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A new procedure has been developed for the in situ FT-IR determination of rosmarinic acid (RA) in suspension cultures of Lavandula officinalis. The method involves sample preparation on ZnSe crystals or microplates from silicon, and measuring absorbance spectra between 4000 and 700 cm(-1). First derivative spectra were analysed after normalisation using partial least square (PLS) algorithm. The correlation between spectral analysis and HPLC measurements of cell extracts shows that the FT-IR procedure is suitable for qualitative and quantitative analyses of RA in cell suspension cultures.

We have applied infrared microspectroscopic imaging for the examination of benign breast tumor tissue sections. The IR spectra of the sections were obtained by classical point microscopy with a movable stage and via a microscope equipped with a focal plane array detector. The infrared microscopic data were analysed using functional group mapping techniques and cluster analysis. The output values of the two procedures were reassembled into infrared images of the tissues, and were compared with standard staining images of the corresponding tissue region. The comparative examination of identical tissue sections by the two IR approaches enabled us to assess potential problems associated with tissue microheterogeneity. It was found that in case of fibroadenoma, a benign lesion located in breast ducts, point microscopy with a spot size of ∼30 μm is a useful practical approach which minimizes the possibility of ‘contamination’ of the spectra because of spectral averaging of all tissue components present in the corresponding microareas. A comparison of the spectra of the benign breast tumor with those of a malignant ductal carcinoma in situ revealed that IR microspectroscopy has the potential to differentiate between these two breast tumor types.

Spatially resolved Fourier transform infrared (FT-IR) spectroscopy of single oral mucosa cells and FT-IR spectroscopy of liver cell fractions produced by sucrose density gradient centrifugation have been applied to acquire structural information of cell organelles. For the spatially resolved measurements, mapping as well as mercury cadmium telluride (MCT) focal plane array (FPA) detector techniques were utilized. Surprisingly, infrared spectra of distinct subcellular structures differed only slightly. Aside from a minor intensity increase of the symmetric and the antisymmetric PO2− bands in the spectra of the nucleus, all normalized infrared spectra exhibited a remarkably high degree of similarity. Considering the fact that more than 98% of the DNA and significant amounts of the RNA are concentrated in the nucleus which occupies in oral mucosa cells only 5% of the total cell volume, these findings may be interpreted as a proof of the hypothesis that DNA is partially “invisible” in infrared spectroscopy. The finding of comparably small IR spectroscopic differences between cell organelles was confirmed by measurements on cell fractions specifically enriched by sucrose density gradient centrifugation. Besides variations of the non-specific overall protein:lipid ratio, the IR spectra of the distinct pellets exhibited only slightly differing absorbance values for the PO2− bands. As found for the nuclei by spatially resolved microspectrometry, IR spectra of the nuclear pellet displayed minor increased absorbances of the PO2− band at 1083 cm−1 when compared to spectra of the mitochondrial or microsomal pellets.

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]

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 (IC50 30 and 3 microM, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a Ki of 14 microM, and a k3 of 1.3 x 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 Hg2+ 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 dinitrosyl-iron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO+ transfer to the ligand-binding protein.

