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1.
Bioorg Med Chem ; 26(9): 2401-2409, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29650461

RESUMO

Mycobacterium tuberculosis is considered one of the most successful pathogens in the history of mankind, having caused 1.7 million deaths in 2016. The amount of resistant and extensively resistant strains has increased; BCG has been the only vaccine to be produced in more than 100 years though it is still unable to prevent the disease's most disseminated form in adults; pulmonary tuberculosis. The search is thus still on-going for candidate antigens for an antituberculosis vaccine. This paper reports the use of a logical and rational methodology for finding such antigens, this time as peptides derived from the Rv3587c membrane protein. Bioinformatics tools were used for predicting mycobacterial surface location and Rv3587c protein structure whilst circular dichroism was used for determining its peptides' secondary structure. Receptor-ligand assays identified 4 high activity binding peptides (HABPs) binding specifically to A549 alveolar epithelial cells and U937 monocyte-derived macrophages, covering the region between amino acids 116 and 193. Their capability for inhibiting Mtb H37Rv invasion was evaluated. The recognition of antibodies from individuals suffering active and latent tuberculosis and from healthy individuals was observed in HABPs capable of avoiding mycobacterial entry to host cells. The results showed that 8 HABPs inhibited such invasion, two of them being common for both cell lines: 39265 (155VLAAYVYSLDNKRLWSNLDT173) and 39266 (174APSNETLVKTFSPGEQVTTY192). Peptide 39265 was the least recognised by antibodies from the individuals' sera evaluated in each group. According to the model proposed by FIDIC regarding synthetic vaccine development, peptide 39265 has become a candidate antigen for an antituberculosis vaccine.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Membrana/imunologia , Mycobacterium tuberculosis/fisiologia , Fragmentos de Peptídeos/imunologia , Vacinas contra a Tuberculose/imunologia , Sequência de Aminoácidos , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/toxicidade , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Linhagem Celular Tumoral , Biologia Computacional , Desenho de Fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas de Membrana/síntese química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/toxicidade , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo , Vacinas contra a Tuberculose/síntese química , Vacinas contra a Tuberculose/metabolismo , Vacinas contra a Tuberculose/toxicidade , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/toxicidade
2.
Anticancer Drugs ; 29(3): 253-261, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29346131

RESUMO

Fibroblast activation protein-α (FAPα) is a promising tumor-associated target expressed by reactive stromal fibroblasts in tumor tissue. FAPα has a postprolyl peptidase activity and can specifically cleave N-terminal benzyloxycarbonyl (Z)-blocked peptides, such as the substrate Z-Gly-Pro-AMC. Doxorubicin (DOX) is an effective antitumor drug, but its application is greatly limited by toxic adverse effects owing to poor tumor selectivity. Based on these facts, we previously designed a FAPα-targeting prodrug of doxorubicin (FTPD) which can be selectively hydrolyzed by FAPα. FTPD can retain potent antitumor efficacy and has favorable tumor targeting. The present study aimed to further evaluate the toxicological profile and the safety pharmacological property of FTPD in vitro and in vivo. The cytotoxicity assay showed that FTPD displayed markedly lower cytotoxicity to 3T3 cells and HEK-293 cells compared with DOX. In the short-term toxicity study, mice treated with 25 mg/kg of FTPD showed no obvious change in the appearance and general behavior, and no case of mortality was observed within 14 days. Unlike DOX, FTPD exhibited reduced toxicity to heart, liver, kidney, spleen as well as peripheral white blood cells in mice. Moreover, open file test and general pharmacology study were also conducted correspondingly in mice and beagle dogs. It was found that FTPD may not produce significant pharmacological effects on spontaneous locomotor activity and cardiovascular-respiratory system except for a transient decreasing in systolic blood pressure. Taken together, the results of this work suggest that FTPD has more favorable toxicological profile and better drug safety compared with its parent drug DOX.


