RESUMO
BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.
Assuntos
COVID-19/patologia , Nasofaringe/ultraestrutura , SARS-CoV-2/ultraestrutura , Antígenos Virais/metabolismo , COVID-19/diagnóstico , COVID-19/virologia , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Humanos , Aumento da Imagem , Microscopia , Microvilosidades/ultraestrutura , Mucosa Nasal/ultraestrutura , Mucosa Nasal/virologia , Nasofaringe/virologia , SARS-CoV-2/isolamento & purificação , Vírion/ultraestruturaRESUMO
The eradication of hepatitis C virus (HCV) infection is a public health priority. Despite the efficiency of treatment with direct-acting antivirals, the high cost of the therapy and the lack of accurate data about the HCV-infected population worldwide constitute important factors hampering this task. Hence, an affordable preventive vaccine is still necessary for reducing transmission and the future disease burden globally. In this work, chimeric proteins (EnvCNS3 and NS3EnvCo) encompassing conserved and immunogenic epitopes from the HCV core, E1, E2 and NS3 proteins were produced in Escherichia coli, and their immunogenicity was evaluated in BALB/c mice. The impact of recombinant HCV E2.680 protein and oligodeoxynucleotide 39M (ODN39M) on the immune response to chimeric proteins was also assessed. Immunization with chimeric proteins mixed with E2.680 enhanced the antibody and cellular response against HCV antigens and chimeric proteins. Interestingly, the combination of NS3EnvCo with E2.680 and ODN39M as adjuvant elicited a potent antibody response characterized by an increase in antibodies of the IgG2a subclass against E2.680, NS3 and chimeric proteins, suggesting the induction of a Th1-type response. Moreover, a cytotoxic T lymphocyte response and a broad response of IFN-γ-secreting cells against HCV antigens were induced with this formulation as well. This T cell response was able to protect vaccinated mice against challenge with a surrogate model based on HCV recombinant vaccinia virus. Overall, the vaccine candidate NS3EnvCo/E2.680/ODN39M might constitute an effective immunogen against HCV with potential for reducing the likelihood of viral persistence.
Assuntos
Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Proteínas Recombinantes/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos , Clonagem Molecular , Epitopos , Feminino , Regulação da Expressão Gênica/imunologia , Antígenos da Hepatite C/imunologia , Imunidade Celular , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , PlasmídeosRESUMO
Chronic infection with HCV is a leading cause of cirrhosis, hepatocellular carcinoma and liver failure. One of the least understood steps in the HCV life cycle is the morphogenesis of new viral particles. HCV infection alters the lipid metabolism and generates a variety of microenvironments in the cell cytoplasm that protect viral proteins and RNA promoting viral replication and assembly. Lipid droplets (LDs) have been proposed to link viral RNA synthesis and virion assembly by physically associating these viral processes. HCV assembly, envelopment, and maturation have been shown to take place at specialized detergent-resistant membranes in the ER, rich in cholesterol and sphingolipids, supporting the synthesis of luminal LDs-containing ApoE. HCV assembly involves a regulated allocation of viral and host factors to viral assembly sites. Then, virus budding takes place through encapsidation of the HCV genome and viral envelopment in the ER. Interaction of ApoE with envelope proteins supports the viral particle acquisition of lipids and maturation. HCV secretion has been suggested to entail the ion channel activity of viral p7, several components of the classical trafficking and autophagy pathways, ESCRT, and exosome-mediated export of viral RNA. Here, we review the most recent advances in virus morphogenesis and the interplay between viral and host factors required for the formation of HCV virions.
