Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue and for the inhibition of platelet aggregation and platelet adhesion attributed to endothelium-derived relaxing factor. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay, chemiluminescence or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.

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These data indicate a possible role for a tomato sauce constituent, possibly lycopene, in the treatment of prostate cancer and warrant further testing with a larger sample of patients, including a control group.

A variety of gram-positive and gram-negative intact bacterial cells have been analysed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and shown to provide fingerprint mass spectra with discrete peaks being observed over the mass range from 3 to 40 kDa. The spectra show both more peaks and peaks at a higher mass/charge ratio than have hitherto been reported for these micro-organisms and would appear to provide a profile of cellular proteinaceous material. The spectra are shown to be reproducible over variable time periods of up to three months and factors affecting reproducibility are discussed. The procedure, which requires minimal sample preparation, yields results in 30-40 minutes and allows visual identification of species-and strain-specific biomarkers for the characterization of the organisms. The importance of accurately defining sample preparation methodologies is central to the ability of the technique to generate reliable and reproducible data.

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The ability to rapidly identify the taxonomic class of the wide variety of microorganisms involved in human and animal disease is becoming increasingly important, especially with the increasing development of resistance to the antibiotics which form the main defence against them. A number of groups have recognised the utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry MALDI-TOF in the analysis of these microorganisms. However, no consistent methodology has been developed which is in general use. In particular the use of different solvent extraction systems and mass spectrometric matrices can have significant effects on the quality of the data obtained. We have now studied a number of the commonly used matrices and a range of solvent systems of widely varying polarity in an attempt to devise an optimum analytical strategy for the rapid characterisation of these organisms by MALDI-TOFMS. The E. coli ATCC 9637 organisms were initially washed to remove growth medium contaminants, followed by extraction with one of a range of solvents prior to admixing with a number of different single matrices or binary and ternary combinations of these matrices. The results obtained indicate that a binary combination of 2-(4-hydroxyphenylazo)benzoic acid and 2-mercaptobenzothiazole (1:1) as matrix provides the best data after the proteinaceous material from the organism cell surface was extracted with 17% formic acid, 33% isopropyl alcohol and 50% water, (solvent 2 in this work).

Two mass spectrometric techniques, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) have been used to study the intact humanized monoclonal antibody CAMPATH 1H, its fully and partially deglycosylated species, and 13 fragments prepared from it. The transformed ESI mass spectra of the glycosylated species gave complex patterns of molecular masses (M(r's). These have been substantially assigned to the presence of a mixture of glycoforms, each resulting from the combination of a single protein species with specific glycans of four distinct masses. The MALDI mass spectra of the glycosylated species, with the exception of that of the smallest fragment Fc/2, which indicated the presence of three of the glycans, gave single M(r) values comparable to the mean M(r) calculated from the ESI results. The M(r) values for the 10 prepared nonglycosylated species support the validity of the published amino acid sequence for the antibody and define the cleavage sites for the enzymic fragmentations. It is concluded that mass measurement of the Fc/2 fragment using ESI techniques provides a convenient means of preliminary assessment of the major glycosylated entities.