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1.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202728

RESUMO

The prognosis of patients with oral squamous carcinoma (OSCC) largely depends on the stage at diagnosis, the 5-year survival rate being approximately 30% for advanced tumors. Early diagnosis, including the detection of lesions at risk for malignant transformation, is crucial for limiting the need for extensive surgery and for improving disease-free survival. Saliva has gained popularity as a readily available source of biomarkers (including cytokines) useful for diagnosing specific oral and systemic conditions. Particularly, the close interaction between oral dysplastic/neoplastic cells and saliva makes such fluid an ideal candidate for the development of non-invasive and highly accurate diagnostic tests. The present review has been designed to answer the question: "Is there evidence to support the role of specific salivary cytokines in the diagnosis of OSCC?" We retrieved 27 observational studies satisfying the inclusion and exclusion criteria. Among the most frequent cytokines investigated as candidates for OSCC biomarkers, IL-6, IL-8, TNF-α are present at higher concentration in the saliva of OSCC patients than in healthy controls and may therefore serve as basis for the development of rapid tests for early diagnosis of oral cancer.


Assuntos
Biomarcadores , Carcinoma de Células Escamosas/metabolismo , Citocinas/metabolismo , Neoplasias Bucais/metabolismo , Saliva/metabolismo , Biomarcadores Tumorais , Carcinoma de Células Escamosas/diagnóstico , Humanos , Biópsia Líquida/métodos , Neoplasias Bucais/diagnóstico , Prognóstico
2.
Biochim Biophys Acta Gen Subj ; 1865(5): 129843, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33444726

RESUMO

Among their various functions, the members of the cerato-platanin family can stimulate plants' defense responses and induce resistance against microbial pathogens. Recent results suggest that conserved loops, also involved in chitin binding, might be a structural motif central for their eliciting activity. Here, we focus on cerato-platanin and its orthologous cerato-populin, searching for a rationale of their diverse efficiency to elicit plants' defense and to interact with oligosaccharides. A 3D model of cerato-populin has been generated by homology modeling using the NMR-derived cerato-platanin structure as template, and it has been validated by fitting with residual dipolar couplings. Loops ß1-ß2 and ß2-ß3 have been indicated as important for some CPPs members to express their biological function. When compared to cerato-platanin, in cerato-populin they present two mutations and an insertion that significantly modify their electrostatic surface. NMR relaxation experiments point to a reduced conformational plasticity of cerato-populin loops with respect to the ones of cerato-platanin. The different electrostatic surface of the loops combined with a distinct network of intra-molecular interactions are expected to be factors that, by leading to a diverse spatial organization and dissimilar collective motions, can regulate the eliciting efficacy of the two proteins and their affinity for oligosaccharides.


Assuntos
Ceratocystis/metabolismo , Proteínas Fúngicas/metabolismo , Oligossacarídeos/metabolismo , Doenças das Plantas/microbiologia , Ceratocystis/química , Proteínas Fúngicas/química , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
3.
Metabolites ; 10(8)2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32781584

RESUMO

The detection of salivary molecules associated with pathological and physiological alterations has encouraged the search of novel and non-invasive diagnostic biomarkers for oral health evaluation. While genomic, transcriptomic, and proteomic profiles of human saliva have been reported, its metabolic composition is a topic of research: metabolites in submandibular/sublingual saliva have never been analyzed systematically. In this study, samples of whole, parotid, and submandibular/sublingual saliva from 20 healthy donors, without dental or periodontal diseases, were examined by nuclear magnetic resonance. We identified metabolites which are differently distributed within the three saliva subtypes (54 in whole, 49 in parotid, and 36 in submandibular/sublingual saliva). Principal component analysis revealed a distinct cluster for whole saliva and a partial overlap for parotid and submandibular/sublingual metabolites. We found exclusive metabolites for each subtype: 2-hydroxy-3-methylvalerate, 3-methyl-glutarate, 3-phenylpropionate, 4-hydroxyphenylacetate, 4-hydroxyphenyllactate, galactose, and isocaproate in whole saliva; caprylate and glycolate in submandibular/sublingual saliva; arginine in parotid saliva. Salivary metabolites were classified into standard and non-proteinogenic amino acids and amines; simple carbohydrates; organic acids; bacterial-derived metabolites. The identification of a salivary gland-specific metabolic composition in healthy people provides the basis to invigorate the search for salivary biomarkers associated with oral and systemic diseases.

