RESUMO
Two silklike proteins, [TGRGDSPAGG(GAGAGS)3AS]5 (FS5) and [TGRGDSPA-(GVPGV)2GG(GAGAGS)3AS]8 (FES8) were designed to demonstrate the superior performance as biomaterials of silklike proteins. The former protein consists of the crystalline domain sequence, (GAGAGS)n from Bombyx mori silk fibroin and cell-adhesive sequence TGRGDSPA coming from fibronectin-containing RGD triplet. The additional sequence (GVPGV)n from elastin was included in the latter protein. The considerably higher cell-adhesion activities of these proteins for NHDF and VERO cells were observed by comparing with those of silklike materials without RGD sequences and also the crystalline fraction of B. mori silk fibroin. This tendency was independent of the treatments, 4.5M LiClO4 or formic acid (FA), on silklike proteins. Their activities are also higher than those of commercial Fibronectin F for NHDF cell. Their structural characterization was studied using 13C solid-state NMR. Although the overlapped peaks in usual 13C CP/MAS NMR spectra make the detailed structural analysis difficult, the methyl resonance regions observed using dipolar dephasing NMR were very useful for the analysis. The presence of both random coil and beta-sheet structures was observed in these proteins clearly. The content of beta-sheet structure in both proteins increases after FA treatment when compared with the lyophilized samples. The production of electrospun nanofibers from their hexafluoroacetone solution was also tried. The silklike protein FES8 could prepare nonwoven silk fibers although FS5 could not.
Assuntos
Bombyx/química , Elastina/química , Elastina/genética , Fibroínas/química , Fibroínas/genética , Fibronectinas/química , Fibronectinas/genética , Seda/química , Seda/genética , Acetona/análogos & derivados , Acetona/química , Animais , Sequência de Bases , Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Chlorocebus aethiops , Escherichia coli/metabolismo , Fluorocarbonos/química , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Nanotubos , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alicerces Teciduais , Células VeroRESUMO
There are a variety of silkworms and silk fibroins produced by them. Silks have many inherent suitable properties for biomaterials. In this paper, a novel silk-like hybrid protein, [DGG(A)(12)GGAASTGRGDSPAAS](5), which consists of polyalanine region of silk fibroin from a wild silkworm, Samia cynthia ricini, and cell adhesive region including Arg-Gly-Asp (RGD) sequence, derived from fibronectin, was designed and produced. The genes encoding the hybrid protein were constructed and expressed in Escherichia coli. The main conformation of the polyalanine region, that is, either alpha-helix or beta-sheet, could be easily controlled by treatment with different acidic solvents, trifluoroacetic acid or formic acid, respectively. This structural change was monitored with 13C CP/MAS NMR. Higher cell adhesive and growth activities of the hybrid protein compared with those of collagen were obtained.
Assuntos
Materiais Biocompatíveis/metabolismo , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Teste de Materiais/métodos , Polímeros/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Seda , Animais , Biopolímeros , Bombyx/genética , Bombyx/metabolismo , Chlorocebus aethiops , Elasticidade , Fibroínas/genética , Fibroínas/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Células VeroRESUMO
For dental outpatients undergoing conscious sedation, recovery from sedation must be sufficient to allow safe discharge home, and many researchers have defined "recovery time" as the time until the patient was permitted to return home after the end of dental treatment. But it is frequently observed that patients remain in the clinic after receiving permission to go home. The present study investigated "clinical recovery time," which is defined as the time until discharge from the clinic after a dental procedure. We analyzed data from 61 outpatients who had received dental treatment under conscious sedation at the Hiroshima University Dental Hospital between January 1998 and December 2000 (nitrous oxide-oxygen inhalation sedation [n = 35], intravenous sedation with midazolam [n = 10], intravenous sedation with propofol [n = 16]). We found that the median clinical recovery time was 40 minutes after nitrous oxide-oxygen sedation, 80 minutes after midazolam sedation, and 52 minutes after propofol sedation. The clinical recovery time was about twice as long as the recovery time described in previous studies. In a comparison of the sedation methods, clinical recovery time differed (P = .0008), being longer in the midazolam sedation group than in the nitrous oxide-oxygen sedation group (P = .018). These results suggest the need for changes in treatment planning for dental outpatients undergoing conscious sedation.