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New ionization and detection techniques in mass spectrometry have been successfully applied for efficient analyses of complex biological systems. It is, however, still difficult to trace structural changes of a specific molecular species in such systems. In the present study, a molecular probe strategy in combination with tandem electrospray ionization mass spectrometry has been examined using synthetic deuterium-labeled phosphatidylcholine hydroperoxide (PC-OOH/D3) and ethyl-labeled phosphatidylcholine having docosahexaenoic acid side chain (DHA-PC/Et). Administration of a mixture of PC-OOH/D3 and DHA-PC/Et to human blood and human skin surface, followed by extraction and analysis with collision-induced tandem electrospray ionization mass spectrometry demonstrated that metabolites of both molecular probes can be detected simultaneously with strict selectivity. The present method is also found to be useful in tracing chemical changes of the unstable docosahexaenoyl group on the surface of processed fish. The activity of phospholipase A2 can also be assessed using a phospholipid molecular probe with a linoleoyl and a deuteriomethyl group via selective detection of the lyso-phospholipid product by mass spectrometry. The advantage of the present method is that no chromatographic separation is required and analysis can be performed under strictly the same condition for different molecular probes, affording multiple data by one experiment. The present strategy may be useful for tracing time-dependent phenomena in dynamic phospholipid biochemistry, and can be widely used for any biological and food systems.

We have achieved the synthesis of 1-aryl-2-substituted 4-oxoquinoline and 4-oxo-1,8-naphthyridine derivatives, which cannot be synthesized by known methods, via two useful synthons, 2-formyl-4-oxoquinoline and 2-methylsulfonyl-4-oxo-1,8-naphthyridine. We also succeeded in the synthesis of 1-aryl-3-fluoro-4-oxoquinoline by fluorocyclization of N-arylenaminone with Select-fluor ® and potassium carbonate in DMF in a one-pot procedure. To the best of our knowledge, this is the first synthesis of 3-fluoro-4-oxoquinoline derivatives. We confirmed that these protocols were mutually applicable to the synthesis of 4-oxoquinoline and 4-oxo-1,8-naphthyridine derivatives.

An ethyl-labeled phosphatidylcholine hydroperoxide (PC-OOH/Et 2) was synthesized as a molecular probe for naturally occurring PC-OOH 1. Applying the precursor ion scan mode in tandem ESI mass spectrometry at m/z 198, a signal of the PC-OOH/Et 2 alone could be selectively detected even in the presence of a large excess of a complex mixture of phospholipids in the blood. Furthermore, molecular species that formed from PC-OOH/Et 2 by its degradation in the blood were also observed in the same spectrum. Since the molecular probe-and-mass spectrometry-assisted analytical method presented herein requires no separation process by HPLC or TLC and is speedy, requiring less than 1 h, it may be useful in lipid analysis.

Various 2-substituted 4-oxoquinoline and 4-oxo-1,8-naphthyridine derivatives are prepared from the key intermediates (I) and (VIII), respectively.

The complete glycerol-3-phosphate dehydrogenase gene of Drosophila virilis isolated by screening with αGPDHM cDNA of the adult fly was sequenced. The gene contains eight exons spread over a total of approximate 8 kb DNA. Its exon/intron organization is identical to that of D. melanogaster. A single transcription initiation site was determined by primer extension. The stop codons are located at the 3′ end of each of the exons 6 to 8. TATA and CAAT boxes are present upstream of the transcriptional start site. Adult αGPDH protein is encoded by exons 1 to 6 and exon 8. Comparison of the sequence with that of D. melanogaster showed that the homology of the nucleotide sequence of the coding region is 85% and that the homology of the amino acid sequence is 98%. On the contrary, the non-coding region is quite different in length and nucleotide sequence.

Streptothricin antibiotics, racemomycin-A and -C(RM-A and -C), which contain one and two β-lysine moieties in the molecule, were found to show antimicrobial activity on plant-pathogenic microorganisms and phytogrowth-inhibitory activity.RN-A and -C showed antimicrobial activities against all the plant-pathogenic microorganisms tested. In particular, RM-C showed strong antifungal activity on Fusarium oxysporum f. sp. lycopersici IFO-6531 (minimal inhibitory concentration (MIC) : 3.0 μg/ml). Both antibiotics storongly inhibited the growth of the root of Brassica rapa L. even at the low concentration of 50 ppm. These activities of RM-C were slightly stronger than those of RM-A. This would indicated that the β-lysine moiety in the molecule plays some role in the activity of streptothricin antibiotics.On the other hand, racemomycinic-A-acid (RM-A-acid), produced by opening the lactam ring of the streptolidine moiety of RM-A, did not show any of the above-mentioned activities. This would suggest a close relationship betweem the streptolidine moiety in the molecule of streptothricin antibiotics and the activities.

The amino-acid sequence of cytoplasmic glycerol-3-phosphate dehydrogenase ( sn -glycerol-3-phosphate: NAD + 2-oxidoreductase, EC 1.1.1.8) (αGPDH m form) from Drosophila virilis was determined by the sequencing of peptides produced by trypsin, chymotrypsin, and Staphylococcus aureus V8 proteinase digestion after cyanogen bromide cleavage. In addition, carboxyl terminal residues were determined by analysis of amino acids produced by carboxypeptidase treatment. The enzyme is composed of two identical subunits, each of which has 352 amino-acid residues and a molecular weight of 38925. The N-terminal residue of the enzyme is blocked. The comparison of Drosophila and rabbit muscle enzymes revealed that both enzymes share similar properties, such as dimeric structure, blocked N-terminal and molecular weight. Furthermore, the amino-acid sequencing showed that the rabbit muscle and Drosophila glycerol-3-phosphate dehydrogenases are homogeneous because they are 63% identical; this value is comparable to the similarity between cytochromes c from both animals, a protein known to be slow in evolutionary rate. There is, however, a marked difference in C-terminal sequence, the D. virilis enzyme having an extra tripeptide (-Gln-Thr-Leu) at the C-terminal end.

Three acid phosphatase (EC 3.1.3.2) allozymes (ACPH1, ACPH2, and ACPH4) of Drosophila virilis show different activities as measured by electrophoretic techniques. Recently, it was suggested that these differences are attributable to the variable ability of the allozymes to be incorporated into lysosomes (Narise, S., Genet. Res. Cambr., 45:143, 1985). Immunoelectrophoresis demonstrated that the activity differences between these electrophoretic variants coincided with differences in the amount of the enzyme protein in soluble fractions but not in whole cell-free extracts. These results support the idea that acid phosphatase allozymes in D. virilis are cell-localization variants. We examined the problem by structural analysis of both the protein and the carbohydrate moieties of these allozyme glycoproteins, since lysosomal enzymes are known to become localized in lysosomes through their carbohydrate moieties. The three ACPH allozymes were purified to homogeneity from their respective homozygotes and compared with respect to amino acid composition and carbohydrate content and composition. Amino acid compositions were similar, while content and compositions of neutral sugars were significantly different. The neutral sugar content of ACPH1 was 9.2%; that of ACPH2, 21.0%; and that of ACPH4, 7.3%. A trace of hexosamines, but no N-acetylneuraminic acid, was found in the ACPH allozymes. Isoelectric points varied corresponding to their electrophoretic mobilities, which were not changed by treatment with alkaline phosphatase and neuraminidase.