1. In the present study we examined whether interleukin-1 beta (IL-1 beta) increases the activity of adenylyl cyclase in vascular smooth muscle cells and determined its role in the cytokine-induced expression of the inducible nitric oxide synthase (iNOS) and activation of nuclear transcription factor-kappa B (NF-kappa B). In addition the interaction between cyclic AMP- and cyclic GMP-elevating agonists on the IL-1 beta-stimulated expression of iNOS was examined. 2. Exposure of vascular smooth muscle cells to IL-1 beta stimulated the formation of cyclic AMP but not of cyclic GMP. The intracellular level of cyclic AMP reached a maximum within 1 h and then gradually declined over the next 5 h. This IL-1 beta (60 u ml-1)-stimulated formation of cyclic AMP was modest (about 3 fold at 60 u ml-1 for 1 h) compared to that evoked by isoprenaline (about 9 fold at 3 x 10(-6) M for 2 min). 3. The IL-1 beta (60 u ml-1 for 24 h)-stimulated accumulation of nitrite, which was taken as an index of NO production, was concentration-dependently increased by preferential inhibitors of cyclic AMP-dependent phosphodiesterases (rolipram and trequinsin). This effect was reproduced by a specific activator of the cyclic AMP-dependent protein kinase(s) A, Sp-8-CPT-cAMPS (10(-4) M) but was prevented by a specific inhibitor of cyclic AMP-dependent protein kinase(s) A, Rp-8-CPT-cAMPS (10(-4) M). These compounds alone [rolipram (10(-6) M), trequinsin (3 x 10(-6) M) and Sp-8-CPT-cAMPS (10(-4) M)] slightly but significantly increased the release of nitric oxide while Rp-8-CPT-cAMPS elicited no such effect. 4. Inducible NOS protein was expressed in IL-1 beta (30 u ml-1, 24 h)-stimulated smooth muscle cells as assessed by Western blot analysis. The level of iNOS protein was markedly increased in smooth muscle cells which had been exposed to IL-1 beta in combination with either rolipram (3 x 10(-6) M) or Sp-8-CPT-cAMPS (10(-4) M) but was reduced in those exposed to IL-1 beta and Rp-8-CPT-cAMPS (10(-4) M). A weak expression of iNOS protein was found in smooth muscle cells which had been exposed to either Sp-8-CPT-cAMPS or rolipram alone for 24 h while Rp-8-CPT-cAMPS elicited no such effect. 5. Exposure of smooth muscle cells to IL-1 beta (30 u ml-1) for 30 min increased the level of NF-kappa B-DNA complexes in nuclear extracts as detected by electrophoretic mobility shift assay. Similar levels of NF-kappa B-DNA complexes were found in cells which had been exposed to IL-1 beta in combination with either Sp-8-CPT-cAMPS (10(-4) M), trequinsin (10(-6) M) or rolipram (10(-6) M). None of the modulators alone affected the basal level of NF-kappa B binding activity. 6. NO-donors [sodium nitroprusside (SNP) 10(-4) M; dinitrosyl-iron-di-L-cysteine-complex (DNIC), 10(-4) M; 3-morpholino-sydnonimine (SIN-1), 10(-4) M] and atrial natriuretic factor (10(-6) M) significantly increased the IL-1 beta (30 or 60 u ml-1, 24 h)-stimulated expression of iNOS protein and activity as assessed indirectly by the conversion of oxyhaemoglobin to methaemoglobin. In the absence of IL-1 beta, SNP (10(-4) M, 24 h) but not the other cyclic GMP-dependent vasodilators caused a modest expression of iNOS protein. No such effect was found in smooth muscle cells exposed to SNP in combination with Rp-8-CPT-cAMPS (10(-4) M) while an increased level of iNOS protein was found in those exposed to SNP in combination with either Sp-8-CPT-cAMPS (10(-4) M) or rolipram (3 x 10(-6) M). 7. Exposure of vascular smooth muscle cells to either S-nitroso-L-cysteine (Cys-SNO, 10(-4) M), SNP (10(-4) M) or SIN-1 (10(-4) M) for 35 min affected minimally the basal activation of NF-kappa B but abolished that evoked by IL-1 beta (30 u ml-1 added during the last 30 min). However, addition of Cys-SNO following the stimulation with IL-1 beta (during the last 5 min of the 30 min exposure period) reduced the level of NF-kappa B-DNA complexes only slightly. 8. These data indicate that the cyclic AMP-dependent pathway plays a decisi