4.
Data Brief ; 29: 105355, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32190721

RESUMO

The presented data were obtained with the lipocalin allergen Mus m 1.0102 and its cysteine mutants MM-C138A, MM-C157A and MM-C138,157A, whose structural features and unfold reversibility investigations are presented in the research article entitled "The allergen Mus m 1.0102: cysteine residues and molecular allergology" [1]. The data were obtained by means of a Dynamic Light Scattering-based thermal stability assay, a Fluorescence-based ligand-binding assay and a basophil degranulation test, and describe proteins' fold stability, ligand binding ability and allergenic potential, respectively. Analysis of the collected data produced the temperatures corresponding to the onset of the protein unfolding, the dissociation constants for N-Phenyl-1-naphthylamine ligand and the profiles of ß-hexosaminidase release from RBL SX-38 cells, sensitized with the serum of selected allergic patients and incubated with increasing antigens concentrations. These data allow for comparison of the lipocalin allergen Mus m 1.0102 with its conserved cysteines mutants and, with regard to their potential application in allergy diagnostics and immunotherapy, they contribute to the process of recombinant allergen characterization and standardization.

5.
Mol Immunol ; 120: 1-12, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32044430

RESUMO

Mus m 1.0102 is a member of the mouse Major Urinary Protein family, belonging to the Lipocalins superfamily. Major Urinary Proteins (MUPs) are characterized by highly conserved structural motifs. These include a disulphide bond, involved in protein oxidative folding and protein structure stabilization, and a free cysteine residue, substituted by serine only in the pheromonal protein Darcin (MUP20). The free cysteine is recognized as responsible for the onset of inter- or intramolecular thiol/disulphide exchange, an event that favours protein aggregation. Here we show that the substitution of selected cysteine residues modulates Mus m 1.0102 protein folding, fold stability and unfolding reversibility, while maintaining its allergenic potency. Recombinant allergens used for immunotherapy or employed in allergy diagnostic kits require, as essential features, conformational stability, sample homogeneity and proper immunogenicity. In this perspective, recombinant Mus m 1.0102 might appear reasonably adequate as lead molecule because of its allergenic potential and thermal stability. However, its modest resistance to aggregation renders the protein unsuitable for pharmacological preparations. Point mutation is considered a winning strategy. We report that, among the tested mutants, C138A mutant acquires a structure more resistant to thermal stress and less prone to aggregation, two events that act positively on the protein shelf life. Those features make that MUP variant an attractive lead molecule for the development of a diagnostic kit and/or a vaccine.


Assuntos
Alérgenos/química , Alérgenos/imunologia , Proteínas/química , Proteínas/imunologia , Alérgenos/genética , Substituição de Aminoácidos , Animais , Linhagem Celular , Cisteína/química , Humanos , Testes Imunológicos , Ligantes , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier
6.
Int J Mol Sci ; 19(12)2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30487461

RESUMO

The synthetic peptide T11F (TCRVDHRGLTF), derived from the constant region of human IgM antibodies, proved to exert a significant activity in vitro against yeast strains, including multidrug resistant isolates. Alanine substitution of positively charged residues led to a decrease in candidacidal activity. A more dramatic reduction in activity resulted from cysteine replacement. Here, we investigated the conformational properties of T11F and its alanine-substituted derivatives by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Peptide interaction with Candida albicans cells was studied by confocal and scanning electron microscopy. T11F and most of its derivatives exhibited CD spectra with a negative band around 200 nm and a weaker positive band around 218 nm suggesting, together with NMR coupling constants, the presence of a polyproline II (PPII) helix, a conformational motif involved in a number of biological functions. Analysis of CD spectra revealed a critical role for phenylalanine in preserving the PPII helix. In fact, only the F11A derivative presented a random coil conformation. Interestingly, the loss of secondary structure influenced the rate of killing, which turned out to be significantly reduced. Overall, the obtained results suggest that the PPII conformation contributes in characterising the cell penetrating and fungicidal properties of the investigated peptides.