Assuntos
Doxorrubicina/administração & dosagem , Doxorrubicina/toxicidade , Gelatinases/administração & dosagem , Gelatinases/toxicidade , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/toxicidade , Pró-Fármacos/administração & dosagem , Pró-Fármacos/toxicidade , Serina Endopeptidases/administração & dosagem , Serina Endopeptidases/toxicidade , Células 3T3 , Animais , Cães , Feminino , Células HEK293 , Humanos , Masculino , Camundongos
3.
J Mol Biol ; 429(12): 1800-1816, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28501587

RESUMO

In previous work, we have generated the engineered Escherichia coli strains SuptoxD and SuptoxR, which upon co-expression of the effector genes djlA or rraA, respectively, are capable of suppressing the cytotoxicity caused by membrane protein (MP) overexpression and of producing dramatically enhanced yields for a variety of recombinant MPs of both prokaryotic and eukaryotic origin. Here, we investigated the functional requirements for DnaJ-like protein A (DjlA)- and regulator of ribonuclease activity A (RraA)-mediated enhancement of recombinant MP production in these strains and show that: (i) DjlA and RraA act independently, that is, the beneficial effects of each protein on recombinant MP production occur through a mechanism that does not involve the other, and in a non-additive manner; (ii) full-length and membrane-bound DjlA is required for exerting its beneficial effects on recombinant MP production in E. coli SuptoxD; (iii) the MP production-promoting properties of DjlA in SuptoxD involve the action of the molecular chaperone DnaK but do not rely on the activation of the regulation of capsular synthesis response, a well-established consequence of djlA overexpression; (iv) the observed RraA-mediated effects in E. coli SuptoxR involve the ribonucleolytic activity of RNase E, but not that of its paralogous ribonuclease RNase G; and (v) DjlA and RraA are unique among similar E. coli proteins in their ability to promote bacterial recombinant MP production. These observations provide important clues about the molecular requirements for suppressed toxicity and enhanced MP accumulation in SuptoxD/SuptoxR and will guide future studies aiming to decipher the exact mechanism of DjlA- and RraA-mediated enhancement of recombinant MP production in these strains.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Membrana/genética , Proteínas de Membrana/toxicidade , Engenharia Metabólica , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade
4.
Am J Pathol ; 182(5): 1718-26, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23499467

RESUMO

Dystrophic neurites surrounding ß-amyloid (Aß) plaques precede neuronal death in Alzheimer disease. These neuritic alterations may be one of the initial stages for synaptic loss and dysfunction. However, intracellular pathways that cause local disruption of neuronal processes by Aß remain to be fully elucidated. The identification of Aß-induced genes that mediate neuritic pathology would provide considerable insight into the mechanisms of Alzheimer's disease. Previously, we reported that selective up-regulation of calsyntenin-3 (Cst-3) by Aß and accumulation of neurotoxic Cst-3 in dystrophic neurites surrounding Aß plaques may lead to local disruption of these neurites. Like amyloid precursor protein, Cst-3 undergoes two-step proteolytic processing: the primary cleavage with α-secretase generates an N-terminal ectodomain and a C-terminal fragment (CTF). The CTF is subsequently cleaved into p3 peptide and an intracellular domain via γ-secretase. It would be interesting to know whether accumulated Cst-3 in dystrophic neurites surrounding Aß plaques is the full-length version or a CTF. Herein, we show that the CTF but not full-length Cst-3 accumulated in dystrophic neurites surrounding Aß plaques in Tg2576 mouse and Alzheimer disease brains. In vitro experiments with Cst-3 fragments have revealed that only the CTF resulted in acceleration of neuronal death. These results indicate that accumulation of the neurotoxic CTF in neurites surrounding Aß plaques may lead to local disruption of neuronal processes and development of dystrophic neurites.


Assuntos
Doença de Alzheimer/patologia , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/toxicidade , Proteínas de Membrana/toxicidade , Neuritos/patologia , Placa Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Feminino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Fármacos Neuroprotetores/farmacologia , Placa Amiloide/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ratos
5.
Acta Ophthalmol ; 90(7): e512-23, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22994140

