RESUMO
The effects of leptin-replacement therapy on the plasma proteome of three unique adults with genetically based leptin deficiency were studied longitudinally during the course of recombinant human leptin-replacement treatment. Quantitative proteomics analysis was performed in plasma samples collected during four stages: before leptin treatment was initiated, after 1.5 and 6 years of leptin-replacement treatment, and after 7 weeks of temporary interruption of leptin-replacement therapy. Of 500 proteins reliably identified and quantitated in those four stages, about 100 were differentially abundant twofold or more in one or more stages. Synchronous dynamics of abundances of about 90 proteins was observed reflecting both short- and long-term effects of leptin-replacement therapy. Pathways and processes enriched with overabundant synchronous proteins were cell adhesion, cytoskeleton remodeling, cell cycle, blood coagulation, glycolysis, and gluconeogenesis. Plausible common regulators of the above synchronous proteins were identified using transcription regulation network analysis. The generated network included two transcription factors (c-Myc and androgen receptor) that are known to activate each other through a double-positive feedback loop, which may represent a potential molecular mechanism for the long-term effects of leptin-replacement therapy. Our findings may help to elucidate the effects of leptin on insulin resistance.
Assuntos
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Leptina/deficiência , Leptina/genética , Adulto , Proteínas Sanguíneas/análise , Adesão Celular/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Feminino , Terapia Genética , Gluconeogênese/genética , Glicólise/genética , Terapia de Reposição Hormonal , Humanos , Resistência à Insulina , Masculino , Redes e Vias Metabólicas/genética , Proteoma/análise , Proteoma/metabolismoRESUMO
The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-A pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Teóricos , Dados de Sequência Molecular , Peso Molecular , Redes Neurais de Computação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Mapeamento de Peptídeos/estatística & dados numéricos , Proteômica/métodos , Proteômica/estatística & dados numéricos , TripsinaRESUMO
Interaction of potent photodynamic agents, sulfonated aluminum phthalocyanines (AlPcSn where n is a number of sulfonic groups), with biological membranes was studied here using model systems: sensitized photoinactivation of gramicidin channels in planar lipid bilayers and adsorption on lipid monolayers. Fluoride anions known to form complexes with aluminum were found to inhibit both the adsorption of aluminum phthalocyanines on lipid monolayers, as measured with a Langmuir trough by surface pressure and surface potential changes, and photodynamic efficacy of the dyes, as studied by gramicidin channel photoinactivation. The similar effects were caused by the alkalinization of the medium. Fluoride anions appeared to be much more effective in the case of AlPcS4 as compared to AlPcS3. The suppression of the photodynamic potency of aluminum phthalocyanines was attributed to desorption of the dyes from lipid bilayers induced by fluoride or hydroxyl ions. With AlPcS4 an enhancement of the dye aggregation leading to a decrease in the sensitizing activity was probably involved in the fluoride effect as revealed by absorption and fluorescence spectral measurements. Capillary electrophoresis was employed to understand the mechanism of the dye desorption. The results of these experiments indicated that the reduction in the membrane affinity was associated with an increase in the negative charge of the dye molecules due to the binding of fluoride or hydroxyl ions.
Assuntos
Fluoretos/farmacologia , Gramicidina/efeitos da radiação , Gramicidina/química , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Indóis/farmacologia , Isoindóis , Bicamadas Lipídicas/química , Fotoquímica , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologiaRESUMO
The complexes of Fe(III), Co(III), Mn(III), Al(III), Cu(II), Ni(II), Cd(II) and Zn(II) with N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) were separated by ion exchange in different modes: ion chromatography (IC) and ion electrokinetic chromatography (IEKC). In column IC these complexes were separated on an IonPac AS4a anion-exchange column (Dionex, USA). Parameters of the background electrolyte that were examined in IEKC mode include polymer, competing ion concentration and pH. The use of poly(diallyldimethylammonium chloride) (PDADMACl) as a modifier in IEKC provides separation selectivity only slightly different from that observed in IC on the IonPac AS4a column. Optimal separation conditions were found to be: 0.1 mM HBED, 50 mM PDADMAOH, 10 mM Na2 B4 O7, pH adjusted to 10 with acetic acid. The use of an aromatic ligand allowed a 10-fold decrease in detection limits of metal ions in comparison with previously studied EDTA. A separation efficiency up to 400,000 theoretical plates was demonstrated for IEKC.