Assuntos
Anticorpos/química , Peptídeos Penetradores de Células/química , Fungicidas Industriais/química , Peptídeos/química , Candida albicans/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Dicroísmo Circular , Fungicidas Industriais/farmacologia , Microscopia Confocal , Microscopia Eletrônica de Varredura , Ressonância Magnética Nuclear Biomolecular , Peptídeos/farmacologia
7.
Ann Hematol ; 97(10): 1909-1917, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29881883

RESUMO

The upholding of red blood cells (RBC) quality and the removal of leukocytes are two essential issues in transfusion therapy. Leukodepletion provides optimum results, nonetheless there are cases where irradiation is recommended for some groups of hematological patients such as the ones with chronic graft-vs-host disease, congenital cellular immunodeficiency, and hematopoietic stem cell transplant recipients. The European guidelines suggest irradiation doses from 25 to 50 Gray (Gγ). We evaluated the effect of different prescribed doses (15 to 50 Gγ) of X-ray irradiation on fresh leukodepleted RBCs bags using a novel protocol that provides a controlled irradiation. Biochemical assays integrated with RBCs metabolome profile, assessed by nuclear magnetic resonance spectroscopy, were performed on RBC units supernatant, during 14 days storage. Metabolome analysis evidenced a direct correlation between concentration increase of three metabolites, glycine, glutamine and creatine, and irradiation dose. Higher doses (35 and 50 Gγ) effect on RBC mean corpuscular volume, hemolysis, and ammonia concentration are considerable after 7 and 14 days of storage. Our data show that irradiation with 50 Gγ should be avoided and we suggest that 35 Gγ should be the upper limit. Moreover, we suggest for leukodepleted RBCs units the irradiation with the prescribed dose of 15 Gγ, value at center of bag, and ranging between 13.35-15 Gγ, measured over the entire bag volume, may guarantee the same benefits of a 25 Gγ dose assuring, in addition, a better quality of RBCs.


Assuntos
Eritrócitos/metabolismo , Eritrócitos/efeitos da radiação , Metaboloma/efeitos da radiação , Raios X , Adulto , Preservação de Sangue/métodos , Transplante de Medula Óssea/métodos , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Transfusão de Eritrócitos/métodos , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Leucemia/terapia , Masculino , Pessoa de Meia-Idade , Doses de Radiação
8.
Biopolymers ; 2018 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-29359791

RESUMO

Sticholysin II (StII) is a pore-forming actinoporin from the sea anemone Stichodactyla helianthus. A mechanistic model of its action has been proposed: proteins bind to cell membrane, insert their N-termini into the lipid core and assemble into homo-tetramer pores responsible for host-cell death. Because very likely the first 10 residues of StII N-terminus are critical for membrane penetration, to dissect the molecular details of that functionality, we studied two synthetic peptides: StII1-30 and StII16-35 . They show diverse haemolytic and candidacidal activity that correlate with distinct orientations in SDS micelles. NMR shows that StII1-30 partly inserts into the micelle, while StII16-35 lays on the micelle surface. These results justify the diverse concentration dependence of their candidacidal activity supposing a different mechanism of action and providing new hints on StII lytic activity at molecular level. Biotechnological application of these peptides, focused on the development of therapeutic immunocomplexes, may be envisaged.