RESUMO

PURPOSE: To evaluate the effect of ZY1, a novel 21-amino acid peptide from human placenta growth factor-1 (PlGF-1), against ocular neovascularization, and to study its possible toxicity to the retina and the underlying mechanism of antiangiogenic effect. METHODS: MTS assays, a modified Boyden chamber and Matrigel(™) were used to evaluate the effect of ZY1 on the proliferation, migration and tube formation of RF/6A rhesus macaque choroid-retina endothelial cells induced by vascular endothelial growth factor (VEGF) in vitro. The antiangiogenic effect of ZY1 was also studied with corneal micropocket angiogenesis assays and oxygen-induced retinopathy (OIR) assays in mice. Electrophysiological tests and histological examinations were used to study the possible toxicity of ZY1 against mouse neuroretina. Competitive ELISA and Western blotting were performed to elucidate the underlying mechanism of ZY1. RESULTS: ZY1 inhibited VEGF-induced RF/6A proliferation, migration and tube formation. It also inhibited ocular neovascularization when applied to the corneal micropocket angiogenesis assays and OIR assays in mice. Electrophysiological tests and histological examinations revealed no evident functional or morphologic abnormalities in mouse neuroretina after ZY1 injection. ZY1 competed for binding to VEGFR-1 against PlGF and VEGF and inhibited VEGFR-1/ERK/AKT activation. CONCLUSION: It is concluded that the novel peptide ZY1 is an effective inhibitor of ocular pathologic angiogenesis and may provide a promising alternative for ocular antiangiogenic therapy.


Assuntos
Inibidores da Angiogênese/farmacologia , Neovascularização da Córnea/prevenção & controle , Proteínas de Membrana/farmacologia , Fragmentos de Peptídeos/farmacologia , Retina/efeitos dos fármacos , Neovascularização Retiniana/prevenção & controle , Inibidores da Angiogênese/toxicidade , Animais , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Eletrorretinografia , Endotélio Vascular/citologia , Ensaio de Imunoadsorção Enzimática , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Macaca mulatta , Masculino , Proteínas de Membrana/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Proteína Oncogênica v-akt/metabolismo , Oxigênio/toxicidade , Fragmentos de Peptídeos/toxicidade , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
6.
Biophys J ; 103(4): 738-47, 2012 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-22947935

RESUMO

The temperature-induced misfolding pathway of PDZ3, the third PDZ domain of the PSD95 neuronal protein, is populated by a trimeric ß-sheet-rich intermediate state that leads to a stepwise and reversible formation of supramacromolecular structures. Using FTIR, we have found that misfolding of this pathway is not due to different ensembles of a variety of precursors, but comes mainly from the interconversion of a flexible ß-sheet of the domain to wormlike fibrils. The appearance of the wormlike fibril FTIR component is also accompanied by a slight decrease of the band that corresponds to loops in the native state, whereas the rest of the regular elements of secondary structure are fairly well maintained upon misfolding. Transmission electron microscope micrographs have confirmed the presence of wormlike fibrils upon heating at 60°C, where the trimeric intermediate is maximally populated. Toxicity assays in the human neuroblastoma cell line SH-SY5Y show that cytotoxicity increases as the aggregation pathway proceeds. NMR analysis of chemical shifts as a function of temperature has revealed, as one of the main conformational aspects of such an interconversion at the residue level, that the ß-sheet arrangement around strand ß3 promotes the change that drives misfolding of the PDZ3 domain.


Assuntos
Proteínas de Membrana/química , Domínios PDZ , Dobramento de Proteína , Multimerização Proteica , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/toxicidade , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína
7.
Int J Mol Sci ; 12(4): 2125-37, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21731431

RESUMO

Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-X(L), caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.


Assuntos
Apoptose/efeitos dos fármacos , Plaquetas/citologia , Dibucaína/toxicidade , Proteínas de Membrana/toxicidade , Plaquetas/efeitos dos fármacos , Caspase 3/metabolismo , Adesão Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Fosfatase 2 de Especificidade Dupla/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Selectina-P/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Ristocetina/farmacologia , Trombina/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo
8.
Trends Endocrinol Metab ; 22(6): 211-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21592818

RESUMO

In many species the lactating mammary gland is one of the most lipogenic organs of the body. The majority of the lipid produced during lactation is secreted into milk by a novel process of membrane envelopment of cytoplasmic lipid droplets (CLDs). Adipophilin (ADRP/ADPH/PLIN2), a member of the perilipin (PAT) family of lipid droplet proteins, is hypothesized to play a pivotal role in both formation and secretion of milk lipids. Production of milk lipids is the only known example of CLD secretion, and the only process in which PAT family members undergo secretion. This review discusses emerging data on the structural and functional properties of adipophilin that determine its physiological actions and mediate its effects on milk lipid formation and secretion.


