ABSTRACT
Aptamers are composed of single-stranded oilgonucleotides that can selectively bind desired molecules. It has been reported that RNA or DNA could act as not only a genetic messenger but also a catalyst in metabolic pathways. RNA aptamers (average sizes 40-50 bp) are smaller than antibodies and have strong binding capacities to target molecules, similar to antigen-antibody interactions. Once an aptamer was selected, it can be readily produced in large quantities at low cost. The objectives of this study are to screen and develop aptamers specific to oral pathogens such as Porphyromonas gingivalis, Treponema denticola, and Streptococcus mutans. The bacterial cell pellet was fixed with formaldehyde as a target molecule for the screening of aptamers. The SELEX method was used for the screening of aptamers and a modified western blot analysis was used to verify their specificities. Through SELEX, 40 kinds of aptamers were selected and the specificity of the aptamers to the bacterial cells was confirmed by modified western blot analysis. Through the SELEX method, 40 aptamers that specifically bind to oral pathogens were screened and isolated. The aptamers showed possibility as effective candidates for the detection agents of oral infections.
Subject(s)
Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/pharmacology , Porphyromonas gingivalis/drug effects , SELEX Aptamer Technique , Streptococcus mutans/drug effects , Treponema denticola/drug effects , Aptamers, Nucleotide/therapeutic use , Biosensing Techniques , Humans , Nucleic Acid Conformation , Porphyromonas gingivalis/isolation & purification , Sensitivity and Specificity , Streptococcus mutans/isolation & purification , Streptococcus oralis/drug effects , Streptococcus oralis/isolation & purification , Streptococcus oralis/pathogenicity , Streptococcus sanguis/drug effects , Streptococcus sanguis/isolation & purification , Streptococcus sanguis/pathogenicity , Treponema denticola/isolation & purification , Treponema denticola/pathogenicityABSTRACT
During the search for naturally occurring cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) inhibitors, it was found that the extracts from Sophora flavescens exhibit potent inhibitory activity against cGMP PDE5 prepared from rat diaphragm. Therefore, the inhibitory activities of five flavonoids, kushenol H (1), kushenol K (2), kurarinol (3), sophoflavescenol (4) and kuraridine (5), isolated from S. flavescens were measured against cGMP PDE5 to identify potent cGMP PDE5 inhibitory constituents. Among tested compounds, sophoflavescenol (4), a C-8 prenylated flavonol, showed the most potent inhibitory activity (IC(50)=0.013 microM) against cGMP PDE5 with 31.5- and 196.2-fold selectivity over PDE3 and PDE4, respectively. Kinetic analysis revealed that sophoflavescenol was a mixed inhibitor of PDE5 with a K(i) value of 0.005 microM.