Assuntos
Hepacivirus/ultraestrutura , Hepatite C/virologia , Vírion/ultraestrutura , Montagem de Vírus/genética , Genoma Viral , Hepacivirus/genética , Hepatite C/genética , Humanos , Gotículas Lipídicas/metabolismo , RNA Viral/genética , Vírion/genética , Replicação Viral/genéticaRESUMO
B23/NPM is a multifunctional nucleolar protein frequently overexpressed, mutated, or rearranged in neoplastic tissues. B23/NPM is involved in diverse biological processes and is mainly regulated by heteroligomer association and posttranslational modification, phosphorylation being a major posttranslational event. While the role of B23/NPM in supporting and/or driving malignant transformation is widely recognized, the particular relevance of its CK2-mediated phosphorylation remains unsolved. Interestingly, the pharmacologic inhibition of such phosphorylation event by CIGB-300, a clinical-grade peptide drug, was previously associated to apoptosis induction in tumor cell lines. In this work, we sought to identify the biological processes modulated by CIGB-300 in a lung cancer cell line using subtractive suppression hybridization and subsequent functional annotation clustering. Our results indicate that CIGB-300 modulates a subset of genes involved in protein synthesis (ES = 8.4, p < 0.001), mitochondrial ATP metabolism (ES = 2.5, p < 0.001), and ribosomal biogenesis (ES = 1.5, p < 0.05). The impairment of these cellular processes by CIGB-300 was corroborated at the molecular and cellular levels by Western blot (P-S6/P-4EBP1, translation), confocal microscopy (JC-1, mitochondrial potential), qPCR (45SrRNA/p21, ribosome biogenesis), and electron microscopy (nucleolar structure, ribosome biogenesis). Altogether, our findings provide new insights on the potential relevance of the CK2-mediated phosphorylation of B23/NPM in cancer cells, revealing at the same time the potentialities of its pharmacological manipulation for cancer therapy. Finally, this work also suggests several candidate gene biomarkers to be evaluated during the clinical development of the anti-CK2 peptide CIGB-300.
Assuntos
Caseína Quinase II/genética , Neoplasias/genética , Proteínas Nucleares/metabolismo , Ribossomos/genética , Apoptose/efeitos dos fármacos , Caseína Quinase II/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metabolismo Energético/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteínas Nucleares/genética , Nucleofosmina , Peptídeos Cíclicos/administração & dosagem , Fosforilação/efeitos dos fármacos , Ribossomos/metabolismoRESUMO
Growth hormone releasing peptide, GHRP-6, a hexapeptide (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2, MW = 872.44 Da) that belongs to a class of synthetic growth hormone secretagogues, can stimulate growth hormone secretion from somatotrophs in several species including humans. In the present study, we demonstrate that GHRP-6 dispersed in aqueous solution, at pH 7.0, room temperature of 22 °C, is able to form long nanotubes, which is evidenced by combining small angle X-ray scattering (SAXS), transmission electron microscopy and molecular dynamics simulation results. Such nanotubes possess inner and outer cross-sections equal to 6.7(2) nm and 13.4(5) nm, respectively. The mechanism of peptide self-assembly was determined by molecular dynamics simulations revealing that the peptides self-assemble like amphiphilic molecules in aqueous solution in a partially interdigitated structure. In this case, the position of the positively charged amino terminus is located at the peptide-water interface, whereas the neutral NH2-capped carboxy terminus remains buried at the hydrophobic core. In contrast, the long side chain of Lys-6 stretches out of the hydrophobic core positioning its positive charge near the cylinder surface. The peptide configuration in the nanotube wall comes from the interplay between the hydrophobic interactions of the aromatic side chains of GHRP-6 and the electrostatic repulsion of its cationic charges. On increasing the peptide concentration, the long nanotubes self-arrange in solution displaying a bi-dimensional hexagonal-like packing in the SAXS curves, with a center-to-center distance of â¼15 nm. Further, we also show that the nanostructure formed in solution is quite stable and is preserved following transfer to a solid support.
Assuntos
Nanotubos/química , Oligopeptídeos/química , Sequência de Aminoácidos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo , Água/químicaRESUMO
Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy.