Assuntos
Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Lactação/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/fisiologia , Organelas/metabolismo , Animais , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/toxicidade , Modelos Biológicos , Perilipina-2
10.
EMBO J ; 30(10): 2057-70, 2011 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-21441896

RESUMO

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a ß-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various ß-sheet-rich (ß) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid ß-peptide, (iii) yeast prion proteins or (iv) designed ß-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with ß-conformers. Interestingly, a secreted version of N-PrP associated with ß-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various ß-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana/toxicidade , Proteínas PrPC/metabolismo , Proteínas PrPC/toxicidade , Doenças Priônicas/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Morte Celular , Humanos , Proteínas de Membrana/química , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Proteínas PrPC/química , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/toxicidade
11.
Clin Vaccine Immunol ; 17(11): 1781-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20861327

RESUMO

Staphylococcus aureus and Streptococcus pyogenes secrete exotoxins that act as superantigens, proteins that cause hyperimmune reactions by binding the variable domain of the T-cell receptor beta chain (Vß), leading to stimulation of a large fraction of the T-cell repertoire. To develop potential neutralizing agents, we engineered Vß mutants with high affinity for the superantigens staphylococcal enterotoxin B (SEB), SEC3, and streptococcal pyrogenic exotoxin A (SpeA). Unexpectedly, the high-affinity Vß mutants generated against SEB cross-reacted with SpeA to a greater extent than they did with SEC3, despite greater sequence similarity between SEB and SEC3. Likewise, the Vß mutants generated against SpeA cross-reacted with SEB to a greater extent than with SEC3. The structural basis of the high affinity and cross-reactivity was examined by single-site mutational analyses. The cross-reactivity seems to involve only one or two toxin residues. Soluble forms of the cross-reactive Vß regions neutralized both SEB and SpeA in vivo, suggesting structure-based strategies for generating high-affinity neutralizing agents that can cross-react with multiple exotoxins.


Assuntos
Antitoxinas/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Enterotoxinas/antagonistas & inibidores , Exotoxinas/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Staphylococcus aureus/patogenicidade , Streptococcus pyogenes/patogenicidade , Superantígenos/metabolismo , Animais , Antitoxinas/efeitos adversos , Antitoxinas/genética , Proteínas de Bactérias/toxicidade , Reações Cruzadas , Enterotoxinas/toxicidade , Exotoxinas/toxicidade , Proteínas de Membrana/toxicidade , Ligação Proteica , Coelhos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Análise de Sobrevida , Fatores de Virulência/antagonistas & inibidores
12.
J Neurochem ; 115(4): 1045-56, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20831617

RESUMO

NMDA receptors (NMDARs) mediate excitatory synaptic transmission during repetitive or prolonged glutamate release, playing a critical role in synaptic plasticity or cell death, respectively. Evidence indicates that a major pathway of NMDAR signaling to cell death in cortical and hippocampal neurons requires the scaffolding protein post-synaptic density 95 (PSD-95) and activation of neuronal nitric oxide synthase. However, it is not known if this PSD-95-dependent pathway contributes to excitotoxicity in other brain regions. It is also unclear whether the neuroprotective effects of Tat-NR2B9c, a membrane-permeant peptide that disrupts PSD-95/NMDAR binding, correlate with uncoupling NR2B- and/or NR2A-type NMDARs from PSD-95. In this study, we used cultured hippocampal and striatal neurons to test the potency of Tat-NR2B9c on uncoupling NR2 subunits from PSD-95 and protecting against NMDA-induced excitotoxicity. We found that the concentration of Tat-NR2B9c required to dissociate 50% of PSD-95 was fourfold lower for NR2B than NR2A in cultured hippocampal and striatal neurons, and that this concentration correlated tightly with protection against NMDA-induced toxicity in hippocampal neurons without altering NMDAR current. In contrast, NMDAR signaling to cell death in cultured striatal neurons occurred independently of the NR2B/PSD-95 interaction or neuronal nitric oxide synthase activation. These results will facilitate development of neuronal type-specific protective therapies.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Morte Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/toxicidade , Proteínas de Membrana/metabolismo , Proteínas de Membrana/toxicidade , Camundongos , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo I/fisiologia , Peptídeos/toxicidade , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologia , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Trends Biochem Sci ; 35(4): 228-35, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20036556

RESUMO

The power of fruit fly genetics is being deployed against some of the most intractable and economically significant problems in modern medicine, the neurodegenerative diseases. Fly models of Alzheimer's disease can be exposed to the rich diversity of biological techniques that are available to the community and are providing new insights into disease mechanisms, and assisting in the identification of novel targets for therapy. Similar approaches might also help us to interpret the results of genome-wide association studies of human neurodegenerative diseases by allowing us to triage gene "hits" according to whether a candidate risk factor gene has a modifying effect on the disease phenotypes in fly model systems.


