RESUMEN
For many secondary metabolites, heterologous synthesis is the definitive step to determine their required biosynthetic genes. Using a multivector expression system in Saccharomyces cerevisiae, we reconstituted not only two natural statins from two fungal species, i.e., lovastatin from Aspergillus terreus and FR901512 from Xylaria grammica, but also new statin structures by mixing their genes. Combinatorial gene exchange experiments revealed the functional promiscuity of two polyketide synthases in A. terreus, lovB, and lovF; they could synthesize FR901512 with Xylaria genes. Key structure determinants of statins are essential accessory genes that are irreplaceable across species.
Asunto(s)
Aspergillus/genética , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Xylariales/genética , Cromatografía Líquida de Alta Presión , Inhibidores de Hidroximetilglutaril-CoA Reductasas/análisis , Lovastatina/análisis , Lovastatina/biosíntesis , Espectrometría de Masas , Plásmidos/genética , Plásmidos/metabolismo , Sintasas Poliquetidas/genéticaRESUMEN
We investigated the effects of olanzapine on cisplatin-induced pica (the consumption of non-nutrient materials such as kaolin) and glucose homeostasis in rats to clarify the effects of olanzapine when used as an anti-emetic drug. Rats were injected intraperitoneally (i.p.) with either 5 mg/kg cisplatin or saline. Additionally, 2 or 10 mg/kg olanzapine were administered i.p. to the rats 10 min before the administration of cisplatin and subsequently administered every 24 h for 3 d. Kaolin and food intake was measured using an automatic monitoring apparatus. Plasma glucose levels were measured by an enzyme electrode method. The plasma levels of insulin and intact proinsulin were measured by enzyme-linked immunosorbent assay (ELISA). The proinsulin-to-insulin (P/I) ratio was calculated. Cisplatin significantly increased kaolin intake, but decreased food intake and body weight up to 72 h. Olanzapine had no effect on these parameters. Neither olanzapine nor cisplatin alone had a significant effect on the plasma levels of glucose, insulin, or proinsulin. However, a combination of olanzapine and cisplatin significantly decreased plasma insulin levels, but increased plasma intact proinsulin levels and the P/I ratio. Our results suggest that an additive deterioration of insulin-secreting beta-cell function and disturbance of glucose homeostasis should be considered during treatment of patients with olanzapine for cisplatin-induced nausea and vomiting.
Asunto(s)
Antieméticos/farmacología , Antineoplásicos/efectos adversos , Benzodiazepinas/farmacología , Glucemia/análisis , Cisplatino/efectos adversos , Animales , Ingestión de Alimentos , Homeostasis/efectos de los fármacos , Caolín , Masculino , Náusea/inducido químicamente , Náusea/prevención & control , Olanzapina , Pica/inducido químicamente , Pica/prevención & control , Proinsulina/sangre , Ratas Wistar , Vómitos/inducido químicamente , Vómitos/prevención & controlRESUMEN
Ustiloxin B is a secondary metabolite known to be produced by Ustilaginoidea virens. In our previous paper, we observed the production of this compound by Aspergillus flavus, and identified two A. flavus genes responsible for ustiloxin B biosynthesis (Umemura et al., 2013). The compound is a cyclic tetrapeptide of Tyr-Ala-Ile-Gly, whose tyrosine is modified with a non-protein coding amino acid, norvaline. Although its chemical structure strongly suggested that ustiloxin B is biosynthesized by a non-ribosomal peptide synthetase, in the present study, we observed its synthesis through a ribosomal peptide synthetic (RiPS) pathway by precise sequence analyses after experimental validation of the cluster. The cluster possessed a gene (AFLA_094980), termed ustA, whose translated product, UstA, contains a 16-fold repeated peptide embedding a tetrapeptide, Tyr-Ala-Ile-Gly, that is converted into the cyclic moiety of ustiloxin B. This result strongly suggests that ustiloxin B is biosynthesized through a RiPS pathway and that UstA provides the precursor peptide of the compound. The present work is the first characterization of RiPS in Ascomycetes and the entire RiPS gene cluster in fungi. Based on the sequence analyses, we also proposed a biosynthetic mechanism involving the entire gene cluster. Our finding indicates the possibility that a number of unidentified RiPSs exist in Ascomycetes as the biosynthetic genes of secondary metabolites, and that the feature of a highly repeated peptide sequence in UstA will greatly contribute to the discovery of additional RiPS.