9.
J Pharm Biomed Anal ; 147: 485-492, 2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-28648253

RESUMO

BACKGROUND: Time dependent quantification of endogenous metabolites in biological samples (blood, urine, biological tissues extracts) in normal and pathological conditions as well as following therapeutic protocols is well established. In the clinical practice, such a dynamic flux of information allows the physician to identify and appreciate alterations associated to biochemical pathways of specific organs. In the years, many biochemical assays have been developed to detect, selectively, this vast array of molecules. METHODS: The Proton Nuclear Magnetic Resonance (1H NMR) spectrum allows the identification and quantification of more than 30 RBC-associated metabolites with minimum manipulation of the sample. To validate the use of 1H NMR spectroscopy for quality control purposes in transfusion medicine, a series of statistical tools have been employed to analyse and compare accuracy and precision of the 1H NMR results with respect to the ones obtained by standard biochemical assays. RESULTS: Among the many metabolites that can be detected and quantified by 1H NMR spectroscopy we selected creatinine and lactate, since they are routinely quantified by standard biochemical assays and because they are characterized by a wide concentration dynamic range. We show that 1D 1H NMR spectroscopy is an accurate a precise method for metabolite quantification. CONCLUSION: These results validate the use of 1H NMR spectroscopy in transfusion medicine as a method to evaluate the quality of RBC packed units and to develop novel and more efficient RBCs storage protocols.


Assuntos
Transfusão de Sangue/normas , Eritrócitos/química , Espectroscopia de Ressonância Magnética/normas , Controle de Qualidade , Adulto , Bioensaio/normas , Bioensaio/estatística & dados numéricos , Transfusão de Sangue/estatística & dados numéricos , Humanos , Espectroscopia de Ressonância Magnética/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Prótons
10.
Biochim Biophys Acta ; 1864(11): 1548-57, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27519162

RESUMO

BACKGROUND: The species Mus musculus experiences an obligate proteinuria: predominant are the Major Urinary Proteins (MUPs), that, collectively known as the major mouse allergen Mus m 1, are among the most important aeroallergens for mouse allergic patients. The production of a soluble and stable hypoallergenic form of Mus m 1 is essential for the development of immunotherapeutic protocols to treat allergic symptoms. METHODS: We introduced the substitution C138S in recombinant Mus m 1.0102, an allergenic isoform of Mus m 1. Solubility, conformation, stability and ability to refold after chemical denaturation were investigated with dynamic light scattering, circular dichroism, fluorescence and NMR spectroscopy. An in vitro degranulation assay was used to evaluate the protein allergenic potential, and compare it with Mus m 1.0102 and with an hypoallergenic variant bearing the substitution Y120A. RESULTS: Mus m 1.0102-C138S retains a native-like fold revealing, however, local conformational alterations that influence some of its physical and allergenic properties: it is monodispersed, thermostable up to 56°C, able to reversibly unfold and it exhibits an enhanced allergenicity. CONCLUSIONS: The unique free thiol group affects the solution structural stability of the native protein. Because the mutant C138S does not aggregate over time it is a good lead protein to develop diagnostic and therapeutic applications. GENERAL SIGNIFICANCE: We elucidated the relationship between unfolding reversibility and sulphydryl reactivity. We ascribed the enhanced allergenicity of the mutant C138S to an increased accessibility of its allergenic determinants, an enticing feature to further investigate the structural elements of the allergen-IgE interface.


Assuntos
Alérgenos/química , Asma/induzido quimicamente , Conjuntivite Alérgica/induzido quimicamente , Imunoglobulina E/química , Rinite Alérgica/induzido quimicamente , Adulto , Alérgenos/genética , Alérgenos/imunologia , Substituição de Aminoácidos , Animais , Asma/imunologia , Asma/fisiopatologia , Clonagem Molecular , Conjuntivite Alérgica/imunologia , Conjuntivite Alérgica/fisiopatologia , Feminino , Expressão Gênica , Humanos , Imunoglobulina E/metabolismo , Masculino , Camundongos , Modelos Moleculares , Pichia/genética , Pichia/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Rinite Alérgica/imunologia , Rinite Alérgica/fisiopatologia , Relação Estrutura-Atividade
11.
Front Mol Biosci ; 3: 13, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27148539

