RESUMEN
The combinatorial synthesis and biological evaluation of destruxins are described herein. First, the total synthesis of destruxin E was achieved, and its absolute configuration was successfully determined to be (S). In addition, the preparation of a combinatorial library based on the structure of destruxins was carried out by the split-and-pool method. Biological evaluation of the resulting analogs against osteoclast-like multinuclear cells (OCLs) revealed that the N-methyl-alanine residue was crucial to inducing morphological changes in OCLs. In particular, functionalization at the ß-position of the proline (Pro) residue was found to be tolerant of the desired biological activity of destruxin E, suggesting that the ß-position of the Pro residue should be a promising site for the introduction of a chemical tag toward the preparation of a molecular probe.
Asunto(s)
Depsipéptidos/farmacología , Proteínas Fúngicas/farmacología , Osteoclastos/efectos de los fármacos , Técnicas Químicas Combinatorias , Depsipéptidos/síntesis química , Depsipéptidos/química , Proteínas Fúngicas/síntesis química , Proteínas Fúngicas/química , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The solid-phase combinatorial synthesis of cyclodepsipeptide destruxinâ E has been demonstrated. The combinatorial synthesis of cyclization precursors 8 was achieved by using a split and pool method on SynPhase Lanterns. The products were successfully macrolactonized in parallel in the solution phase by using 2-methyl-6-nitrobenzoic anhydride and 4-(dimethylamino)pyridine N-oxide to afford macrolactones 9, and the subsequent formation of an epoxide in the side chain gave 18 member destruxinâ E analogues 6. Biological evaluation of analogues 6 indicated that the N-MeAla residue was crucial to the induction of morphological changes in osteoclast-like multinuclear cells (OCLs). Based on structure-activity relationships, azido-containing analogues 15 were then designed for use as a molecular probe. The synthesis and biological evaluation of analogues 15 revealed that 15 b, in which the Ile residue was replaced with a Lys(N3 ) residue, induced morphological changes in OCLs at a sufficient concentration, and modification around the Ile residue would be tolerated for attachment of a chemical tag toward the target identification of destruxinâ E (1).
Asunto(s)
Anhídridos/química , Óxidos N-Cíclicos/química , Depsipéptidos/síntesis química , Proteínas Fúngicas/síntesis química , Nitrobenzoatos/química , Evolución Biológica , Ciclización , Depsipéptidos/química , Proteínas Fúngicas/química , Técnicas de Síntesis en Fase Sólida , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Total synthesis of biologically active cyclodepsipeptide destruxin E using solid- and solution-phase synthesis is described. The solid-phase synthesis of destruxin E was initially investigated for the efficient synthesis of destruxin analogues. Peptide elongation from polymer-supported ß-alanine was efficiently performed using DIC/HOBt or PyBroP/DIEA, and subsequent cleavage from the polymer-support under weakly acidic conditions furnished a cyclization precursor in moderate yield. Macrolactonization of the cyclization precursor was smoothly performed using 2-methyl-6-nitrobenzoic anhydride (MNBA)/4-(dimethylamino)pyridine N-oxide (DMAPO) to afford macrolactone in moderate yield. Finally, formation of the epoxide in the side chain via three steps provided destruxin E, and the stereochemistry of the epoxide was determined to be S. Its diastereomer, epi-destruxin E, was also synthesized in the same manner used to synthesize the natural product. The stereochemistry of the epoxide was critical for the V-ATPase inhibition; natural product destruxin E exhibited 10-fold more potent V-ATPase inhibition than epi-destruxin E. Next, the scalable synthesis of destruxin E for in vivo study was also performed via solution-phase synthesis. The scalable synthesis of a key component, (S)-HA-Pro-OH, was achieved using osmium-catalyzed diastereoselective dihydroxylation with (DHQD)2PHAL as a chiral ligand; peptide synthesis using Cbz-protected amino acid derivatives furnished the cyclization precursor on a gram-scale. Macrolactonization smoothly provided the macrolactone without forming a dimerized product, even at 6 mM, and the synthesis of destruxin E was achieved via three steps on a gram scale in high purity (>98%).
