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1.
FEMS Yeast Res ; 13(7): 659-73, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23859640

RESUMO

The immunobiological efficacy of synthetically prepared mannooligosaccharides and a glucooligosaccharide mimicking the structure of Candida albicans cell wall glycans was assessed in vivo and in vitro to exploit immune responses. The exposure of mice splenocytes to BSA-based conjugates of synthetic oligomannosides and oligoglucoside revealed intense influence on T-cell subset polarization. The conjugates biased the immune responses towards Th1 and Th17 with respect to the prevalence of interferon-gamma (IFN-γ) and interleukin (IL)-17 (IL-17) over IL-4 and IL-10 levels. The inflammatory activity of the conjugates has been evaluated based on the induction of pro-inflammatory cytokines. Postvaccination, antimannooligosaccharide and antiglucooligosaccharide antisera were subjected to an evaluation of the structure-immunomodulation activity relationship. Clinical isolates of C. albicans CCY 29-3-32 and C. albicans CCY 29-3-164 were applied to study interactions between Candida cells and anti-oligosaccharide antibodies. In situ recognition of parietal oligomannosyl and oligoglucosyl sequences in C. albicans cell wall by the antisera raised against BSA-based conjugates of synthetic oligomannosides and oligoglucoside revealed the effective recognition of specific distribution of natural oligosaccharide sequences in the cell wall of C. albicans serotype A. With respect to these results, it can be concluded that new, synthetically prepared oligosaccharides mimicking Candida cell wall structures represent prospective immunobiologically effective components for further immunopharmacologically relevant Candida vaccine design.


Assuntos
Antígenos de Fungos/imunologia , Candida albicans/imunologia , Parede Celular/imunologia , Interações Hospedeiro-Patógeno , Oligossacarídeos/imunologia , Animais , Anticorpos Antifúngicos/imunologia , Candida albicans/química , Parede Celular/química , Citocinas/metabolismo , Camundongos , Oligossacarídeos/síntese química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
2.
Bioconjug Chem ; 22(12): 2507-18, 2011 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-22035070

RESUMO

A panel of phosphorescent oligoarginine conjugates of tetracarboxylic Pt(II)-coproporphyrin I dye (PtCP), monosubstituted with long peptides or tetra-substituted with short peptides and having different linkers and peripheral groups, is described. Their photophysical properties, cell loading efficiency, and mechanisms of transport into the cell were investigated and compared. The conjugates were seen to rely on endocytotic mechanisms of cell entry, which are different from that of the unconjugated oligoarginine peptide, and show diverse patterns of intracellular distribution. On the basis of this study, the tetra-substituted PtCP conjugate displaying whole cell distribution was selected for the sensing of intracellular O(2). This probe has been tested in biological experiments on a fluorescence plate reader, including the monitoring of in situ oxygenation of respiring cells and their responses to metabolic stimulation. Similar conjugates of the phosphorescent Pd(II)-coprorphyrin and fluorescent coproporphyrin-ketone were also synthesized and assessed for the sensing of low levels intracellular O(2) and ratiometric pH-sensing, respectively. The results produced and the structure-activity relationships determined can facilitate the rational design of new bioconjugates of porphyrin dyes tailored to specific applications.


Assuntos
Arginina/química , Técnicas Biossensoriais , Coproporfirinas/química , Corantes Fluorescentes/química , Oligopeptídeos/química , Oxigênio/análise , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Técnicas Biossensoriais/métodos , Linhagem Celular , Linhagem Celular Tumoral , Coproporfirinas/metabolismo , Endocitose , Corantes Fluorescentes/metabolismo , Humanos , Medições Luminescentes/métodos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Ratos
3.
FEBS J ; 277(22): 4651-61, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20883447

RESUMO

Research on cell-penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans-activator of transcription (TAT)-derived peptides and, to a lesser extent, on proline-rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen-sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15-24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH-SY5Y and HeLa, via cell-type-dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time-resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen-sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O(2) levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.


Assuntos
Anti-Infecciosos/metabolismo , Técnicas Biossensoriais , Corantes Fluorescentes/metabolismo , Oxigênio/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Anti-Infecciosos/química , Linhagem Celular , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Fragmentos de Peptídeos/genética , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Ratos
4.
Luminescence ; 18(3): 182-92, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12830817

RESUMO

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin-I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies.


Assuntos
Coproporfirinas/química , Imunoconjugados/química , Indicadores e Reagentes/síntese química , Luminescência , Paládio/química , Animais , Anticorpos Monoclonais/química , Coproporfirinas/síntese química , Reagentes de Ligações Cruzadas/química , Fluorometria/métodos , Imunoglobulina G/química , Isotiocianatos/química , Camundongos , Modelos Químicos , Estrutura Molecular , Coloração e Rotulagem
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