The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO) 2 Fe(L-cysteine) 2 ], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO) 2 Fe(L-cysteine) 2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NO x -species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyliron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO) 2 Fe(Lcysteine) 2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyliron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

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The biological signal molecule nitric oxide (NO) exists in a free and carrier-bound form. Since the structure of the carrier is likely to influence the interaction of NO with macromolecular targets, we assessed the interaction of a dinitrosyl-iron-dithiolate complex carrying different thiol ligands with glutathione reductase. The enzyme was irreversibly inhibited by dinitrosyl-iron-di-L-cysteine and dinitrosyl-iron-di-glutathione in a concentration-and time-dependent manner (IC 50 30 and 3 M, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a K i of 14 M, and a k 3 of 1.3 ؋ 10 ؊3 s ؊1. A participation of catalytic site thiols in the inhibitory mechanism was indicated by the findings that only the NADPH-reduced enzyme was inhibited by dinitrosyl-iron complex and that blockade of these thiols by Hg 2؉ afforded protection against irreversible inhibition. This inhibition was not accompanied by formation of a protein-bound dinitrosyl-iron complex and/or S-nitrosation of active site thiols (Cys-58 and Cys-63). However, one NO moiety exhibiting an acid lability similar to a secondary N-nitrosamine was present per mol of inhibited monomeric enzyme. These findings suggest specifically N-nitrosation of glutathione reductase as a likely mechanism of inhibition elicited by dinitrosyliron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO ؉ transfer to the ligand-binding protein.Nitrosyl transfer from endogenous nitric oxide (NO) 1 carriers such as S-nitrosoglutathione (1) and dinitrosyl-iron complex (DNIC) (2) to macromolecular targets is regarded as one major mechanism of biological NO signaling (3). Evidence has been provided that endogenous low mass S-nitrosothiols (predominantly S-nitrosoglutathione) and proteinaceous S-nitrosothiols (S-nitroso-hemoglobin, S-nitroso-serum albumin, and other yet unidentified proteins) exist in human erythrocytes (4), plasma (5) and bronchial secretion (6). A further NO adduct with GSH, GSNOH, was recently postulated as another transport form of NO (7). S-Nitrosation of protein thiols or subsequent reactions such as ADP-ribosylation (8), formation of protein disulfide (9), and cysteine sulfenic acid (10) may influence protein function by allosteric mechanisms. Furthermore, reversible S-nitrosation of cell membrane-bound proteins may be involved in transmembraneous NO transport (11).A concept has been derived from established chemistry to account for the influence of the redox state of the NO moiety on biological NO transfer reactions. According to this concept nitrosation of nucleophilic targets occurs by attack of nitrosonium (NO ϩ )-like species assumed to be present in "NO carriers" such as N 2 O 3 , S-nitrosothiols, and certain iron-nitrosyl complexes (3, 12). Less attention has been paid to the influence of the carrier structure on the interaction of NO with macromolecular targets. However, it is conceivable that the NO carrier will direct the NO moiety specifically to macromolecules r...

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By use of a Fourier transform infrared (FTIR) spectroscopic imaging technique, we examine the dynamic optical clearing processes occurring in hyperosmotically biocompatible agents penetrating into skin tissue in vitro. The sequential collection of images in a time series provides an opportunity to assess penetration kinetics of dimethyl sulphoxide (DMSO) and glycerol beneath the surface of skin tissue over time. From 2-D IR spectroscopic images and 3-D false color diagrams, we show that glycerol takes at least 30 min to finally penetrate the layer of epidermis, while DMSO can be detected in epidermis after only 4 min of being topically applied over stratum corneum sides of porcine skin. The results demonstrate the potential of a FTIR spectroscopic imaging technique as an analytical tool for the study of dynamic optical clearing effects when the bio-tissue is impregnated by hyperosmotically biocompatible agents such as glycerol and DMSO.

IR microspectroscopic imaging is a relatively new approach for the examination of tissue sections. In contrast to standard light microscopy based procedures, the IR approach requires neither sample staining nor fixation. The IR spectra of breast tumor tissue sections are obtained via a microscope equipped with a focal plane array detector. This enabled the simultaneous collection of individual mid-IR spectra from thousands of different sample positions with a spatial resolution near the diffraction limit. The analysis of the IR data reveals a high sensitivity of the IR approach toward changes in tissue biochemistry and variations in breast tissue architecture. Moreover, the data demonstrate the need for collecting spectra with high spatial resolution at the level of individual cells. This minimizes problems associated with tissue microheterogeneity and is an essential prerequisite for future studies aimed at developing IR microspectroscopic imaging as a complement to present diagnostic tools for breast cancer.

A highly loaded compressor cascade, which features a chord length ten times larger than in real turbomachinery, is used to perform an investigation of the influence of technical surface roughness. The surface structure of a precision forged blade was engraved in two 0.3-mm-thick sheets of copper with the above-mentioned enlarging factor (Leipold and Fottner, 1996). To avoid additional effects due to thickening of the blade contour, the sheets of copper are applied as inlays to the pressure and suction side. At the high-speed cascade wind tunnel, the profile pressure distribution and the total pressure distribution at the exit measurement plane were measured for the rough and the smooth blade for a variation of inlet flow angle and inlet Reynolds number. For some interesting flow conditions, the boundary layer development was investigated with laser-two-focus anemometry and one-dimensional hot-wire anemometry. At low Reynolds numbers and small inlet angles, a separation bubble is only slightly reduced due to surface roughness. The positive effect of a reduced separation bubble is overcompensated by a negative influence of surface roughness on the turbulent boundary layer downstream of the separation bubble. At high Reynolds numbers, the flow over the rough blade shows a turbulent separation leading to high total pressure loss coefficients. The laser-two-focus measurements indicate a velocity deficit close to the trailing edge, even at flow conditions where positive effects due to a reduction of the suction side separation have been expected. The turbulence intensity is reduced close downstream of the separation bubble but increased further downstream due to surface roughness. Thus the rear part of the blade but not the front part reacts sensitively on surface roughness. [S0889-504X(00)01302-7]

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