The nature of an L-arginine-derived relaxing factor released from vascular smooth muscle cells cultured on microcarrier beads and stimulated for 20 h with interleukin 1 beta was investigated. Unlike the unstable relaxation elicited by authentic nitric oxide (NO) in a cascade superfusion bioassay system, the effluate from vascular smooth muscle cells induced a stable relaxation that was susceptible to inhibition by oxyhemoglobin. Three putative endogenous NO carriers mimicked this stable relaxing effect: S-nitroso-L-cysteine, low molecular weight dinitrosyl-iron complexes (DNICs), and the adduct of NG-hydroxy-L-arginine (HOArg) with NO. Inactivation of S-nitroso-L-cysteine by Hg2+ ions or trapping of DNICs with agarose-bound bovine serum albumin abolished their relaxing effects, whereas that of the vascular smooth muscle cell effluate remained unaffected. In addition, neither S-nitrosothiols nor DNICs were detectable in the effluate from these cells, as judged by UV and electron spin resonance (ESR) spectroscopy. The HOArg-NO adduct was instantaneously generated upon reaction of HOArg with authentic NO under bioassay conditions. Its pharmacological profile was indistinguishable from that of the vascular smooth muscle cell effluate, as judged by comparative bioassay with different vascular and nonvascular smooth muscle preparations. Moreover, up to 100 nM HOArg was detected in the effluate from interleukin 1 beta-stimulated vascular smooth muscle cells, suggesting that sufficient amounts of HOArg are released from these cells to spontaneously generate the HOArg-NO adduct. This intercellular NO carrier probably accounts for the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells and also from other NO-producing cells, such as macrophages and neutrophils.

The combination of the two classical biophysical methods, microscopy and infrared spectroscopy, has led to the development of a potent analytical technology termed infrared microspectroscopy. It combines high lateral resolution as obtained by microscopy and the chemical identification of the sample components by infrared spectroscopy. The two approaches mainly utilized in microspectroscopy are the mapping and the imaging techniques, which are introduced and presented. Especially, since the development of so called focal plane array detectors, which are implemented in the imaging methods (microspectroscopic imaging) has become a promising bio-analytical tool for ultrastructural medical diagnostics, due to the fact that the time required for analyzing a sample has been reduced dramatically and the lateral resolution improved to ∼4 μm. Mid infrared microscopy allows a direct access to spatially resolved molecular and structural information of the analyzed area. The image contrast is generated on the basis of the tissue's intrinsic biochemical composition. The current investigation shows how mid infrared microspectroscopic mapping and imaging is used for the bio-analytical characterization and identification of specific molecular components of a tissue sample at high lateral resolution of a few microns (approaching the mid infrared diffraction limit). Furthermore, the potential of these methods for monitoring the penetration and distribution of drugs within biological tissues are presented. Due to the fact, that mid infrared microspectroscopy is a noninvasive, nondestructive technique for the analyzed sample, requiring no complicated and time consuming staining procedures, it is a convenient method for histological and pathological investigations, allowing the generation of a huge amount of biochemical information not yet available with other nonvibrational techniques. The strength of the presented microscopic technique is the fact that the infrared images are directly comparable to outcomes of classical histological staining procedures and can be interpreted by nonspectroscopists. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.

For the first time FTIR spectroscopy has been applied to the measurement of enantiomeric purity. The underlying concept is based on the use of pseudo-enantiomers that are (13)C-labeled at appropriate positions. Upon applying Lambert-Beer's law in the determination of the concentrations of both enantiomers, the ee values are accessible, accuracy to within +/-5 % of the true values being possible. The application of a commercially available high-throughput FTIR system results in a slightly decreased accuracy (+/-7% for the ee values), but this allows a throughput of up to 10000 samples per day. The method is of interest in the area of combinatorial symmetric catalysis and directed evolution of enantioselective enzymes.