Asunto(s)
Productos Biológicos/síntesis química , Depsipéptidos/síntesis química , Proteínas Fúngicas/síntesis química , Adenosina Trifosfatasas/antagonistas & inhibidores , Anhídridos/química , Productos Biológicos/farmacología , Catálisis , Óxidos N-Cíclicos/química , Ciclización , Depsipéptidos/farmacología , Compuestos Epoxi/química , Proteínas Fúngicas/farmacología , Lactonas , Nitrobenzoatos/química , Osmio/química , Polímeros/química , Técnicas de Síntesis en Fase Sólida , Estereoisomerismo , beta-Alanina/químicaRESUMEN
Hydrophobins are natural surfactant proteins endowed with exceptional surface activity and film-forming capabilities and their use as effective "fluorine-free fluorosurfactants" has been recently reported. In order to increase their fluorophilicity further, here we report the preparation of a unique fluorous-modified hydrophobin, named F-HFBI. F-HFBI was found to be more effective than its wild-type parent protein HFBI at reducing interface tension of water at both air/water and oil/water interfaces, being particularly effective at the fluorous/water interface. F-HFBI was also found to largely retain the exceptionally good capability of forming strong and elastic films, typical of the hydrophobin family. Further studies by interface shear rheology and isothermal compression, alongside Quartz Crystal Microbalance and Atomic Force Microscopy, demonstrated the tendency of F-HFBI to form thicker films compared to the wild-type protein. These results suggest that F-HFBI may function as an effective compatibilizer for biphasic systems comprising a fluorous phase.
Asunto(s)
Flúor/química , Proteínas Fúngicas/química , Trichoderma/química , Adsorción , Proteínas Fúngicas/síntesis química , Halogenación , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía de Fuerza Atómica , Modelos Moleculares , Tecnicas de Microbalanza del Cristal de Cuarzo , Reología , Tensión Superficial , Agua/químicaRESUMEN
The enormous potential of nanogel scaffolds for protein encapsulation has been widely recognized. However, constructing stable polymeric nanoscale networks in a facile, mild, and controllable fashion still remains a technical challenge. Here, we present a novel nanogel formation strategy using horseradish peroxidase (HRP) catalyzed crosslinking on phenolic derivatized dendritic polyglycerol (dPG) in the presence of H2O2 in an inverse miniemulsion. This "enzymatic nanogelation" approach was efficient to produce stable 200 nm dPG nanogel particles, and was performed under physiological conditions, thus making it particularly beneficial for encapsulating biological proteins. Purification of the nanogels was easy to handle and practical because there was no need for a post-quenching step. Interestingly, the use of dPG resulted in higher HRP laden nanogels than for linear polyethylene glycol (PEG) analogs, which illustrates the benefits of dendritic backbones in nanogels for protein encapsulation. In addition, the mild immobilization contributed to the enhanced thermal stability and reusability of HRP. The nanogel preparation could be easily optimized to achieve the best HRP activity. Furthermore, a second enzyme, Candida antarctica lipase B (CalB), was successfully encapsulated and optimized for activity in dPG nanogels by the same enzymatic methodology, which shows the perspective applications of such techniques for encapsulation of diverse proteins.
Asunto(s)
Química Farmacéutica/métodos , Proteínas Fúngicas/síntesis química , Glicerol/síntesis química , Peroxidasa de Rábano Silvestre/síntesis química , Lipasa/síntesis química , Polietilenglicoles/síntesis química , Polietileneimina/síntesis química , Polímeros/síntesis química , Catálisis , Activación Enzimática , Proteínas Fúngicas/metabolismo , Glicerol/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Lipasa/metabolismo , Nanogeles , Polietilenglicoles/metabolismo , Polietileneimina/metabolismo , Polímeros/metabolismoRESUMEN
Protein redesign methods aim to improve a desired property by carefully selecting mutations in relevant regions guided by protein structure. However, often protein structural requirements underlying biological characteristics are not well understood. Here, we introduce a methodology that learns relevant mutations from a set of proteins that have the desired property and demonstrate it by successfully improving production levels of two enzymes by Aspergillus niger, a relevant host organism for industrial enzyme production. We validated our method on two enzymes, an esterase and an inulinase, creating four redesigns with 5-45 mutations. Up to 10-fold increase in production was obtained with preserved enzyme activity for small numbers of mutations, whereas production levels and activities dropped for too aggressive redesigns. Our results demonstrate the feasibility of protein redesign by learning. Such an approach has great potential for improving production levels of many industrial enzymes and could potentially be employed for other design goals.