Asunto(s)
Aspergillus flavus/genética , Familia de Multigenes , Péptidos Cíclicos/genética , Ribosomas/metabolismo , Vías Biosintéticas , Genes Fúngicos/fisiología , Péptidos Cíclicos/biosíntesis , Análisis de Secuencia de ADNAsunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Queratinas/metabolismo , Piel/metabolismo , Calcio/metabolismo , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Humanos , Lipooxigenasa/metabolismo , ARN Mensajero/metabolismo , Receptores de Superficie Celular/metabolismo , Piel/embriologíaAsunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Apoptosis/fisiología , Ictiosis Lamelar , Queratinocitos/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Animales , Células Cultivadas , Humanos , Ictiosis Lamelar/genética , Ictiosis Lamelar/patología , Ictiosis Lamelar/fisiopatología , Queratinocitos/patología , Ratones , Ratones MutantesRESUMEN
Biomaterial scaffolds are categorized into artificial or natural polymers, or combinations of the two. Artificial polymers often undergo serum protein adsorption, elicit foreign body and encapsulation immune responses post-implantation. Large pore bovine electrospun collagen I was therefore screened as a candidate for human keratinocyte and fibroblast cell scaffolds. Human HaCaT keratinocyte and dermal fibroblasts were seeded on electrospun denatured collagen I microfiber (DCM) scaffolds and after 72 h Livedead(®) assays performed to determine adhesive cell, survival and scaffold penetration. Both keratinocytes and fibroblasts attached to and survived on DCM scaffolds, however only fibroblasts migrated over and into this biomaterial. HaCaT keratinocytes remained largely stationary on the scaffold surface in discrete islands of monolayered cells. For this reason, normal human epidermal keratinocyte (NHEK) scaffold interactions were assessed using scanning and transmission electron microscopy (EM) that demonstrated DCM scaffolds comprised networks of interlocking and protruding collagen fibers with a mean diameter of 2-5 µm, with a mean inter-fiber pore size of 6.7 µm (range 3-10 µm) and scaffold thickness 50-70 µm. After 72 h the keratinocytes and fibroblasts on DCM scaffolds had attached, flattened and spread over the entire scaffold with assembly of lamellapodia and focal adhesion (FA)-like junctions. Using transmission EM, NHEKs and HaCaT keratinocytes assembled desmosomes, lamellapodia and FA junctions, however, neither hemidesmosomes nor basal lamina were present. In long term (21 day) co-culture fibroblasts migrated throughout the scaffold and primary keratinocytes (and to a lesser extend HaCaTs) stratified on the scaffold surface forming a human skin equivalent (HSE). In vivo testing of these HSEs on immunocompetent (BalbC) and immunodeficient (SCID) excisionally wounded model mice demonstrated scaffold wound biocompatibility and ability to deliver human cells after scaffold biodegradation.
Asunto(s)
Materiales Biocompatibles/química , Colágeno/química , Fibroblastos/citología , Queratinocitos/citología , Microfibrillas/ultraestructura , Trasplante de Piel/métodos , Andamios del Tejido/química , Animales , Bovinos , Células Cultivadas , Técnicas de Cocultivo , Fibroblastos/fisiología , Fibroblastos/trasplante , Humanos , Queratinocitos/fisiología , Queratinocitos/trasplante , Ensayo de Materiales , Ratones , Ratones SCID , Desnaturalización Proteica , Piel/ultraestructura , Ingeniería de Tejidos/métodosRESUMEN
Harlequin ichthyosis (HI) is caused by loss-of-function mutations in the keratinocyte lipid transporter ABCA12. The patients often die in the first 1 or 2 weeks of life, although HI survivors' phenotypes improve within several weeks after birth. In order to clarify the mechanisms of phenotypic recovery, we studied grafted skin and keratinocytes from Abca12-disrupted (Abca12(-/-)) mice showing abnormal lipid transport. Abca12(-/-) neonatal epidermis showed significantly reduced total ceramide amounts and aberrant ceramide composition. Immunofluorescence and immunoblotting of Abca12(-/-) neonatal epidermis revealed defective profilaggrin/filaggrin conversion and reduced protein expression of the differentiation-specific molecules, loricrin, kallikrein 5, and transglutaminase 1, although their mRNA expression was up-regulated. In contrast, Abca12(-/-) skin grafts kept in a dry environment exhibited dramatic improvements in all these abnormalities. Increased transepidermal water loss, a parameter representing barrier defect, was remarkably decreased in grafted Abca12(-/-) skin. Ten-passage sub-cultured Abca12(-/-) keratinocytes showed restoration of intact ceramide distribution, differentiation-specific protein expression and profilaggrin/filaggrin conversion, which were defective in primary-cultures. Using cDNA microarray analysis, lipid transporters including four ATP-binding cassette transporters were up-regulated after sub-culture of Abca12(-/-) keratinocytes compared with primary-culture. These results indicate that disrupted keratinocyte differentiation during the fetal development is involved in the pathomechanism of HI and, during maturation, Abca12(-/-) epidermal keratinocytes regain normal differentiation processes. This restoration may account for the skin phenotype improvement observed in HI survivors.