of newly infected individuals. All except one remained seropositive for anti-S antibodies for >9 months on average. Significant (>15%) declines in anti-SARS-CoV-2 antibody concentrations were observed in only 18% of individuals (9/50). Our data on the high seropositivity rate and stability of anti-SARS-CoV-2 antibody levels in healthcare personnel working outside of the "red zone" indicate their regular exposition to SARS-CoV-2/an increased risk of infection, while a low frequency of vaccine-induced antibody response acquired after the start of vaccination points to vaccine hesitancy.
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A microarray-based assay to detect IgG and IgM antibodies against betacoronaviruses (SARS-CoV-2, SARS, MERS, OC43, and HKU1), other respiratory viruses and type I interferons (IFN-Is) was developed. This multiplex assay was applied to track antibody cross-reactivity due to previous contact with similar viruses and to identify antibodies against IFN-Is as the markers for severe COVID-19. In total, 278 serum samples from convalescent plasma donors, COVID-19 patients in the intensive care unit (ICU) and patients who recovered from mild/moderate COVID-19, vaccine recipients, prepandemic and pandemic patients with autoimmune endocrine disorders, and a heterogeneous prepandemic cohort including healthy individuals and chronically ill patients were analyzed. The anti-SARS-CoV-2 microarray results agreed well with the ELISA results. Regarding ICU patients, autoantibodies against IFN-Is were detected in 10.5% of samples, and 10.5% of samples were found to simultaneously contain IgM antibodies against more than two different viruses. Cross-reactivity between IgG against the SARS-CoV-2 nucleocapsid and IgG against the OC43 and HKU1 spike proteins was observed, resulting in positive signals for the SARS-CoV-2 nucleocapsid in prepandemic samples from patients with autoimmune endocrine disorders. The presence of IgG against the SARS-CoV-2 nucleocapsid in the absence of IgG against the SARS-CoV-2 spike RBD should be interpreted with caution.
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Anticuerpos Antivirales/inmunología , Interferón Tipo I/inmunología , SARS-CoV-2/inmunología , Virus/inmunología , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , COVID-19/inmunología , Prueba Serológica para COVID-19 , Reacciones Cruzadas , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Análisis por Matrices de Proteínas , Enfermedades Respiratorias/inmunología , Enfermedades Respiratorias/virología , Virus/clasificaciónRESUMEN
In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC100 below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.
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Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.
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Acetilcisteína/farmacología , Adyuvantes Inmunológicos/farmacología , Eflornitina/farmacología , Hepatitis C/inmunología , Inmunidad Activa/efectos de los fármacos , Proteínas no Estructurales Virales/inmunología , Animales , Proliferación Celular , Células Cultivadas , Femenino , Inmunogenicidad Vacunal/efectos de los fármacos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Ratones , Ratones Endogámicos DBA , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/inmunología , Óxido Nítrico/metabolismo , Oligodesoxirribonucleótidos/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
Enhanced production of reactive oxygen species (ROS) triggered by various stimuli, including viral infections, has attributed much attention in the past years. It has been shown that different viruses that cause acute or chronic diseases induce oxidative stress in infected cells and dysregulate antioxidant its antioxidant capacity. However, most studies focused on catalase and superoxide dismutases, whereas a family of peroxiredoxins (Prdx), the most effective peroxide scavengers, were given little or no attention. In the current review, we demonstrate that peroxiredoxins scavenge hydrogen and organic peroxides at their physiological concentrations at various cell compartments, unlike many other antioxidant enzymes, and discuss their recycling. We also provide data on the regulation of their expression by various transcription factors, as they can be compared with the imprint of viruses on transcriptional machinery. Next, we discuss the involvement of peroxiredoxins in transferring signals from ROS on specific proteins by promoting the oxidation of target cysteine groups, as well as briefly demonstrate evidence of nonenzymatic, chaperone, functions of Prdx. Finally, we give an account of the current state of research of peroxiredoxins for various viruses. These data clearly show that Prdx have not been given proper attention despite all the achievements in general redox biology.
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Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in majority of tumors. Several TERT-based cancer vaccines are currently in clinical trials, but immune correlates of their antitumor activity remain largely unknown. Here, we characterized fine specificity and lytic potential of immune response against rat TERT in mice. BALB/c mice were primed with plasmids encoding expression-optimized hemagglutinin-tagged or nontagged TERT or empty vector and boosted with same DNA mixed with plasmid encoding firefly luciferase (Luc DNA). Injections were followed by electroporation. Photon emission from booster sites was assessed by in vivo bioluminescent imaging. Two weeks post boost, mice were sacrificed and assessed for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) production by T-cells upon their stimulation with TERT peptides and for anti-TERT antibodies. All TERT DNA-immunized mice developed cellular and antibody response against epitopes at the N-terminus and reverse transcriptase domain (rtTERT) of TERT. Photon emission from mice boosted with TERT/TERT-HA+Luc DNA was 100 times lower than from vector+Luc DNA-boosted controls. Bioluminescence loss correlated with percent of IFN-γ/IL-2/TNF-α producing CD8+ and CD4+ T-cells specific to rtTERT, indicating immune clearance of TERT/Luc-coexpressing cells. We made murine adenocarcinoma 4T1luc2 cells to express rtTERT by lentiviral transduction. Expression of rtTERT significantly reduced the capacity of 4T1luc2 to form tumors and metastasize in mice, while not affecting in vitro growth. Mice which rejected the tumors developed T-cell response against rtTERT and low/no response to the autoepitope of TERT. This advances rtTERT as key component of TERT-based therapeutic vaccines against cancer.
