RESUMEN
L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of l-[³H]-leucine, one of the substrates for LAT1 uptake, was investigated. l-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of l-[³H]-leucine, while P(NIPAAm-co-DMAAm) and l-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that l-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that l-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Imagen Molecular/métodos , Neoplasias/diagnóstico por imagen , Polímeros/metabolismo , Acrilamidas/química , Colorantes Fluorescentes/química , Células HeLa , Calor , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Transportador de Aminoácidos Neutros Grandes 1/química , Transportador de Aminoácidos Neutros Grandes 1/efectos de los fármacos , Leucina/metabolismo , Neoplasias/metabolismo , Polímeros/químicaRESUMEN
A shiga toxin-producing Escherichia coli (STEC) O26 strain resistant to cefotaxime (CTX) and cefpodoxime (but not ceftazidime) was isolated from the faecal sample of a 17-year-old outpatient with diarrhea. The double disk synergy test, twin test, polymerase chain reaction and sequence analysis confirmed that the strain produced CTX-M-3 type extended-spectrum beta-lactamase (ESBL). Conjugation experiment results suggested that the CTX resistance in this strain was determined by an approximately 85kbp plasmid that was readily transferable to a susceptible recipient E. coli strain. This is the first report from Japan of CTX-M-3type ESBL-producing STEC O26.