RESUMO

Blood transfusion is a fundamental therapy in numerous pathological conditions. Regrettably, many clinical reports describe adverse transfusion's drawbacks due to red blood cells alterations during storage. Thus, the possibility for a blood bank to ameliorate the quality of the erythrocyte concentrates units is crucial to improve clinical results and reduce transfusion adverse occurrences. Leukodepletion is a pre-storage treatment recognized to better preserve the quality of red blood cells with respect to leukoreduction. Aim of this work is to unravel the biochemical and biophysical basis that sustain the good clinical outcomes associated to the use of leukodepleted erythrocytes units. Erythrocytes concentrates were prepared as leukoreduced (n = 8) and pre-storage leukodepleted (n = 8) and then studied during 6 weeks in blood bank conditions. Overall, the data indicate that leukodepletion not only provide red blood cells with an appropriate amount of nutrients for a longer time but also selects red blood cells characterized by a more resilient plasma membrane fit to prolong their viability. We believe these results will stimulate new ideas to further optimize the current storage protocols.

12.
Antimicrob Agents Chemother ; 60(4): 2435-42, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26856836

RESUMO

Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Regiões Determinantes de Complementaridade/farmacologia , Imunoglobulina G/farmacologia , Peptídeos/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antifúngicos/síntese química , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Regiões Determinantes de Complementaridade/química , Humanos , Imunoglobulina G/química , Larva/efeitos dos fármacos , Larva/microbiologia , Testes de Sensibilidade Microbiana , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Peptídeos/síntese química , Fosfatidilserinas/análise , Fosfatidilserinas/metabolismo , Relação Estrutura-Atividade , Análise de Sobrevida
14.
Blood Transfus ; 12(4): 548-56, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24960643

RESUMO

BACKGROUND: Blood transfusion is an established therapeutic practice. The characteristics of blood components at different storage times are expected to affect the efficacy of transfusion therapy. Metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy requires little or no sample treatment and allows identification of more than 50 soluble metabolites in a single experiment. The aim of this study was to assess the metabolic behaviour of red blood cells during 42 days of storage in blood bank conditions. MATERIALS AND METHODS: Red blood cells (RBC), collected from eight healthy male donors, aged 25-50 years, were prepared as prestorage leukoreduced erythrocyte concentrates and stored under standard blood bank conditions. Samples taken at various storage times were separated in two fractions: the supernatant, recovered after centrifugation, and the red blood cell lysate obtained after protein depletion by ultrafiltration. The metabolic profile of the red blood cells was determined from analysis of (1)H-NMR spectra. RESULTS: The red blood cell supernatant was studied to track the consumption of the preservative additives and to detect and quantify up to 30 metabolites excreted by the erythrocytes. The NMR spectra of the RBC lysate provided complementary information on some biochemical pathways and set the basis for building a time-dependent red blood cell metabolic profile. DISCUSSION: We proved the analytical power of (1)H-NMR spectroscopy to study red blood cell metabolism under blood bank conditions. A potential biomarker able to provide information on the level of cellular oxidative stress protection was identified. Our data support the hypothesis that a more detailed knowledge of metabolic modifications during storage opens the way to the development of new and more effective protocols for red blood cell conservation and patient-oriented transfusion therapy.


Assuntos
Preservação de Sangue , Eritrócitos/metabolismo , Estresse Oxidativo , Adulto , Biomarcadores/metabolismo , Eritrócitos/citologia , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Fatores de Tempo
15.
Biomol NMR Assign ; 8(2): 405-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24091893

RESUMO

Plant pathogenic fungi secrete several non-catalytic proteins involved in various aspects of the pathogenesis process. Amongst these, cerato-populin (Pop1) produced by Ceratocystis populicola; a protein orthologous of cerato-platanin (CP), the core member of the CP family. These two proteins interact with host and non-host plants. In plane leaves they induce synthesis of phytoalexins, disruption of intercellular and intracellular leaf tissue, cell plasmolysis, programmed cell death, over-expression of defence-related genes, H2O2 and NO production, activation of MAPK cascade and plant resistance. All these features point to CP and Pop1 as defence inducers, though Pop1 shows a reduced efficiency. Pop1/CP similarity is 73%. CD spectroscopy highlights some secondary structure differences between Pop1 and CP. Indeed, the region between the first two cysteines (C20-C57), that in CP includes the ß2-strand and it is involved in GlcNAc (N-acetyl-D-glucosamine) interaction, in Pop1 is predicted to be fully disordered.


