Inhibition of gingipains and Porphyromonas gingivalis growth and biofilm formation by prenyl flavonoids.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is considered a major pathogen of chronic periodontitis, which also may be implicated with systemic diseases such as atherosclerosis. Secreted cysteine proteases, gingipains Rgp and Kgp, are essential for P. gingivalis virulence. Some polyphenols and flavonoids are known to inhibit gingipain activity and interfere with biofilm formation by P. gingivalis. Many bioactive compounds have been isolated from Epimedium species, but availability of these compounds on gingipains and P. gingivalis is still unclear. Therefore, the aim of this study was to evaluate natural products from medical plants to develop a new therapeutic agent against periodontal disease. MATERIAL AND METHODS: Prenylated flavonoids were isolated from Epimedium species plant using column chromatographies. The inhibitory effect of the prenylated flavonoids against protease activity of gingipains were examined using purified gingipains and fluorogenic substrates. Anti-P. gingivalis activity was evaluated to analyze planktonic growth and biofilm formation in brain heart infusion medium in the presence of the prenylated flavonoids. RESULTS: We isolated 17 prenylated flavonoids (Limonianin, Epimedokoreanin B, etc.) from Epimedium species. We found that some prenylated flavonoids inhibited gingipain activity in a non-competitive manner with Ki values at µm order. The prenylated flavonoids also hindered growth and biofilm formation of P. gingivalis, in a manner independent of gingipain inhibition by the compounds. CONCLUSION: The results indicated an inhibitory effect of the prenylated flavonoids against P. gingivalis and would provide useful information for future development of periodontitis treatment that suppresses gingipains, P. gingivalis growth and biofilm formation.

Isolation and characterization of a wound-inducible ribonuclease from Nicotiana glutinosa leaves.

A wound-inducible ribonuclease (RNase NW) was purified from leaves of Nicotiana glutinosa. The purified RNase NW has an optimum pH around 5 and 7, and its base specificity is suggested based on the relative rates of hydrolysis of homopolyribonucleotides to be a preference for guanine base. The complete amino acid sequence of RNase NW was deduced by a combination of protein and cDNA sequencings. The cDNA sequence includes the entire coding sequence for a polypeptide with 229 amino acids including a putative secretion signal peptide at the N-terminus composed of 25 amino acids. The amino acids identified by the protein chemical methods are unambiguously localized within the deduced amino acid sequence from the cDNA sequence. Comparison of the sequence of RNase NW with those of other known plant RNases showed that it was identical except for eight residues to that of N. alata RNase NE, which is present in the style and pollen under normal conditions and is induced in roots in response to phosphate starvation [Dodds et al., Plant. Mol. Biol., 31, 227-238 (1996)]. RNase NW shows considerable sequence similarity to other known RNases, sharing 57% to 84% identical residues. Northern blot analysis using an RNase NW cDNA fragment as a probe showed that the RNase NW transcript was not detected in leaves without wounding, but it was induced within 4 h after wounding and then gradually decreased during 20 h.

Cloning and nucleotide sequence of the gene cluster encoding ribosomal proteins S12 and S7 from Mycobacterium bovis BCG.

A pair of oligonucleotide primers, based on the experimentally determined amino terminal sequence of Mycobacterium bovis BCG ribosomal protein S12 (MboS12) and a highly conserved sequence found in all mycobacterial ribosomal S12 proteins, was used for polymerase chain reaction (PCR) with M. bovis genomic DNA as template. The nucleotide sequence of the 338 bp fragment thus produced confirmed its origin in MboS12 gene. A 5.0 kb EcoRI fragment of M. bovis DNA hybridizing to this fragment was cloned. Its sequencing analysis revealed the presence of two open reading frames in the same strand. Their amino acid sequences deduced from DNA sequence showed high homology with Escherichia coli ribosomal proteins S12 and S7. However, the intercistronic region between S12 and S7 genes, which plays an important role for autoregulation for the str operon in E. coli, is completely absent in M. bovis.