Assuntos
Proteínas do Capsídeo/metabolismo , Vacinas contra Dengue , Vírus da Dengue/classificação , Vírus da Dengue/imunologia , Regulação Viral da Expressão Gênica/fisiologia , Proteínas do Envelope Viral/metabolismo , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Clonagem Molecular , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Escherichia coli , Estrutura Terciária de Proteína , Proteínas Recombinantes/imunologia , Sorotipagem , Proteínas do Envelope Viral/genéticaRESUMO
OBJECTIVE: Subendothelial retention of proatherogenic lipoproteins by proteoglycans is critical in atherosclerosis. The aim of this study was to characterize the recognition and antiatherogenic properties of a chimeric monoclonal antibody (mAb) that reacts with sulfated molecules. METHODS AND RESULTS: chP3R99 mAb recognized sulfated glycosaminoglycans, mainly chondroitin sulfate (CS), by ELISA. This mAb blocked ≈70% of low-density lipoprotein (LDL)-CS association and ≈80% of LDL oxidation in vitro, and when intravenously injected to Sprague-Dawley rats (n=6, 1 mg/animal), it inhibited LDL (4 mg/kg intraperitoneally, 1 hour later) retention and oxidation in the artery wall. Moreover, subcutaneous immunization of New Zealand White rabbits (n=19) with chP3R99 mAb (100 µg, 3 doses at weekly intervals) prevented Lipofundin-induced atherosclerosis (2 mL/kg, 8 days) with a 22-fold reduction in the intima-media ratio (P<0.01). Histopathologic and ultrastructural studies showed no intimal alterations or slight thickening, with preserved junctions between endothelial cells and scarce collagen fibers and glycosaminoglycans. In addition, immunization with chP3R99 mAb suppressed macrophage infiltration in aorta and preserved redox status. The atheroprotective effect was associated with the induction of anti-CS antibodies in chP3R99-immunized rabbits, capable of blocking CS-LDL binding and LDL oxidation. CONCLUSION: These results support the use of anti-sulfated glycosaminoglycan antibody-based immunotherapy as a potential tool to prevent atherosclerosis.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Aterosclerose/prevenção & controle , Sulfatos de Condroitina/antagonistas & inibidores , Glicosaminoglicanos/antagonistas & inibidores , Imunização , Animais , Especificidade de Anticorpos , Aterosclerose/induzido quimicamente , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Transporte Biológico , Linhagem Celular , Sulfatos de Condroitina/imunologia , Modelos Animais de Doenças , Combinação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Células Espumosas/imunologia , Células Espumosas/metabolismo , Glicosaminoglicanos/imunologia , Lipoproteínas LDL/metabolismo , Camundongos , Oxirredução , Estresse Oxidativo , Fosfolipídeos , Coelhos , Ratos , Ratos Sprague-Dawley , SorbitolRESUMO
Coronaviruses constitute a global threat to the human population; therefore, effective pan-coronavirus antiviral drugs are required to tackle future re-emerging virus outbreaks. Protein kinase CK2 has been suggested as a promising therapeutic target in COVID-19 owing to the in vitro antiviral activity observed after both pharmacologic and genetic inhibition of the enzyme. Here, we explored the putative antiviral effect of the anti-CK2 peptide CIGB-325 on bovine coronavirus (BCoV) infection using different in vitro viral infected cell-based assays. The impact of the peptide on viral mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. Finally, pull-down experiments followed by Western blot and/or mass spectrometry analysis were performed to identify CIGB-325-interacting proteins. We found that CIGB-325 inhibited both the cytopathic effect and the number of plaque-forming units. Accordingly, intracellular viral protein levels were clearly reduced after treatment of BCoV-infected cells, with CIGB-325 determined by immunocytochemistry. Pull-down assay data revealed the physical interaction of CIGB-325 with viral nucleocapsid (N) protein and a group of bona fide CK2 cellular substrates. Our findings evidence in vitro antiviral activity of CIGB-325 against bovine coronavirus as well as some molecular clues that might support such effect. Altogether, data provided here strengthen the rationale of inhibiting CK2 to treat betacoronavirus infections.
Assuntos
Coronavirus Bovino , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Caseína Quinase II/metabolismo , Bovinos , Peptídeos/farmacologia , FosforilaçãoRESUMO
Large cell lung carcinoma (LCLC) is one form of NSCLC that spreads more aggressively than some other forms, and it represents an unmet medical need. Here, we investigated for the first time the effect of the anti-CK2 CIGB-300 peptide in NCI-H460 cells as an LCLC model. NCI-H460 cells were highly sensitive toward CIGB-300 cytotoxicity, reaching a peak of apoptosis at 6 h. Moreover, CIGB-300 slightly impaired the cell cycle of NCI-H460 cells. The CIGB-300 interactomics profile revealed in more than 300 proteins that many of them participated in biological processes relevant in cancer. Interrogation of the CK2 subunits targeting by CIGB-300 indicated the higher binding of the peptide to the CK2α' catalytic subunit by in vivo pull-down assays plus immunoblotting analysis and confocal microscopy. The down-regulation of both phosphorylation and protein levels of the ribonuclear protein S6 (RPS6) was observed 48 h post treatment. Altogether, we have found that NCI-H460 cells are the most CIGB-300-sensitive solid tumor cell line described so far, and also, the findings we provide here uncover novel features linked to CK2 targeting by the CIGB-300 anticancer peptide.