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HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of "non-AIDS-defining" malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.
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Adenocarcinoma/virología , Neoplasias de la Mama/virología , Infecciones por VIH/metabolismo , Transcriptasa Inversa del VIH/metabolismo , VIH-1/metabolismo , Neoplasias Mamarias Experimentales/virología , Proteína 1 Relacionada con Twist/metabolismo , Animales , Carcinogénesis , Procesos de Crecimiento Celular , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Femenino , Infecciones por VIH/patología , Transcriptasa Inversa del VIH/genética , VIH-1/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Metástasis de la Neoplasia , Especies Reactivas de Oxígeno/metabolismo , Proteína 1 Relacionada con Twist/genética , Regulación hacia ArribaRESUMEN
Tumorigenesis is accompanied by the metabolic adaptation of cells to support enhanced proliferation rates and to optimize tumor persistence and amplification within the local microenvironment. In particular, cancer cells exhibit elevated levels of biogenic polyamines. Inhibitors of polyamine biosynthesis and inducers of their catabolism have been evaluated as antitumor drugs, however, their efficacy and safety remain controversial. Our goal was to investigate if drug-induced modulation of polyamine metabolism plays a role in dedifferentiation using differentiated human hepatocyte-like HepaRG cell cultures. N¹,N11-diethylnorspermine (DENSpm), a potent inducer of polyamine catabolism, triggered an epithelial-mesenchymal transition (EMT)-like dedifferentiation in HepaRG cultures, as shown by down-regulation of mature hepatocytes markers and upregulation of classical EMT markers. Albeit the fact that polyamine catabolism produces H2O2, DENSpm-induced de-differentiation was not affected by antioxidants. Use of a metabolically stable spermidine analogue showed furthermore, that spermidine is a key regulator of hepatocyte differentiation. Comparative transcriptome analyses revealed, that the DENSpm-triggered dedifferentiation of HepaRG cells was accompanied by dramatic metabolic adaptations, exemplified by down-regulation of the genes of various metabolic pathways and up-regulation of the genes involved in signal transduction pathways. These results demonstrate that polyamine metabolism is tightly linked to EMT and differentiation of liver epithelial cells.
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Virally induced liver cancer usually evolves over long periods of time in the context of a strongly oxidative microenvironment, characterized by chronic liver inflammation and regeneration processes. They ultimately lead to oncogenic mutations in many cellular signaling cascades that drive cell growth and proliferation. Oxidative stress, induced by hepatitis viruses, therefore is one of the factors that drives the neoplastic transformation process in the liver. This review summarizes current knowledge on oxidative stress and oxidative stress responses induced by human hepatitis B and C viruses. It focuses on the molecular mechanisms by which these viruses activate cellular enzymes/systems that generate or scavenge reactive oxygen species (ROS) and control cellular redox homeostasis. The impact of an altered cellular redox homeostasis on the initiation and establishment of chronic viral infection, as well as on the course and outcome of liver fibrosis and hepatocarcinogenesis will be discussed The review neither discusses reactive nitrogen species, although their metabolism is interferes with that of ROS, nor antioxidants as potential therapeutic remedies against viral infections, both subjects meriting an independent review.
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Hepatitis B/metabolismo , Hepatitis C/metabolismo , Neoplasias Hepáticas/virología , Estrés Oxidativo , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Transducción de SeñalRESUMEN
It is generally acknowledged that reactive oxygen species (ROS) play crucial roles in a variety of natural processes in cells. If increased to levels which cannot be neutralized by the defense mechanisms, they damage biological molecules, alter their functions, and also act as signaling molecules thus generating a spectrum of pathologies. In this review, we summarize current data on oxidative stress markers associated with human immunodeficiency virus type-1 (HIV-1) infection, analyze mechanisms by which this virus triggers massive ROS production, and describe the status of various defense mechanisms of the infected host cell. In addition, we have scrutinized scarce data on the effect of ROS on HIV-1 replication. Finally, we present current state of knowledge on the redox alterations as crucial factors of HIV-1 pathogenicity, such as neurotoxicity and dementia, exhaustion of CD4+/CD8+ T-cells, predisposition to lung infections, and certain side effects of the antiretroviral therapy, and compare them to the pathologies associated with the nitrosative stress.
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Infecciones por VIH/virología , VIH-1/patogenicidad , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Fármacos Anti-VIH/efectos adversos , Relación CD4-CD8 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Interacciones Huésped-Patógeno , Humanos , Oxidantes/uso terapéutico , Oxidación-Reducción , Transducción de Señal , Resultado del Tratamiento , Replicación ViralRESUMEN
Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 µg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 µg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.
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Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Virus de la Hepatitis Delta/inmunología , Antígenos de Hepatitis delta/inmunología , Animales , Anticuerpos Antivirales/sangre , Línea Celular , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Virus de la Hepatitis Delta/genética , Antígenos de Hepatitis delta/biosíntesis , Humanos , ARN Viral/genética , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N(3)-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N(1) position.