PHOTOSENSORY BEHAVIOR OF A BACTERIORHODOPSIN-DEFICIENT MUTANT, ET-15, OF Halobacterium halobium

– Halobacterium halobium , strain ET-15, which does not contain detectable amounts of bacteriorhodopsin (BR) shows behavioral responses to UV and yellow-green light. Attractant stimuli. i.e. light-increases in the yellow-green range or light-decreases in the UV, suppress the spontaneous reversals of the swimming direction for a certain time. Repellent stimuli, i.e. light-decreases in the yellow-green range or light-increases in the UV, elicit an additional reversal response after a few seconds. Action spectra of both sensory photosystems, PS 370 and PS 565, were measured with attractant as well as with repellent stimuli. As in BR-containing cells, maximal sensitivity was always found at 370 nm for the UV-system and at 565 nm for the long-wavelength system. Fluence-response curves at 370 and 565 nm obtained with strain ET-15 and with a BR-containing strain show that the sensitivity of both photosystems is not reduced in the absence of BR. It is concluded that BR is required neither for PS 565 nor for PS 370. Instead retinal-containing pigments different from BR have to be assumed to mediate photosensory behavior.     

Übergangsmetall-substituierte phosphane,arsane und stibane : XXV. C5H5(CO)2[P(OMe)3]M-substituierte dioxaphospholane und dioxarsolane; stereochemische evidenz für den intermolekularen ablauf einer isomerisierung unter methylgruppenverschiebung

2-C₅H₅(CO)₂[P(OMe)₃]Mo(W)-substituted 4,4,5,5-tetramethyl-1,3,2-dioxarsolanes and -dioxaphospholanes (Ia–Ic) are obtained from C₅H₅(CO)₃M derivatives via CO/P(OMe)₃ exchange. In all cases the phosphite ligand appears trans to the σ-bonded arsenic heterocycle, which prefers a conformation with the transition metal group in an axial position. The dioxaphospholane-metal (Ia) undergoes isomerization with alkyl migration at 0°C, which due to stereochemical reasons can only occur by an intermolecular mechanism.     

Electronic and molecular properties of an adsorbed protein monolayer probed by two-color sum-frequency generation spectroscopy

Two-color sum-frequency generation spectroscopy (2C-SFG) is used to probe the molecular and electronic properties of an adsorbed layer of the green fluorescent protein mutant 2 (GFPmut2) on a platinum (111) substrate. First, the spectroscopic measurements, performed under different polarization combinations, and atomic force microscopy (AFM) show that the GFPmut2 proteins form a fairly ordered monolayer on the platinum surface. Next, the nonlinear spectroscopic data provide evidence of particular coupling phenomena between the GFPmut2 vibrational and electronic properties. This is revealed by the occurrence of two doubly resonant sum-frequency generation processes for molecules having both their Raman and infrared transition moments in a direction perpendicular to the sample plane. Finally, our 2C-SFG analysis reveals two electronic transitions corresponding to the absorption and fluorescence energy levels which are related to two different GFPmut2 conformations: the B (anionic) and I forms, respectively. Their observation and wavelength positions attest the keeping of the GFPmut2 electronic properties upon adsorption on the metallic surface.     

Immunoaffinity screening with capillary electrochromatography

Highly efficient capillary electrochromatographic separations of cardiac glycosides and other steroids are presented. Employing butyl-derivatized silica particles as stationary phase resulted in a nearly three times faster electroosmotic flow (EOF) compared to capillary electrochromatography (CEC) with octadecyl silica particles. On-column focusing with a preconcentration factor of 180 was performed and separation efficiencies of up to 240,000 plates per meter were obtained. Using label-free standard UV absorbance, detection limits of 10-80 nM were reached for all steroids tested. For screening of cardiac glycosides, e.g., digoxin and digitoxin in mixtures of steroids, CEC was combined with immunoaffinity extraction using immobilized polyclonal anti-digoxigenin antibodies and F(ab) fragments. Simply adding small amounts of antibody carrying particles to the samples and comparing chromatograms before and after antibody addition allowed screening for high affinity antigens in mixtures with moderate numbers of compounds. Under conditions of competing antigens, affinity fingerprints of immobilized anti-digoxigenin and anti-digitoxin antibodies were obtained, reflecting the cross-reactivity of eleven steroids. The method provides high selectivity due to the combination of bioaffinity interaction with highly efficient CEC separation and UV detection at several wavelengths in parallel. This selectivity was exploited for the detection of four cardiac glycosides in submicromolar concentrations in an untreated urine sample.     

Determination of sub-nanogram amounts of dihydroergotamine in plasma and urine using liquid chromatography and fluorimetric detection with off-line and on-line solid-phase drug enrichment

A highly sensitive and selective high-performance liquid chromatographic method, involving sample pre-treatment, column switching and fluorimetric detection, is described for the determination of dihydroergotamine in plasma and urine samples. The pre-chromatographic sample treatment consists of extraction by means of an Extrelut column for plasma samples, and pre-separation with enrichment steps on a Sep-Pak column for urine samples. The samples are then injected onto a pre-separation column (Aquapore), and the fraction containing dihydroergotamine are automatically diverted onto an analytical column (ODS reversed phase). An acetonitrile-ammonium carbamate gradient is used as the mobile phase. High recovery of dihydroergotamine from both plasma (87%) and urine (100%) and a detection limit as low as 100 pg/ml were achieved, with a linear response up to 5 ng/ml. The assay demonstrated a high degree of selectivity with regard to the extensive metabolism of dihydroergotamine especially to the main metabolite 8'-hydroxydihydroergotamine. The assay was successfully applied for more than one year to the determination of plasma and urine concentrations of dihydroergotamine after parenteral administration.     

Dual-label autoradiographic analysis of human skin fibroblast and myoblast proteins by two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension

Horizontal two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two-dimensional separations of skin fibroblast proteins were obtained using pH 4-10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4-7 and pH 7-10 immobilised pH gradient gels respectively in the first dimension. Two-dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two-dimensional electrophoresis, using pH 4-7 and pH 7-10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual-labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two-dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two-dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.