RESUMO
In this study, we evaluate in mice a novel formulation containing nucleocapsid-like particles of dengue-2 virus (recNLP) co-immunized with a chimeric protein composed of the dengue-4 envelope domain III fused twice within the meningococcal P64k protein of Neisseria meningitidis (PD24). The animals receiving the PD24-recNLP mixture showed the highest levels of antiviral antibodies. Similar results were obtained for IFNγ secretion levels, indicating a functional Th1 cellular response. Consistently, the percentage of mice surviving after viral challenge was significantly higher for those immunized with the mixture than for those inoculated with PD24 protein alone. In addition, in vivo depletion experiments demonstrated the decisive role of CD4(+) and CD8(+) cells in the protection conferred by immunization with PD24-recNLP. In conclusion, this report demonstrates for the first time the adjuvant capacity of dengue-2 virus recNLP. Additionally, the evidence presented highlights the potential of these particles for enhancing the immune response against heterologous recombinant proteins.
Assuntos
Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Nucleocapsídeo/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Proteínas da Membrana Bacteriana Externa/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Dengue/imunologia , Feminino , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Células Th1/imunologia , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genéticaRESUMO
Novel therapeutic peptides are increasingly making their way into clinical application. The cationic and amphipathic properties of certain peptides allow them to cross biological membranes in a non-disruptive way without apparent toxicity increasing drug bioavailability. By modifying the primary structure of the Limulus-derived LALF(32-51) peptide we designed a novel peptide, L-2, with antineoplastic effect and cell-penetrating capacity. Interestingly, L-2 induced cellular cytotoxicity in a variety of tumor cell lines and systemic injection into immunocompetent and nude mice bearing established solid tumor, resulted in substantial regression of the tumor mass and apoptosis. To isolate the gene transcripts specifically regulated by L-2 in tumor cells, we conducted suppressive subtractive hybridization (SSH) analysis and identified a set of genes involved in biological processes relevant to cancer biology. Our findings describe a novel peptide that modifies the gene expression of the tumor cells and exhibits antitumor effect in vivo, indicating that peptide L-2 is a potential candidate for anticancer therapy.
Assuntos
Alanina/química , Antineoplásicos/farmacologia , Neoplasias Experimentais/patologia , Biblioteca de Peptídeos , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificaçãoRESUMO
The capsid protein is one of the three structural proteins of flaviviruses and is the building block of the nucleocapsid. It has also a predominant role in the replication of dengue virus. To obtain nucleocapsid-like particles from recombinant dengue-2 capsid protein produced in E. coli, a purification process using cation exchange chromatography was established. The purified protein exhibited a molecular mass corresponding to a dimer; therefore, similar to that reported for alphaviruses, an in vitro assembly reaction using single-stranded DNA was performed. In all cases, particles were obtained independently of the specificity and the length of the oligonucleotides used. The present work is the first report of in vitro assembly of the recombinant dengue capsid protein, which could constitute a powerful tool in the development of vaccine candidates.
Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Dengue/fisiologia , Virossomos/metabolismo , Montagem de Vírus , Proteínas do Capsídeo/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Microscopia Eletrônica de Transmissão , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Virossomos/genética , Virossomos/ultraestruturaRESUMO
In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine pro file in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-gamma producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Interferon gama/biossíntese , Interleucina-4/biossíntese , Fragmentos de Peptídeos/imunologia , Baço/imunologia , Proteínas do Core Viral/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Feminino , Hepatite C/prevenção & controle , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/administração & dosagem , Baço/citologia , Células Th2/imunologia , Proteínas do Core Viral/administração & dosagemRESUMO
Protein Kinase (casein kinase 2, CK2) is a serine-threonine kinase that is frequently dysregulated in many human tumors. Therefore we hypothesized that peptides capable of binding to the CK2 acidic domain may exhibit potential anticancer properties. By screening a random cyclic peptide phage display library, we have identified a novel peptide, P15, that abrogated CK2 phosphorylation by blocking the substrate in vitro. To verify its potential antineoplastic effect, P15 was fused to the cell-penetrating peptide derived from the HIV-Tat protein. Interestingly, P15-Tat induced apoptosis as evidenced by rapid caspase activation and cellular cytotoxicity in a variety of tumor cell lines. Furthermore, direct injection of P15-Tat into C57BL6 mice bearing day 7-established solid tumors, resulted in substantial regression of the tumor mass. Our findings describe a new proapoptotic cyclic peptide that blocks the CK2 phosphorylation and exhibits antitumor effect in vivo, indicating that the P15 peptide may potentially be used clinically to treat solid tumors or as an adjuvant for cancer therapy.
