Oxygen-regulated mRNAs for light-harvesting and reaction center complexes and for bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus during the shift from anaerobic to aerobic growth.
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The stability and regulation by oxygen of mRNAs for the photosynthetic apparatus in Rhodobacter capsulatus have been studied by using proflavin to inhibit transcription and by shifting cells from anaerobic to aerobic conditions. The results from the inhibition experiments show that the mRNA for the light-harvesting LH-II polypeptides (beta, alpha) is more stable than that for the light-harvesting LH-I polypeptides (beta, alpha) during anaerobic growth, whereas the mRNAs for the reaction center polypeptides L (RC-L), M (RC-M), and H (RC-H) are less stable than both the LH-I and LH-II mRNAs. When photosynthetic cells are shifted from anaerobic to aerobic conditions, an immediate decrease in the levels of mRNA for the LH-I, LH-II, RC-L, RC-M, and RC-H proteins was observed. The level of mRNA for the LH-II proteins, however, is more sensitive to oxygen and is reduced faster than the level of mRNA for the LH-I proteins. These results suggest that oxygen represses the expression of genes coding for the light-harvesting antenna and reaction center complexes and may selectively accelerate the degradation of mRNA for the LH-II proteins. The mRNAs for several enzymes in the bacteriochlorophyll biosynthetic pathway are regulated by oxygen in a similar manner. The mRNAs for carotenoid biosynthetic enzymes, however, are regulated by oxygen in a different way. We have found that the amounts of mRNAs for carotenoid biosynthetic enzyme, relative to the amounts of mRNAs for LH and RC, increased during the shift from anaerobic to aerobic conditions. We have particularly shown that although the expression of most photosynthetic genes in R. capsulatus is repressed by oxygen, the crtA gene, located in the BamHI H fragment of the R' plasmid pRPS404 and responsible for the oxidation of spheroidene to spheroidenone, responds to oxygen in an opposite fashion. This exzymatic oxidation may protect the photosynthetic apparatus from photooxidative damage. (+info)
Calcium regulation of pigment transport in vitro.
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Calcium has been implicated in the regulation of many cellular motility events. In this study we have examined the role of different Ca2+ concentrations on the in vitro transport of pigment within cultured chromatophores. Cells treated with Brij detergent for 1-2 min were stripped of their plasma membranes, leaving their cytoskeleton and associated pigment granules exposed to the external milieu. We found that retrograde pigment transport (aggregation) is induced upon addition of 1 mM MgATP2- with 10(-7) M free Ca2+, while an orthograde transport (redispersal) of pigment results from lowering the concentration of free Ca2+ to 10(-8) M while maintaining 1 mM MgATP2-. These Ca2+-regulated movements are ATP dependent but are apparently independent of cAMP and insensitive to calmodulin inhibitors. The observations reported here provide novel evidence that the concentration of free Ca2+ acts to regulate the direction of intracellular organelle transport. (+info)
Chromatophore motor fields in the squid, Lolliguncula brevis.
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Chromatophore motoneurones in Lolliguncula brevis are known to originate in the suboesophageal lobes of the brain and to project directly to the mantle and fin through bilateral stellate ganglia and fin nerves. The chromatophore motor fields of stellar and fin nerves were investigated by stimulation of the cut end of individual nerves in a semi-intact preparation. This elicited expansion of yellow and brown chromatophores in distinct motor fields. Brown chromatophores extended over the entire mantle, whereas yellow chromatophores were limited to the dorsal and lateral mantle areas. Combined nerve stimulation and lesions demonstrated substantial overlap between adjacent chromatophore motor fields and innervation of individual chromatophores by different stellar nerves. (+info)
Improved screening for beta-lactam antibiotics. A sensitive, high-throughput assay using DD-carboxypeptidase and a novel chromophore-labeled substrate.
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The very sensitive and specific method for the detection of beta-lactam antibiotics using DD-carboxypeptidase (DDCase) from Actinomadura strain R39 has been improved to meet the requirements of a high-throughput beta-lactam screening from culture broths of microorganisms. The method is based on a novel chromophor-labeled substrate N alpha-acetyl-N epsilon-4-(7-nitrobenzofurazanyl)-L-lysyl-D-al anyl-D-alanine (ANLA2) which is converted by DDCase into ANLA1 with only one D-alanine residue left. Both compounds are intensely yellow as well as highly fluorescent and can be separated by thin-layer chromatography. This allows easy determination of residual DDCase activity after reaction with beta-lactams by simple visual inspection of chromatograms. Also, many assays can be run at a time without sophisticated instrumentation. Details of the method as well as some results of a beta-lactam screening performed with this type of assay are described. (+info)
Polarized pigment granule transport occurs in the absence of microtubules in squirrelfish erythrophores: studies of the effects of estramustine.
