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Fluorescence techniques are widely applied in protein research owing to their specificity and sensitivity, but require prior fluorescent labeling. Here we show a novel approach to optimize labeling protocols by monitoring labeling reactions using fluorescence polarization: the larger molecular mass of the fluorescent protein conjugate compared to the dye alone results in an increase in fluorescence anisotropy during the reaction. Thereby, labeling of lysozyme with fluorescein isothiocyanate or carboxyfluorescein succinimidyl ester could be monitored and the influence of parameters such as the pH could be quantitatively assessed. Moreover, the impact and kinetics of side reactions such as hydrolysis were determined. This new method is rapid, easy to implement, and generically applicable.

Combining virtual screening with biophysical studies of protein-ligand complexes is an effective tool for designing new peptidomimetics. When a three-dimensional structure of the target protein is known, automated docking of chemical databases can be used as a powerful filter to reduce the number of molecules that will be further tested. Easy screening of potential hits can then be performed using fluorescence polarization techniques, if a fluorescent-labeled ligand already exists for the target. In addition, thermodynamic properties of protein-ligand complexes can be measured by circular dichroism spectroscopy.

In this study the morphological changes in the shape and structure of the ulna were investigated following resection of a mid-shaft segment in the radius. These morphological alterations are called in German "Martin'sche Umbauzone" (MUZ; Martin's transformation zone). Various explanations for the MUZ can be found in the literature; Wolff's law, in particular, offers hints about the development of this phenomenon. The morphological changes in the ulna were investigated by microangiography, polarized light and fluorescence microscopy as well as by micro-radiography. The radius was operated on in 36 mongrel dogs. The development and localization of new bone formation at the endosteal and periosteal envelope of the ulna were studied. The woven bone formation of the envelopes (modelling) and the intracortical remodelling in the distal part of the ulnae were compared with the intact ulnae of the opposite extremities. It is concluded from these morphological alterations that the experimental design chosen provides a valuable, tissue-related model for the investigation of morphogenesis in bones. Further investigations will deal with the correlation between strain and new bone formation.

Ten intensive care patients and five healthy volunteers each received a bolus injection of lidocaine HCl (100 mg, 2%) over an injection period of 5 s. After 0.5, 1, 2, 4, 8, 15 and 25 min arterial, central venous and peripheral venous blood samples were collected. In four of the volunteers, arterial and central venous samples were also taken about 10 s after the end of injection. The fluorescence polarization method by means of the Abbott-TDx system was used, and plasma concentrations of lidocaine were determined. The measurements showed that lidocaine levels in central venous plasma 10 s after the end of administration were higher than those in arterial plasma. By 30 s after administration the opposite situation had developed, so that arterial concentrations were higher than those in central venous plasma. This relation did not change throughout the study, though the two levels became closer, as is shown by the ratios (Table 3, Fig. 2). Concentrations in peripheral venous plasma increased more slowly but remained far below those in arterial and central venous plasma, at least for the first 8 min. After 15 min lidocaine levels were almost the same in all three samples. During the entire study there were no ECG changes, and neither heart rate nor blood pressure showed any significant deviation from the values obtained at the beginning. The volunteers had minor toxic manifestations, such as dizziness, tinnitus and a metallic taste in the mouth; one person had a sensation of pressure in his chest, which improved following oxygen administration.(ABSTRACT TRUNCATED AT 250 WORDS)

A total of 120 samples of serum were obtained from 8 patients with COPD and submitted to a comparative analysis of serum theophylline levels employing the TDX and the EMIT methods of determination. At the same time, two different slow-release preparations of theophylline were compared, their kinetics investigated, and their effects tested using body plethysmography. For this open, randomized cross-over preliminary investigation, numerous inclusion and exclusion criteria were applied.

Histological, histochemical, fluorescence optical and polarization microscopical investigations were performed in 53 placentas of rabbits from the 10th to the 35th day of pregnancy. A "sialomucin coat" could be demonstrated in all cases at the maternal surface of the metabolic barrier of the placenta. Polarization optical measurements after association with N,N'-diethylpseudoisocyanine chloride showed a lower content of sialic acid at the surface of the trophoblast in postmature placentas of rabbits in comparison to normal mature placentas. The immunological function of the placental barrier is discussed with respect to maturity.

The determination of CRP in serum was performed by the fluorescence polarization immunoassay (FPIA) and the kinetic nephelometry (KNM). Both methods showed a good precision and accuracy. The investigation was carried out in 190 sera with a CRP concentration up to 49 mg/dl. The comparison of the two methods in 100 sera with a CRP concentration between 0.6 and 5 mg/dl showed a good correlation (correlation coefficient r = 0.9753). Close correlation (correlation coefficient r = 0.9947) was also found in 90 sera with a CRP concentration between 5 and 49 mg/dl. Discussion of the FPIA and KNM methods.

Long time treatment of asthma in children with theophylline requires therapeutic drug monitoring by determination of serum concentrations. Beside established assays a carrier-bound reagent test has recently been developed, which has been demonstrated to be accurate, precise and cheaper than other assays. It has the advantage to give rapid results within minutes, which makes it suitable for intensive care units as well as for outpatient care.

The article reports threshold ranges and detection limits for the screening of tetracyclic benzodiazepines (adinazolam, alprazolam, brotizolam, estazolam, loprazolam, midazolam, triazolam and many metabolites) using the EMIT-st and TDx system (FPIA). In most cases, the cross reactivities of the two systems are comparable, but there are also remarkable differences. Detection limits are mainly in the range between 0.2 and 0.5 mg/l but, in some cases, the limits are also considerably higher. The within-day precision is 1.1% (TDx) and 16.5% (EMIT-st); day-to-day precision is 4.7% (TDx) and 12.6% (EMIT-st), respectively.

A new method for determining fetal lung maturity is discussed. Molecular mobility can be determined spectrophotometrically by measuring the fluorescent polarisation (FP) in lipid solutions. This method of determining the microviscosity in lipid solutions can now be applied also in the amniotic fluid. Measurement of FP by means of an electro-optical microviscosimeter is proportional to the lipid concentration in the amniotic fluid and is in good correlation with fetal lung maturity. FP was measured between the 17th and 41st weeks of gestation in 147 amniotic fluid samples. During pregnancy the FP value drops significantly from 0.350 to 0.200. A FP value of 0.290 was fixed as the limit value beyond which RDS may be expected to occur. With a limit value of 0.290, the RDS identification probability was calculated at 94%, whereas the calculated probability of RDS was 81% and the exclusion probability of RDS 97%. At the same time, the probabilities, calculated on the parallel determination of the L/S ratio, yielded a slightly more unfavourable result. The drop in the FP level correlates significantly with the increase of the L/S ratio in the investigated amniotic fluid samples (correlation coefficient 0.77). The method, which requires very special equipment, offers many advantages: it is very accurate, simple, well reproducible and can be performed within half an hour at relatively low laboratory cost.