Assuntos
Apoptose/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Caseína Quinase II , Linhagem Celular Tumoral , Produtos do Gene tat/genética , Produtos do Gene tat/farmacocinética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Biblioteca de Peptídeos , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/farmacocinética , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
A combination of antiviral drugs known as antiretroviral therapy (ART) has shown effectiveness against the human immunodeficiency virus (HIV). ART has markedly decreased mortality and morbidity among HIV-infected patients, having even reduced HIV transmission. However, an important current disadvantage, resistance development, remains to be solved. Hope is focused on developing drugs against cellular targets. This strategy is expected to prevent the emergence of viral resistance. In this study, using a comparative proteomic approach in MT4 cells treated with an anti-HIV leukocyte extract, we identified vimentin, a molecule forming intermediate filaments in the cell, as a possible target against HIV infection. We demonstrated a strong reduction of an HIV-1 based lentivirus expressing the enhanced green fluorescent protein (eGFP) in vimentin knockdown cells, and a noteworthy decrease of HIV-1 capsid protein antigen (CAp24) in those cells using a multiround infectivity assay. Electron micrographs showed changes in the structure of intermediate filaments when MT4 cells were treated with an anti-HIV leukocyte extract. Changes in the structure of intermediate filaments were also observed in vimentin knockdown MT4 cells. A synthetic peptide derived from a cytoskeleton protein showed potent inhibitory activity on HIV-1 infection, and low cytotoxicity. Our data suggest that vimentin can be a suitable target to inhibit HIV-1.
Assuntos
Descoberta de Drogas , HIV-1/fisiologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno , Vimentina/metabolismo , Replicação Viral , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Proteína do Núcleo p24 do HIV/análise , Infecções por HIV/tratamento farmacológico , Humanos , Vimentina/antagonistas & inibidoresRESUMO
Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, 'Candidatus Liberibacter asiaticus'. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.
Assuntos
Brassinosteroides/farmacologia , Citrus/microbiologia , Doenças das Plantas/microbiologia , Rhizobiaceae/efeitos dos fármacos , Citrus/efeitos dos fármacos , Folhas de Planta/microbiologiaRESUMO
Neurofibrillary tangles (NFTs) are the neuropathological hallmarks in Alzheimer's disease (AD). Densities of NFTs correlate with the dementia status. NFTs reflect the intracellular accumulation of abnormal paired helical filaments (PHFs) composed of the microtubule-associated protein tau. Hyperphosphorylation and truncation have been proposed as key events leading to the genesis of PHFs. A recent hypothesis involving conformational changes has been emerging. These structural modifications of the tau protein were detected by monoclonal antibodies (mAbs) recognizing discontinuous epitopes along the tau molecule such as Alz-50, Tau-66 and MC1. A new mAb, TG-3, detects an early pathology in AD. The epitope of mAb TG-3 maps to phosphorylated Thr231 when the tau molecule is conformationally altered. In the present study, we used confocal microscopy to analyze the state of tau molecule adopting the TG-3 conformation during tangle formation. We also compared mAb TG-3 immunoreactivity with that of mAb Alz-50. Immunoelectronmicroscopy was also performed. N- and C- termini markers evidenced that the tau molecule is intact when it adopts the TG-3 conformation. In addition to NFT, mAb TG-3 also recognized NFT-not bearing-neurons suggesting an early processing of tau prior to NFT formation. Ultrastructural analysis evidenced the presence of TG-3 and Alz-50 immunoreactive products on organelles including mitochondria and endoplasmic reticulum. Nuclear heterochromatin was densely immunolabelled. These results together with the fact that TG-3 immunoreactivity is related to intact tau suggest that the conformation recognized by TG-3 is early staged in the neuronal pathology of AD. In addition, we document that the earliest changes in tau occur closely associated with organelles and heterochromatin.