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We have re-examined the involvement of microtubules in the process of pigment granule transport in squirrelfish erythrophores in situ (i.e. on scales). Light-microscopic studies revealed that following exposure to 5 microM-nocodazole for 1 h at 4 degrees C erythrophores retained an ability to aggregate and disperse their pigment uniformly, though at reduced rates. Serial thick-section stereo high-voltage electron-microscopic studies showed that the entire microtubule population was removed by drug treatment and that the microtubules were not reassembled as a result of pigment translocation processes in the presence of reduced levels of nocodazole (0.4 microM). Immunofluorescence microscopic studies confirmed that nocodazole (0.5-1 microM) produced rapid disassembly of the microtubules. Whole-mount electron-microscopic studies showed that the pigment granules were suspended in a cross-linking network of 3-10 nm filaments, which appeared to support ordered pigment transport in situ in the absence of microtubules. Drug inhibition studies showed that micromolar levels of estramustine, a novel anti-MAPs (microtubule-associated proteins) drug, reversibly inhibited pigment transport. The results suggest that an estramustine-sensitive cytomatrix component might produce polarized pigment transport in intact erythrophores. (+info)
Photophosphorylation and oxidative phosphorylation in intact cells and chromatophores of an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114.
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Light-induced ATP synthesis was studied in intact cells and chromatophores of Erythrobacter sp. strain OCh114. ATP synthesis was measured by both the pH method and the luciferin-luciferase luminescence method. The rate of ATP synthesis was moderate (a typical value of 0.65 mol of ATP per mol of bacteriochlorophyll per min), and synthesis was inhibited by antimycin A. ATP was synthesized under illumination only under aerobic conditions and not under anaerobic conditions. This characteristic was similar to that of other light-induced energy transduction processes in this bacterial species, such as oxidation of reaction center, oxidation of cytochrome c551, and translocation of H+, which were not observed under anaerobic conditions. This phenomenon was reconciled with the fact that the Erythrobacter sp. could not grow anaerobically even in the light. The characteristics of oxidative phosphorylation and ATP hydrolysis were also investigated. The respiratory ratio of chromatophores was 2.3. Typical rates of oxidative phosphorylation by NADH and by succinate were 2.9 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.22) and 1.1 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.19), respectively. A typical rate of ATP hydrolysis was 0.25 mol of ATP per mol of bacteriochlorophyll per min in chromatophores. ATPase and adenylate kinase are also involved in the metabolism of adenine nucleotides in this bacterium. (+info)
Localization and stimulation of chromatophore motoneurones in the brain of the squid, Lolliguncula brevis.
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The relatively simple chromatophore system of the squid, Lolliguncula brevis, was studied with combined behavioural, morphological and electrophysiological methods in order to understand how the chromatophore patterns in the skin are organized at the level of the posterior chromatophore lobes (PCL). There are nine simple chromatic components of patterning in L. brevis. Retrograde transport of horseradish-peroxidase from chromatophores in the mantle skin established that the chromatophore motoneurones are located in the PCL. Focal threshold stimulation of the PCL in perfused, semi-intact preparations showed that the motor fields of individual chromatophore motoneurones are compact, including 2-60 chromatophores, generally of the same colour. Adjacent motoneurones in the lobe do not necessarily have adjacent motor fields in the skin. (+info)
Restrictions on rotational and translational diffusion of pigment in the membranes of a rhabdomeric photoreceptor.
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Individual, isolated rhabdoms from dark-adapted crayfish (Orconectes, Procambarus) were studied with a laterally incident microbeam that could be placed in single stacks of microvilli. Concentration gradients of metarhodopsin along the lengths of microvilli were produced by local bleaches, accomplished by irradiation with small spots of orange light at pH 9 in the presence of glutaraldehyde or formaldehyde. No subsequent redistribution of pigment was observed in the dark, indicating an absence of translational diffusion. On the basis of comparison with other systems, glutaraldehyde, but not formaldehyde (0.75%), would be expected to prevent diffusion of protein in the membrane. Under the same conditions photodichroism is observed, indicating an absence of free Brownian rotation. Photodichroism is larger in glutaraldehyde than in formaldehyde, suggesting that the bifunctional reagent quiets some molecular motion that is present after treatment with formaldehyde. Quantitative comparison of photodichroism with mathematical models indicates that the pigment absorption vectors are aligned within +/- 50 degrees of the microvillar axes and are tilted into the surface of the membrane at an average value of about 20 degrees. The photoconversion of rhodopsin to metarhodopsin is accompanied by an increase in molar extinction of about 20% at the lambda maxand a reorientation of the absorption vector by several degrees. The transition moment either tilts further into the membrane or loses some of its axial orientation, or both. The change in orientation is 3.5 time larger in formaldehyde than in glutaraldehyde. (+info)