An experimental investigation of the influence of riblet surface structures on the loss behavior of a highly loaded compressor cascade V103-180 featuring a large chord length for high spatial resolution of the flow phenomena was performed. The cascade experiments were carried out at the High Speed Cascade Wind Tunnel of the University of the Armed Forces Munich in order to simulate realistic Mach and Reynolds numbers. The riblets used for the first investigation are of symmetric v-groove type with heights of 0.0762, 0.1143 and 0.1524 mm, respectively [1]. With two total pressure probes simultaneously traversed over one pitch behind the center airfoil, the local total pressure difference between the structured and the smooth blade is determined. From these measurements, the total pressure loss coefficient can be evaluated. For a better understanding of the flow phenomena, the profile pressure distribution is measured for the smooth and the structured blade. Boundary layer calculations were performed in order to optimise the riblet size for the design conditions of the compressor cascade. Resulting from the measurements an optimised riblet configuration (size and shape) has been manufactured and transferred to the cascade. Further flow measurements have been performed in order to evaluate the total pressure loss coefficient. Additional insight into the flow phenomena of the boundary layer has been achieved by laser-two-focus measurements. The experimental results indicate that the riblets mainly influence the suction side boundary layer behaviour. The ideal dimensionless groove height is obtained h+ = 9 leading to a reduction of the loss coefficient of 6–8%. Values of h+ > 20 cause an increase of the loss coefficient due to the development of a turbulent boundary layer separation.Copyright © 2002 by ASME

FT-IR microspectroscopy in combination with digital image reassembling methodologies have been used to characterize distinct tissue structures within thin sections from the human prostate. The spatially resolved microspectroscopic data were collected with a resolution near the diffraction limit (about 8 micrometers ) by using a HgCdTe focal plane array detector-based infrared imaging instrument. While IR imaging based on distinct spectral parameters such as intensities, frequency values, or half-widths (the univariate imaging technique of chemical mapping) often gives unsatisfactory results, multivariate data analysis techniques (e.g. hierarchical clustering or principal component analysis) confirmed standard histopathological techniques and turned out to be helpful to discriminate reliably between different tissues structures.

FT-IR microspectroscopy in combination with digital image reassembling methodologies have been used to characterize distinct tissue structures within thin sections from the human prostate. The spatially resolved microspectroscopic data were collected with a resolution near the diffraction limit (about 8 μm) by using a HgCdTe focal plane array detector-based infrared imaging instrument. While IR imaging based on distinct spectral parameters such as intensities, frequency values, or half-widths (the univariate imaging technique of chemical mapping) often gives unsatisfactory results, multivariate data analysis techniques (e.g. hierarchical clustering or principal component analysis) confirmed standard histopathological techniques and turned out to be helpful to discriminate reliably between different tissues structures.

A highly loaded compressor cascade which features a chord length that is ten times larger than in real turbomachinary 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 0.3mm thick sheets of copper with the above mentioned enlarging factor (Leipold and Fottner, 1998). To avoid additional effects due to thickening of the blade contour the sheets of copper are applied as inlay’s 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 the laser-two-focus anemometry and the 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 not the front part but the rear part of the blade reacts sensitively on surface roughness.

Zur Analyse von Fehlstellen, deren Ausdehnung im Produkt wenige Millimeter oder Mikrometer betragt, dienen mikroskopische Techniken. Die Fourier-Transform- Infrarot-(FT-IR-)Spektroskopie bestimmt die chemische Beschaffenheit von Fehlstellen mit hoher Ortsauflosung.

FTIR-spectroscopy in combination with the attenuated total reflection (ATR)-technique allows the monitoring of binding reactions between molecules over a large range of molecular masses [1, 2]. In this study an extension to this method is described in which the ligand can be added to immobilized molecules by dialysis; thereby, preventing mechanical perturbations at the ATR-crystal surface often encountered with flow injection methods. To demonstrate the broad field of applications the binding of a heterotrimeric G-protein transducin with its membrane bound receptor rhodopsin was investigated as well as the pH-induced conformational change of the photoactivated receptor.

Moderne Flachendetektoren machen die FT-IR-Mikroskopie zu einem neuen Bildgebungsverfahren. Auch grosere Flachen sind innerhalb von Minuten bei sehr hoher Ortsauflosung zu vermessen. Ergebnisse lassen sich in leicht verstandlichen Bildern auswerten.

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)2Fe(L-cysteine)2], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)2Fe(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 NOx-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)2Fe(L-cysteine)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 dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

By use of a Fourier transform infrared FTIR spectroscopic imaging technique, we examine the dynamic optical clearing pro- cesses occurring in hyperosmotically biocompatible agents penetrat- ing 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. © 2008 Society of Photo-Optical Instrumentation Engineers. DOI: 10.1117/1.2899153

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