Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Mechanism of formation of the putative advanced glycosylation end product and protein cross-link 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole

2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) is a fluorescent molecule which was originally discovered in chloroform extract of ammoniacal solution of acid-hydrolyzed glycated proteins and proposed to represent a protein cross-link. The absence of a lysyl residue side chain and other observations promoted a detailed study of its mechanism of formation. Glycated alpha-t-Butoxycarbonyllysine was incubated for 29 days and periodically assayed for FFI and FFI-like fluorescence. Whereas fluorescence increased over time, FFI recovery was unexpectedly highest on day 0 and lowest on day 29, suggesting that FFI was directly derived from Amadori products. FFI was also recovered from hydrolysates of glycated neopentylamine, furosine, and browned poly-L-lysine but was virtually undetectable in similar solutions basified with NaOH, triethylamine, or pyridine instead of ammonia. Gas chromatography-mass spectrometry analysis of FFI from similar hydrolysates basified in the presence of 15N-enriched NH4Cl revealed for all precursors a parent ion peak at 230 instead of 228 m/e units, suggesting that the two imidazole nitrogen atoms had been incorporated from free ammonia into FFI. Spontaneous FFI synthesis occurred when furosine was reacted with aqueous ammonia at room temperature. These results do not support the proposition that FFI is an advanced glycosylation end product or a protein cross-link. They suggest that FFI is formed from ammonia and furosine which are by-products of acid-hydrolyzed glycated proteins.