Asunto(s)
Aspergillus niger/enzimología , Evolución Molecular Dirigida/métodos , Esterasas/síntesis química , Proteínas Fúngicas/síntesis química , Glicósido Hidrolasas/síntesis química , Secuencia de Aminoácidos/genética , Aspergillus niger/genética , Clonación Molecular/métodos , Esterasas/genética , Proteínas Fúngicas/genética , Glicósido Hidrolasas/genética , Estructura Secundaria de ProteínaRESUMEN
The scalable solution-phase synthesis of the cyclodepsipeptide destruxin E (1) has been achieved. Diastereoselective dihydroxylation of the terminal alkene in a 2-alkoxy-4-pentenoic amide, 7, was successfully accomplished utilizing (DHQD)2PHAL as the chiral ligand, and it was found that the use of the l-proline moiety in the substrate as a chiral auxiliary was essential for the induction of high diastereoselectivity to afford the key compound 4 on a gram scale. MNBA-mediated macrolactonization of 3 was also performed without formation of the dimerized product even under higher-dilution conditions, and it is noteworthy that the internal hydrogen bonds and s-cis configuration of the amide bond between N-methylalanine and N-methylvaline in the cyclization precursor 3 would assist in the macrolactonization to provide the macrolactone 2 without forming a dimerized product. Finally, epoxide formation in the side chain afforded destruxin E (1) on a gram scale in high purity (>98%).
Asunto(s)
Depsipéptidos/síntesis química , Compuestos Epoxi/química , Proteínas Fúngicas/síntesis química , Lactonas/química , Osteoclastos/química , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Ciclización , Depsipéptidos/química , Proteínas Fúngicas/química , Enlace de Hidrógeno , Estructura Molecular , EstereoisomerismoRESUMEN
The folding of disulfide proteins is of considerable interest because knowledge of this may influence our present understanding of protein folding. However, sometimes even the disulfide pattern cannot be unequivocally determined by the available experimental techniques. For example, the structures of a few small antifungal proteins (PAF, AFP) have been disclosed recently using NMR spectroscopy but with some ambiguity in the actual disulfide pattern. For this reason, we carried out the chemical synthesis of PAF. Probing different approaches, the oxidative folding of the synthetic linear PAF yielded a folded protein that has identical structure and antifungal activity as the native PAF. In contrast, unfolded linear PAF was inactive, a result that may have implications concerning its redox state in the mode of action.
Asunto(s)
Antifúngicos/síntesis química , Proteínas Fúngicas/síntesis química , Penicillium chrysogenum/metabolismo , Secuencia de Aminoácidos , Antifúngicos/química , Cisteína/química , Disulfuros/química , Proteínas Fúngicas/química , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Pliegue de ProteínaRESUMEN
A hexapeptide (Hexapeptide-11) of structure Phe-Val-Ala-Pro-Phe-Pro (FVAPFP) originally isolated from yeast extracts and later synthesized by solid state synthesis to high purity has demonstrated an ability to influence the onset of senescence in intrinsically aged fibroblasts, extrinsically aged fibroblasts, and extrinsically aged dermal papillae cells in vitro. The mechanism of senescence control is believed to be related to the peptide's ability to reversibly downregulate ataxia telangiectasia mutated (ATM) and p53 protein expression. The importance of p53 as the gatekeeping protein for monitoring cellular DNA damage is strategic for maintaining cellular health. ATM activates p53 by direct phosphorylation, causing cells to move into senescence which effectively moves them out of reproductive processes. Technologies that can influence ATM and p53 expression may offer unique benefits for controlling cellular senescence and effectively delaying cellular aging processes. The influence on ATM and p53 expression is noted to occur in both cell lines at peptide concentrations between 0.1% and 1.0%. The implications of these effects for aging benefits for skin and hair is important as, to date, no known small peptide has been suggested to demonstrate this effect in such a reversible and dose-dependent fashion.
