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.     

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.     

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.     

Axial dispersion in a liquid fluidized bed of particles akin to immobilized enzymes

The axial dispersion of a liquid fluidized bed of controlled pore silica (CPS) particles has been determined by the pulse tracer method. The CPS used was the same as for enzyme immobilization, having an average diameter of 0.436 mm and mean pore size of 37.5 nm. The fluidization liquid is α-amylase liquefied manioc starch, 30% w/v, 45°C pH=4.5. Nominal bed porosities tested were 0.7 and 0.8. The results show that the axial dispersion coefficient increases with greater superficial liquid velocities. Various available correlations tested disagree with each other to a large extent and are unable to represent collected experimental data.     

HALORHODOPSIN and PHOTOSENSORY BEHAVIOR IN Halobacterium halobium MUTANT STRAIN L-33

Halobacterium halobium , strain L-33, which is deficient in bacteriorhodopsin (BR) but synthesizes increased amounts of halorhodopsin (HR), shows behavioral responses upon changes in fluence rate with visible light or with UV light. The observations support the earlier report (Schimz et al. , 1982). that BR is not essential for photosensing in H. halobium. In the UV-range, changes in light intensity elicit the maximal response at λ= 370 nm. In the visible range, changes in light intensity show the maximal response at Δ= 565 nm and a secondary peak at Δ= 590 nm. The latter corresponds to the absorption maximum of HR (Δ_max= 588 nm). This light-energy converting retinal pigment of H. halobium thus appears to contribute to photosensory behavior.