Assuntos
Ascomicetos , Proteínas Fúngicas/química , Ressonância Magnética Nuclear Biomolecular
16.
PLoS One ; 7(3): e34105, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22470523

RESUMO

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.


Assuntos
Anticorpos/química , Antifúngicos/farmacologia , Peptídeos/farmacologia , Animais , Anticorpos/metabolismo , Antifúngicos/química , Antifúngicos/uso terapêutico , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Caspofungina , Cryptococcus neoformans/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Fúngica/efeitos dos fármacos , Equinocandinas/farmacologia , Eritrócitos/efeitos dos fármacos , Feminino , Hemólise , Humanos , Imunoglobulina A/química , Imunoglobulina A/metabolismo , Regiões Constantes de Imunoglobulina , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulina M/química , Imunoglobulina M/metabolismo , Lipopeptídeos , Malassezia/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/uso terapêutico , Triazóis/farmacologia
17.
Biochemistry ; 51(9): 1885-94, 2012 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-22332965

RESUMO

α-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif, including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an α/ß scaffold typical of the members of the α-KTx family. TdK2 and TdK3, all belonging to the same α-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.


Assuntos
Canal de Potássio Kv1.3/química , Venenos de Escorpião/química , Toxinas Biológicas/química , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Canal de Potássio Kv1.3/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Relação Estrutura-Atividade , Toxinas Biológicas/metabolismo
18.
Int Arch Allergy Immunol ; 157(3): 226-37, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22041937

RESUMO

BACKGROUND: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. METHODS: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. RESULTS: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. CONCLUSIONS: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.


Assuntos
Alérgenos/genética , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Imunoterapia , Mutagênese Sítio-Dirigida , Mutação Puntual , Linfócitos T/imunologia , Adulto , Alérgenos/química , Alérgenos/imunologia , Animais , Teste de Degranulação Basófila , Western Blotting , Dicroísmo Circular , Epitopos de Linfócito T/imunologia , Humanos , Hipersensibilidade/diagnóstico , Camundongos , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Vacinas Sintéticas
19.
Future Med Chem ; 3(9): 1209-31, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21806382

RESUMO

The incidence of life-threatening viral and microbial infections has dramatically increased over recent decades. Despite significant developments in anti-infective chemotherapy, many issues have increasingly narrowed the therapeutic options, making it imperative to discover new effective molecules. Among them, small peptides are arousing great interest. This review will focus in particular on a killer peptide, engineered from an anti-idiotypic recombinant antibody that mimics the activity of a wide-spectrum antimicrobial yeast killer toxin targeting ß-glucan cell-wall receptors. The in vitro and in vivo antimicrobial, antiviral and immunomodulatory activities of killer peptide and its ability to spontaneously and reversibly self-assemble and slowly release its active dimeric form over time will be discussed as a novel paradigm of targeted auto-delivering drugs.


Assuntos
Anti-Infecciosos/farmacologia , Sistemas de Liberação de Medicamentos , Fatores Imunológicos/farmacologia , Fatores Matadores de Levedura/farmacologia , Terapia de Alvo Molecular , Anti-Infecciosos/imunologia , Anti-Infecciosos/metabolismo , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/metabolismo , Anticorpos Anti-Idiotípicos/farmacologia , Humanos , Fatores Imunológicos/imunologia , Fatores Imunológicos/metabolismo , Fatores Matadores de Levedura/imunologia , Fatores Matadores de Levedura/metabolismo , Peptídeos/imunologia , Peptídeos/metabolismo , Peptídeos/farmacologia , beta-Glucanas/metabolismo
20.
J Biol Chem ; 286(20): 17560-8, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21454637

RESUMO

Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ψß-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the ß-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ψß-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.


Assuntos
Ascomicetos/química , Proteínas Fúngicas/química , Ascomicetos/genética , Ascomicetos/metabolismo , Sítios de Ligação , Carboidratos/química , Carboidratos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Ligação Proteica , Estrutura Secundária de Proteína
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