Asunto(s)
Factores Biológicos/farmacología , Proteínas de Ciclo Celular/genética , Senescencia Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Dermis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Proteínas Fúngicas/farmacología , Oligopéptidos/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada , Factores Biológicos/síntesis química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Senescencia Celular/genética , Proteínas de Unión al ADN/metabolismo , Dermis/citología , Dermis/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Fúngicas/síntesis química , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Recién Nacido , Oligopéptidos/síntesis química , Fosforilación , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Técnicas de Síntesis en Fase Sólida , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Petasis reactions of substituted styrenylboronic acids and glyoxylic acid, employing tert-butylsulfinamide as the 'amine' component, proceed with high stereoselectivity to produce ß,γ-dehydrohomoarylalanine derivatives. Subsequent asymmetric dihydroxylation under neutral conditions gives the corresponding protected ß,γ-dihydroxyhomoarylalanines with up to 15:1 dr. The method has been exploited in the efficient, stereoselective synthesis of protected ß,γ-dihydroxyhomotyrosine, a component of the antifungal cyclic peptide echinocandin B.
Asunto(s)
Antifúngicos/síntesis química , Equinocandinas/síntesis química , Proteínas Fúngicas/síntesis química , Glioxilatos/química , Tirosina/análogos & derivados , Antifúngicos/química , Equinocandinas/química , Proteínas Fúngicas/química , Estructura Molecular , Estereoisomerismo , Tirosina/síntesis química , Tirosina/químicaRESUMEN
The total synthesis of destruxin E (1) has been achieved for the first time, and the stereochemistry of its chiral center at the epoxide has been determined to be (S). The cyclization precursor 3a was synthesized by solid-phase peptide synthesis. Macrolactonization of 3a utilizing MNBA-DMAPO, followed by formation of the epoxide, then furnished destruxin E. Its diastereomer, epi-destruxin E (2), was also synthesized in the same manner. Furthermore, the biological evaluation indicated that destruxin E exhibits V-ATPase inhibitory activity 10-fold greater than that of epi-destruxin E.
Asunto(s)
Depsipéptidos/síntesis química , Proteínas Fúngicas/síntesis química , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Ciclización , Depsipéptidos/química , Depsipéptidos/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacología , Estructura Molecular , EstereoisomerismoRESUMEN
High concentration of intracellular reactive oxygen species (ROS) plays a role in damaging biological systems. We isolated clitocybin A from the culture broth of Clitocybe aurantiaca and then clitocybin B and C derivatives were synthesized from clitocybin A. IMR-90 lung fibroblast cells were pre-treated or post-treated with clitocybin A, B and C to the addition of 100 muM H(2)O(2). These compounds inhibited the level of intracellular reactive oxygen species (ROS) and H(2)O(2)-induced cell death as judged by hypodiploid cell formation. The inhibitory effect of clitocybins on H(2)O(2)-induced cell death was comparable to that with N-acetylcysteine (NAC), a well-known ROS scavenger. The inhibition of H(2)O(2)-induced cell death by clitocybins was mediated by the reduction of caspase 3 and 9 activation, cytochrome c release from mitochondria and the degradation of IkappaB-alpha and IkappaB-beta, which could be resulted in the prevention of cellular senescence. It suggests that clitocybins are novel compounds scavenging ROS and protect cells from apoptosis and cellular senescence.
Asunto(s)
Agaricales , Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Proteínas Fúngicas/farmacología , Isoindoles/farmacología , Acetilcisteína/farmacología , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular , Citocromos c/metabolismo , Depuradores de Radicales Libres/síntesis química , Proteínas Fúngicas/síntesis química , Humanos , Peróxido de Hidrógeno , Proteínas I-kappa B/metabolismo , Isoindoles/síntesis química , Isoindoles/química , Isoindoles/aislamiento & purificación , Mitocondrias/metabolismo , Inhibidor NF-kappaB alfaRESUMEN
An important biological event in phytopathogens of the genus Phytophthora is sexual reproduction, which is conducted by two mating types, A1 and A2. A factor known as hormone alpha1 is secreted by the A1 mating type and induces the formation of sexual spores (oospores) in the A2 mating type. Here we describe the asymmetric synthesis and assignment of the absolute configuration of hormone alpha1 by oospore-inducing assays of the synthesized isomers.