Assuntos
Doença de Alzheimer/patologia , Anticorpos Monoclonais , Antígenos/imunologia , Citoesqueleto/patologia , Proteínas do Tecido Nervoso/metabolismo , Emaranhados Neurofibrilares/patologia , Idoso , Idoso de 80 Anos ou mais , Especificidade de Anticorpos , Córtex Entorrinal/patologia , Feminino , Hipocampo/patologia , Humanos , Masculino , Microscopia Confocal , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Neurônios/patologia , Fosforilação , Conformação Proteica , Proteínas tauRESUMO
A fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus polyphemus protein linked to human papillomavirus (HPV)-16 E7 antigen (LALF32-51-E7) was expressed in E. coli BL21 (DE3) cells. The recombinant protein in E. coli accounted for approximately 18% of the total cellular protein and purified with a single affinity chromatographic step. Yields of approximately 38 mg purified LALF32-51-E7 per liter of induced culture was obtained with an overall 52% recovery and constitutes a promising setting for the future production and scaling-up. Purified protein was characterized as soluble aggregates with molecular weight larger than 670 kDa, which is considered an important property to increase the immunogenicity of an antigen preparation. The recombinant fusion protein LALF32-51-E7 will be a promising vaccine candidate for the treatment of HPV-16 related malignancies.
RESUMO
Rheumatoid arthritis (RA) is a chronic T-cell mediated autoimmune disease that affects primarily the joints. The induction of immune tolerance through antigen-specific therapies for the blockade of pathogenic CD4+ T cells constitutes a current focus of research. In this focus it is attempted to simultaneously activate multiple regulatory mechanisms, such as: apoptosis and regulatory T cells (Tregs). APL-1 is an altered peptide ligand derived from a novel CD4+ T-cell epitope of human heat-shock protein of 60kDa, an autoantigen involved in the pathogenesis of RA. Previously, we have reported that APL-1 induces CD4+ CD25(high)Foxp3+ Tregs in several systems. Here, we investigated the ability of APL-1 in inducing apoptosis in PBMCs from RA patients, who were classified as active or inactive according to their DAS28 score. APL-1 decreased the viability of PBMCs from active but not from inactive patients. DNA fragmentation assays and typical morphological features clearly demonstrated that APL-1 induced apoptosis in these cells. Activated CD4+ CD25+ T cells but not resting CD4+ CD25- T cells were identified as targets of APL-1. Furthermore, CD4+ T-cell responses to APL-1 were found to be dependent on antigen presentation via the HLA-DR molecule. Thus, APL-1 is a regulatory CD4+ T cell epitope which might modulate inflammatory immune responses in PBMCs from RA patients by inducing CD4+ CD25(high)Foxp3+ Tregs and apoptosis in activated CD4+ T cells. These results support further investigation of this candidate drug for the treatment of RA.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Chaperonina 60/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Adulto , Idoso , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chaperonina 60/imunologia , Fragmentação do DNA/efeitos dos fármacos , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/ultraestrutura , Ligantes , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a disease of the central nervous system characterized by loss of spinal motor neurons, for which no effective treatment exists. Epidermal growth factor (EGF) and growth hormone releasing peptide-6 (GHRP-6) have been considered as good candidates for the treatment of this disease, due to their well documented effects in eliciting pleiotrophic and cell survival mechanisms. The aim of the present work was to evaluate the separate and combined effects of both peptides in an experimental animal model of ALS, the proximal axonopathy induced by 1,2 diacetylbenzene (1,2 DAB) in mice. The evaluations were conducted by means of behavioral tests (trapeze, tail suspension, gait pattern, and open field) and by recording the complex muscle action potential (CMAP) in three different hind limb segments: proximal S1, medial S2, and distal S3. Intraperitoneal daily administration of 1,2 DAB produced significant reduction in body weight, muscle strength, extensor reflex, spontaneous activity, and changes in gait pattern parameters. In parallel 1,2 DAB produced significant prolongation of onset latency and decrease in amplitude of CMAP and in the integrated complex action potential index. Daily administration of the separate compounds did not accelerate the recovery of the affected parameters, except for the gait pattern. The combined treatment produced significant improvement in behavioral parameters, as well as in electrophysiological recovery, particularly in the proximal segment of CMAP. The latter results confirm the proximal character of 1,2 DAB neuropathy, and suggest that combined therapy with EGF and GHRP-6 might be a good therapeutic strategy for the treatment of ALS.