PubMed · 5665708
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P A Bretscher. 1968-08-10. Models for haem-haem interaction.. https://doi.org/10.1038/219606a0
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The photocycles of the wild-type bacteriorhodopsin and the D96N mutant were investigated by the flash-photolysis technique. The M-intermediate formation (400 nm) and the L-intermediate decay (520 nm) were found to be well described by a sum of two exponents (time constants, tau 1 = 65 and tau 2 = 250 microseconds) for the wild-type bR and three exponents (tau 1 = 55 microseconds, tau 2 = 220 microseconds and tau 3 = 1 ms) for the D96N mutant of bR. A component with tau = 1 ms was found to be present in the photocycle of the wild-type bacteriorhodopsin as a lag-phase in the relaxation of photoresponses at 400 and 520 nm. In the presence of Lu3+ ions or 80% glycerol this component was clearly seen as an additional phase of M-formation. The azide effect on the D96N mutant of bR suggests that the 1-ms component is associated with an irreversible conformational change switching the Schiff base from the outward to the inward proton channel. The maximum of the difference spectrum of the 1-ms component of D96N bR is located at 404 nm as compared to 412 nm for the first two components. We suggest that this effect is a result of the alteration of the inward proton channel due to the Asp96-->Asn substitution. Proton release measured with pyranine in the absence of pH buffers was identical for the wild-type bR and D96N mutant and matched the M-->M' conformational transition. A model for M rise in the bR photocycle is proposed.
The Tradescantia-micronucleus (Trad-MCN) bioassay is an efficient short-term test for genotoxicity of pollutants. In order to increase the efficiency and to standardize the micronucleus (MCN) scoring process, an automated scoring system was developed using the principle of image analysis in computer science. This assemblage is called the Tradescantia-micronucleus image analysis (Trad-MCNIA) system. The MCN frequencies scored by this system were compared with those scored by human observation for its proficiency. A set of low MCN frequency (around 5 MCN/100 tetrads) slides prepared from a control group, a set of medium MCN frequency (around 20 MCN/100 tetrads) slides prepared from sodium azide treated plant cuttings and a set of high MCN frequency (around 50 MCN/100 tetrads) slides prepared from X-ray treated materials were used for this study. In the low MCN frequency slides, the Trad-MCNIA system scored about the same value as human observation. In the medium and high frequency slides, MCN frequencies scored by the system were lower than those scored by human observers. This discrepancy was corrected by increasing the power of the objective of the microscope in the system. The MCN frequencies scored by the system attained 90% congruity with those scored by human observers after the correction. The scoring speed of the system was about 3.5 times as fast as that by human observers, and the data could be statistically analyzed immediately after the data scores were recorded. Further improvements can be made by upgrading the video camera and the computer speed.
A peroxidase-independent method for the determination of phagocyte-derived hydrogen peroxide in vitro is described. The method is based on the catalase-inhibitable oxidation of added cysteine. Human blood neutrophils (1 x 10(6)/ml) were coincubated with the myeloperoxidase (MPO) inhibitor, sodium azide (10 micrograms/ml), for 30 min at 37 degrees C followed by addition of phorbol 12-myristate 13-acetate (PMA, 10 ng/ml), a potent activator of membrane-associated oxidative metabolism. After incubation at 37 degrees C the cells were removed and the supernatants aliquoted and incubated with and without catalase (500 U/ml) for 10 min at room temperature followed by addition of cysteine (50 microM). Thereafter the catalase-inhibitable, H2O2-mediated oxidation of added cysteine was assayed spectrophotometrically following the addition of the thiol-reactive agent 5,5'-dithiobis-(2-nitrobenzoic acid), and the H2O2 concentration determined using a standard curve constructed from difference data based on the oxidation of cysteine by H2O2 (0-200 nmol). The average amount of H2O2 produced by 10(6) PMA-activated neutrophils was 47 +/- 7 nmol/30 min. This sensitive assay procedure is likely to be particularly useful for investigating the effects of pharmacological agents and anti-oxidant nutrients on H2O2 production by activated phagocytes, since these agents are generally unsuited for use in peroxidase-based assays.