Asunto(s)
Diterpenos/química , Diterpenos/síntesis química , Proteínas Fúngicas/química , Proteínas Fúngicas/síntesis química , Phytophthora/química , Espectroscopía de Resonancia Magnética/métodos , Conformación Molecular , Tamaño de la Partícula , EstereoisomerismoRESUMEN
Oxidatively modified low-density lipoprotein (OxLDL) is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis. In the present study, in order to clarify the structure-binding activity relationship of Asp-hemolysin-related peptides to OxLDL, we investigated the interaction between Asp-hemolysin-related peptides consisting of 4 to 29 amino acid residues and OxLDL. The incubation of OxLDL with each Asp-hemolysin-related peptide resulted in the formation of an Asp-hemolysin/OxLDL complex. In particular, the tetrapeptide, YKDG (P-4), bound to OxLDL and inhibited the OxLDL-induced macrophage proliferation in a dose-dependent manner. Furthermore, we demonstrated that lysophosphatidylcholine (LysoPC) extracted from OxLDL inhibited the binding of P-21 to OxLDL in a dose-dependent manner and synthetic [14C]LysoPC bound to P-21. We propose here that the YKDG region is one of the important sites for the binding of these peptides to OxLDL, and LysoPC as a typical lipid moiety of OxLDL is attributed to the binding of OxLDL to these peptides.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas Hemolisinas/metabolismo , Lipoproteínas LDL/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Radioisótopos de Carbono , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Proteínas Fúngicas/síntesis química , Proteínas Hemolisinas/síntesis química , Humanos , Lipoproteínas LDL/química , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , Radioinmunoensayo , Relación Estructura-ActividadRESUMEN
Aspergillus niger produced a proteinaceous hemolysin, nigerlysin when incubated on sheep's blood agar (SBA) at both 23 and 37 degrees C. Nigerlysin was purified from tryptic soy broth (TSB) culture filtrate and found to have a molecular weight of approximately 72 kDa, with an isoelectric point of 3.45. Nigerlysin is heat stable up to 65 degrees C but unstable at 75 degrees C when incubated for 10 min. Circular dichroic analysis revealed that nigerlysin has an alpha helical structure. Exposure of mouse primary cortical neuronal cells to 0.1 microg ml(-1) of nigerlysin resulted in the rapid loss of their viability, approximately 50% in 24 h. The IC50 is estimated to be 0.037 microg ml(-1), or between 0.034 and 0.041 microg ml(-1) at the 95% confidence level.
Asunto(s)
Aspergillus niger/metabolismo , Proteínas Fúngicas/síntesis química , Proteínas Fúngicas/farmacología , Proteínas Hemolisinas/farmacología , Neuronas/efectos de los fármacos , Animales , Aspergillus niger/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Proteínas Hemolisinas/química , Ratones , Neuroaspergilosis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
[Chemical reaction: See text] The synthesis of stevastelin B3 (2) and B (5) are described. In a first approach, epoxy cyclodepsipeptide 8 was considered as a promising candidate for the synthesis of the [15]-membered ring members of the stevastelins; however, the oxirane ring opening, required for the completion of the natural stevastelin synthesis, failed. Thus, we synthesized stevastelin B (5), carrying out the oxirane ring opening earlier in the synthesis and following a synthetic scheme capable of delivering analogues. On the other hand, a translactonization reaction of the [15]-membered ring derivative 59 led to the total synthesis of the natural [13]-membered ring component of the stevastelins family, stevastelin B3 (2).
Asunto(s)
Depsipéptidos/síntesis química , Proteínas Fúngicas/síntesis química , Aminoácidos/química , Compuestos Epoxi , Indicadores y Reactivos , Modelos Moleculares , Péptidos/químicaRESUMEN
[Chemical reaction: See text] The synthesis of a series of stevastelin analogues with modification of the susbstituent at the C-2 position of the stearic acid chain (compounds 28 and 31), variation of the amino acids (compounds 41, 42, 73, and 78), or lacking the lipidic chain (compound 91) is described. The replacement of L-valine and L-threonine with other amino acids proceeded without difficulties for the synthesis of analogues 41 and 42; however, the substitution of L-serine with simple amino acids, such as glycine or L-alanine, proved to be elusive, which was adscribed to factors of conformational flexibility. Finally, the substitution with L-valine or L-threonine proceeded without difficulties to provide the analogues 73 and 78 respectively.