Assuntos
Doença de Alzheimer/fisiopatologia , Modelos Animais de Doenças , Drosophila melanogaster/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Animais , Drosophila melanogaster/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/toxicidade , Fenótipo
14.
J Infect Dis ; 200(7): 1162-5, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19702507

RESUMO

The role that the enterococcal surface protein Esp plays in the capacity of Enterococcus faecium to adhere to uroepithelial cells and the role that it plays in urinary tract infection and peritonitis was investigated in vitro and in vivo, respectively, using Esp-expressing E. faecium (E1162) and its isogenic Esp-deficient mutant (E1162 Delta esp). Esp expression enhanced in vitro binding to bladder and kidney epithelial cells. In mice, higher numbers of E1162 were cultured from kidneys and bladders after the induction of urinary tract infection, compared with E1162 Delta esp numbers. This was accompanied by a higher frequency of bacteremia, higher cytokine levels in kidney tissue, and renal insufficiency. Esp had no effect on the course of E. faecium peritonitis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Enterococcus faecium/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/toxicidade , Infecções Urinárias/microbiologia , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Enterococcus faecium/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Membrana/genética , Camundongos , Ligação Proteica
15.
Toxicon ; 54(8): 1031-7, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19576920

RESUMO

Cnidarians are the largest phylum of generally toxic animals, yet their toxins and venoms have not received as much scientific attention as those of many terrestrial (snakes, scorpions, spiders, etc.) and even some marine animals (i.e. cone snails). Approximately 13,000 living cnidarian species have been described by systematists. A major rationale for their study in the past, besides scientific curiosity, was to better treat victims of their envenomation. While that goal remains a high priority, it is now appreciated that the toxins of these mostly marine animals can be very useful molecular probes for the analysis of ion channels involved in electrical signaling, immune responses and other signal transduction processes of biomedical interest. For instance, anaphylaxis was discovered by Richet (1905) during experiments with sea anemone and hydrozoan tentacular extracts. Similarly, it has recently been shown that a toxin from another sea anemone is able to potently inhibit T-lymphocyte proliferation in models of certain autoimmune diseases. Thus, these natural substances continue to be of relevance for understanding and treating human diseases. In addition to introducing phylum Cnidaria (Coelenterata), we provide a short history of early (until about 1990) research on cnidarian toxins and venoms, to provide a perspective for appreciating the scientific advances of the past two decades that are summarized in the ensuing 19 papers in this special Toxicon issue.


Assuntos
Cnidários/química , Venenos de Cnidários , Citotoxinas , Neurotoxinas , Animais , Cnidários/classificação , Cnidários/fisiologia , Venenos de Cnidários/química , Venenos de Cnidários/isolamento & purificação , Venenos de Cnidários/toxicidade , Citotoxinas/química , Citotoxinas/isolamento & purificação , Citotoxinas/toxicidade , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/toxicidade , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Neurotoxinas/toxicidade , Filogenia , Especificidade da Espécie
16.
J Immunol ; 183(5): 3278-84, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19641134

RESUMO

The serine-threonine kinase RIP1 was originally identified through its ability to bind to the death domain of Fas (CD95). RIP1 has been shown to be recruited to the Fas death-inducing signaling complex (DISC) and is required for the induction of necrotic cell death. In this study, we show that in Jurkat T lymphocytes, RIP1 is also necessary for the most efficient activation of downstream caspases by Fas when treated with membrane-bound Fas ligand, but not with agonistic Abs or cross-linked soluble Fas ligand. RIP1 participates in the Fas-associated death domain protein-mediated recruitment of caspase-8 to the Fas receptor complex in a manner that promotes caspase-8 activation. Cross-linking Abs, such as CH11, bypass the requirement for RIP1 in caspase activation by initiating larger, though less efficient, DISC complexes, while membrane-bound Fas ligand initiates a smaller but more efficient DISC that functions, in part, by effectively incorporating more RIP1 into the complex. Consequently, RIP1 is likely a more integral part of physiological signaling through the Fas/CD95 receptor complex than previously recognized; at least when the signal is mediated by full-length membrane-bound FasL. Cross-linked soluble FasL, which also occurs physiologically, behaves similarly to the CH11 Ab, and may therefore be more likely to initiate nonapoptotic Fas signaling due to less RIP1 in the receptor complex. Thus, agonists that bind the same Fas receptor initiate mechanistically distinct pathways resulting in differential cytotoxicity.