Assuntos
Cromatografia Capilar Eletrocinética Micelar/métodos , Ácido Edético/isolamento & purificação , Metais/química , Ânions , Ácido Edético/análogos & derivados , Ácido Edético/química , Concentração de Íons de Hidrogênio , Sensibilidade e EspecificidadeRESUMO
Two aromatic polyaminocarboxylate ligands, ethylenediaminedi(o-hydroxyphenylacetic acid) (EDDHA) and N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED), were applied for the separation of transition and heavy metal ions by the ion-exchange variant of electrokinetic chromatography. EDDHA structure contains two chiral carbon centers. It makes it impossible to use the commercially available ligand. All the studied metal ions showed two peaks, which correspond to meso and rac forms of the ligand. The separation of metal-HBED chelates was performed using poly(diallyldimethylammonium) polycations in mixed acetate-hydroxide form. Simultaneous separation of nine single- and nine double-charged HBED chelates, including In(III), Ga(III), Co(II)-(III) and Mn(II)-(III) pairs demonstrated the efficiency of 40,000-400,000 theoretical plates. The separation of Co(III), Fe(III) complexes with different arrangements of donor groups and oxidation of Co(II), Mn(H), Fe(II) ions in reaction with HBED have been discussed.
Assuntos
Ácido Edético/análogos & derivados , Eletroforese Capilar/métodos , Etilenodiaminas/química , Metais/análise , Cátions , Ácido Edético/química , OxirreduçãoRESUMO
An aliphatic ionene with hydroxyl group (2HP-8 ionene), mixed aliphatic-aromatic ionenes (3-X and 6-X ionenes), aromatic ionene (Ph-X ionene), and viologen (Dp-X ionene) - polymers with quaternary nitrogen atoms in the main chain served as modifiers in synthesising polyelectrolyte sorbents for ion chromatography. The selectivity of produced and several previously prepared anion exchangers was compared with those of aliphatic ionenes. It was found that aromatic ionenes having a rigid structure of polymer chains are similar to their aliphatic analogues with shorter chains with a high charge density. Polyelectrolyte sorbents based on aromatic ionenes show higher selectivity to aromatic acids (e.g., 1-naphthalenesulfonic acid) as compared with aliphatic ionenes due to specific pi-pi interactions.
Assuntos
Cromatografia por Troca Iônica/métodos , Eletrólitos/química , Polímeros/química , Ânions , Cátions , Nitrogênio/químicaRESUMO
The complexes of Mn2+, Cd2+, Fe3+, Pb2+, Ni2+, Co2+, Zn2+ and Cu2+ with EDTA and cyclohexane-1,2-diaminetetraacetic acid (CDTA) were separated and detected in column and ion electrokinetic chromatography with suppressed conductivity and direct UV detection, respectively. In column ion chromatography (IC) these complexes were separated on an IonPac AS4A anion-exchange column (Dionex, USA). Parameters of carrier electrolyte, which were examined in the ion electrokinetic chromatography (IEKC) mode, include polymer and sulfate concentrations. In IEKC separation selectivity of complexes with poly(diallyldimethylammonium) cation as modifier is similar as for an IonPac AS4A column both for EDTA and CDTA chelates. It was shown that the ion-exchange capacity of the electrokinetic system is more than 100-times lower than the capacity of the IC column for the same peak resolution. In comparison with column main advantages of electrokinetic version are high separation efficiency (220,000-390,000 theoretical plates) and the absence of the analyte interaction with the sorbent matrix.