Asunto(s)
Depsipéptidos/síntesis química , Proteínas Fúngicas/síntesis química , Péptidos/síntesis química , Alquilación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Indicadores y Reactivos , Péptidos/química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Ácidos Esteáricos/síntesis químicaRESUMEN
Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis. Immunostimulatory effects of P. brasiliensis DNA and CpG-oligodeoxyribonucleotides (CpG-ODN) have shown a Th2-Th1 immunomodulation of the isogenic murine model of susceptibility, which develops a progressive and disseminating disease. In this study, we investigated the optimum time interval and doses of CpG-ODN which are able to induce Th2-Th1 immunomodulation. The optimum concentrations for the induction of a decrease in antibody production were 0.5 and 1 microg. Mice immunized twice with CpG-ODN and gp43 (5 and 7 days before the challenge) showed a 60% higher chance of survival compared with the control group (nonimmunized), and an increase in Th1 isotype (IgG2a) was also observed. In vitro assays of naive and preimmunized mice showed discrete cellular proliferation when stimulated by suitable concentrations of CpG-ODN. Type 1 cytokines interleukin-12 (IL-12) and interferon-gamma were increased in cell culture supernatants, but no significant difference was found in Th2 IL-4 cytokines in stimulated or nonstimulated cell cultures. Concerning the Th2-Th1 kinetics in experimental PCM models by adjuvant effect of CpG-ODN, there are still many questions to be answered and clarified. However, the gathering of data obtained in this investigation has led us to suggest that the modulation of Th2-Th1 in experimental PCM depends on time and CpG-ODN concentration.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos Fúngicos/genética , Proteínas Fúngicas/genética , Glicoproteínas/genética , Oligodesoxirribonucleótidos/farmacología , Paracoccidioides/genética , Paracoccidioidomicosis/terapia , Células TH1/inmunología , Células Th2/inmunología , Animales , Anticuerpos Antifúngicos/biosíntesis , Anticuerpos Antifúngicos/sangre , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta Inmunológica , Proteínas Fúngicas/síntesis química , Genes Fúngicos/inmunología , Glicoproteínas/síntesis química , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Oligodesoxirribonucleótidos/síntesis química , Paracoccidioides/crecimiento & desarrollo , Paracoccidioides/inmunología , Paracoccidioidomicosis/inmunología , Paracoccidioidomicosis/patología , Bazo/microbiología , Bazo/patología , Células TH1/efectos de los fármacos , Células Th2/efectos de los fármacosRESUMEN
Protein fibers are fundamental building blocks of life playing an essential role in motility, elasticity, scaffolding, stabilization and the protection of cells, tissues and organisms. Despite nearly a century of research into the assembly mechanisms and structures of fibrous proteins, only limited information is still available. Within the past decade, however, insights have been provided into how some fibrous proteins assemble and how they function in biology. In addition, efforts are increasingly being made to employ protein fibers as performance molecules in man-made medical and technical applications.
Asunto(s)
Ingeniería de Proteínas , Proteínas/síntesis química , Animales , Proteínas Bacterianas/síntesis química , Proteínas Fúngicas/síntesis química , Nanotecnología , NanotubosRESUMEN
The first total synthesis of Hypomurocin A1 (HM A1) in solution phase is described. As members of the peptaibol family, hypomurocins are constituted by two groups of peptides: six undecapeptides (undecamers) in the HM A group and six octadecapeptides (18-mers) in the HM B group. The synthesis presented has been successfully achieved by the 'azirine/oxazolone method' to introduce the two Aib-Pro sequences included in this undecapeptaibol in one step with methyl 2,2-dimethyl-2H-azirine-3-prolinate as the building block. The coupling reactions of the Z-protected amino acids or peptide acids involved the use of N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBt), and led to the peptides in good-to-very-good yields. The peptides were purified by reverse-phase HPLC and characterized by NMR spectroscopy (1H, 13C, COSY, TOCSY, HSQC, HMBC, ROESY), ESI-MS, IR, elemental analysis, optical rotation, and X-ray crystallography. An NMR analysis of HM A1 was also carried out in deuterated micelles to perform a structural comparison of the helix in solution and in membranes.