Assuntos
Caspase 8/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteína Ligante Fas/metabolismo , Proteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteínas de Ligação a RNA/fisiologia , Anticorpos/metabolismo , Morte Celular/imunologia , Reagentes para Ligações Cruzadas/metabolismo , Citotoxicidade Imunológica , Ativação Enzimática/imunologia , Proteína Ligante Fas/imunologia , Proteína Ligante Fas/toxicidade , Humanos , Imunidade Inata , Imunoglobulina M/metabolismo , Células Jurkat , Proteínas de Membrana/imunologia , Proteínas de Membrana/toxicidade , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Oligopeptídeos , Peptídeos/imunologia , Proteínas de Ligação a RNA/metabolismo , Receptor fas/agonistas , Receptor fas/imunologia , Receptor fas/metabolismo
17.
Vaccine ; 26(48): 6143-50, 2008 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-18804135

RESUMO

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, PfAMA1 ectodomain (amino acid 25-545, FVO strain) was produced in Pichia pastoris by 35L scale fed batch fermentation under current Good Manufacturing Practice (cGMP). Fermentation was followed by a three-step chromatographic purification procedure resulting in a yield of 5.8g of purified protein. As judged by size exclusion chromatography, the cGMP-product comprised >95% PfAMA1 monomer, the remainder being predominantly PfAMA1 dimer. In SDS-PAGE two main bands of 68 and 70kDa and some minor bands were evident. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident; less than 15% of the protein had this internal cleavage. By mass-spectrometric analysis, all bands analyzed in overloaded SDS-PAGE gels comprised PfAMA1 derived products. The protein was quantitatively bound by immobilized 4G2, a monoclonal antibody reactive with a reduction sensitive conformational determinant. The lyophilized product was stable for over 1 year. Immunopotency did not diminish, and storage did not lead to alterations in the behaviour of the protein upon formulation with adjuvants selected for Phase I clinical evaluation. These formulations also showed no pharmacotoxicity in rabbits. The final product conformed to preset criteria and was judged suitable for use in human clinical trials.


Assuntos
Antígenos de Protozoários/biossíntese , Indústria Farmacêutica/normas , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/normas , Proteínas de Membrana/biossíntese , Proteínas de Membrana/normas , Pichia/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/normas , Adjuvantes Imunológicos , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/toxicidade , Western Blotting , Clonagem Molecular , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Fermentação , Liofilização , Cobaias , Vacinas Antimaláricas/toxicidade , Masculino , Espectrometria de Massas , Proteínas de Membrana/toxicidade , Camundongos , Dados de Sequência Molecular , Pichia/metabolismo , Plasmodium falciparum/imunologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/toxicidade , Controle de Qualidade , Coelhos , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/normas , Vacinas Sintéticas/toxicidade
18.
PLoS One ; 3(8): e2927, 2008 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-18698348

RESUMO

Familial Danish Dementia (FDD) is an autosomal disease, which is distinguished by gradual loss of vision, deafness, progressive ataxia and dementia. Cataract is the first manifestation of the disease. In this article, we demonstrate a specific correlation between the poisoning of the chaperone activity of the rat eye lens alpha-crystallins, loss of lens transparency in organ culture by the pathogenic form of the Danish dementia peptide, i.e. the reduced Danish dementia peptide (redADan peptide), by a combination of ex vivo, in vitro, biophysical and biochemical techniques. The interaction of redADan peptide and lens crystallins are very specific when compared with another chaperone, HSP-70, underscoring the specificity of the pathogenic form of Danish dementia peptide, redADan, for the early onset of cataract in this disease.


Assuntos
Catarata/complicações , Demência/complicações , Cristalino/patologia , alfa-Cristalinas/fisiologia , Envelhecimento , Peptídeos beta-Amiloides/toxicidade , Animais , Ataxia/complicações , Modelos Animais de Doenças , Humanos , Glicoproteínas de Membrana , Proteínas de Membrana/toxicidade , Chaperonas Moleculares/fisiologia , Doenças Neurodegenerativas/epidemiologia , Ratos , Transtornos da Visão/complicações
19.
PLoS One ; 3(8): e2940, 2008 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-18698359

RESUMO

BACKGROUND: Apical Membrane Antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. This is the first reported use in humans of an investigational vaccine, AMA1-C1/Alhydrogel, with the novel adjuvant CPG 7909. METHODS: A phase 1 trial was conducted at the University of Rochester with 75 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine. Participants were sequentially enrolled and randomized within dose escalating cohorts to receive three vaccinations on days 0, 28 and 56 of either 20 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 15), 80 microg of AMA1-C1/Alhydrogel (n = 30), or 80 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 30). RESULTS: Local and systemic adverse events were significantly more likely to be of higher severity with the addition of CPG 7909. Anti-AMA1 immunoglobulin G (IgG) were detected by enzyme-linked immunosorbent assay (ELISA), and the immune sera of volunteers that received 20 microg or 80 microg of AMA1-C1/Alhydrogel+CPG 7909 had up to 14 fold significant increases in anti-AMA1 antibody concentration compared to 80 microg of AMA1-C1/Alhydrogel alone. The addition of CPG 7909 to the AMA1-C1/Alhydrogel vaccine in humans also elicited AMA1 specific immune IgG that significantly and dramatically increased the in vitro growth inhibition of homologous parasites to levels as high as 96% inhibition. CONCLUSION/SIGNIFICANCE: The safety profile of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine is acceptable, given the significant increase in immunogenicity observed. Further clinical development is ongoing. TRIAL REGISTRATION: ClinicalTrials.gov NCT00344539.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/toxicidade , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Adolescente , Adulto , Hidróxido de Alumínio/toxicidade , Animais , Antígenos de Protozoários/toxicidade , Humanos , Proteínas de Membrana/imunologia , Proteínas de Membrana/toxicidade , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/toxicidade , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/toxicidade , Segurança
20.
Toxicon ; 52(2): 285-92, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18602941

RESUMO

Previous in vivo study demonstrated that betagamma-CAT, a newly identified non-lens betagamma-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, possessed potent lethal toxicity on mammals resulted from hypotension and cardiorespiratory arrest. However, the mechanism of cardiac dysfunction induced by the protein is unknown. Here, we report that betagamma-CAT, with dosages of 0.8-3.0 nM, elicited an acute negative inotropic effect in isolated rabbit heart Langendorff preparations, which mimicked acute heart failure. In addition, the effect of betagamma-CAT on the hearts was mediated by endothelium-dependent coronary vasoconstriction (P<0.01, compared between endothelium-intact and removal hearts). After betagamma-CAT (3.0 nM) treatment, the positive signal of tumor necrosis factor-alpha (TNF-alpha) was detected mainly around the endothelial cell layer as detected by in situ indirect immunofluorescence, indicating that the release of TNF-alpha occurred. At the same time, a rapid TNF-alpha release was detected in primary cultured rabbit endocardial endothelial cells (REECs) treated with betagamma-CAT. After addition of betagamma-CAT (3.0 nM) for 10 min and 30 min, the TNF-alpha levels were increased to 57.33+/-3.22 pg/ml and 60.00+/-5.35 pg/ml (P<0.05, compared with the control values of 21.67+/-3.45 pg/ml and 33.70+/-6.24 pg/ml, respectively). At high concentrations, betagamma-CAT interfered with the cell viability of REECs (CC(50) about 25 nM). Taken together, betagamma-CAT was able to induce acute myocardial depression and the toxic effect might be partially explained by the release of TNF-alpha. The finding provides new information to understand the patho-physiological roles of non-lens betagamma-crystallins and trefoil factors.


Assuntos
Anuros/metabolismo , Cristalinas/toxicidade , Células Endoteliais/efeitos dos fármacos , Coração/efeitos dos fármacos , Proteínas de Membrana/toxicidade , Contração Miocárdica/efeitos dos fármacos , Peptídeos/toxicidade , Doença Aguda , Sequência de Aminoácidos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cristalinas/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Endotélio Vascular/lesões , Endotélio Vascular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/fisiopatologia , Masculino , Proteínas de Membrana/química , Dados de Sequência Molecular , Contração Miocárdica/fisiologia , Peptídeos/química , Coelhos , Pele/metabolismo , Fator Trefoil-2 , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo
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