Glutathione initiates the development of Dictyostelium discoideum through the regulation of YakA

Reduced glutathione (GSH) is an essential metabolite that performs multiple indispensable roles during the development of Dictyostelium. We show here that disruption of the gene (gcsA¯) encoding γ-glutamylcysteine synthetase, an essential enzyme in GSH biosynthesis, inhibited aggregation, and that this developmental defect was rescued by exogenous GSH, but not by other thiols or antioxidants. In GSH-depleted gcsA¯ cells, the expression of a growth-stage-specific gene (cprD) was not inhibited, and we did not detect the expression of genes that encode proteins required for early development (cAMP receptor, carA/cAR1; adenylyl cyclase, acaA/ACA; and the catalytic subunit of protein kinase A, pkaC/PKA-C). The defects in gcsA¯ cells were not restored by cAMP stimulation or by cAR1 expression. Further, the expression of yakA, which initiates development and induces the expression of PKA-C, ACA, and cAR1, was regulated by the intracellular concentration of GSH. Constitutive expression of YakA in gcsA¯ cells (YakA^OE/gcsA¯) rescued the defects in developmental initiation and the expression of early developmental genes in the absence of GSH. Taken together, these findings suggest that GSH plays an essential role in the transition from growth to development by modulating the expression of the genes encoding YakA as well as components that act downstream in the YakA signaling pathway.     

Feedback regulation of photosynthetic electron transport by NADP(H) redox poise

When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought), and can result in the production of reactive oxygen species (ROS). Here, we present evidence for a novel protective process — feedback redox regulation via the redox poise of the NADP(H) pool. Photosynthetic electron transport was studied in two transgenic tobacco (Nicotiana tabacum) lines — one having reduced levels of ferredoxin NADP⁺-reductase (FNR), the enzyme responsible for reducing NADP⁺, and the other reduced levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the principal consumer of NADPH. Both had a similar degree of inhibition of carbon fixation and impaired electron transport. However, whilst FNR antisense plants were obviously stressed, with extensive bleaching of leaves, GAPDH antisense plants showed no visible signs of stress, beyond having a slowed growth rate. Examination of electron transport in these plants indicated that this difference is due to feedback regulation occurring in the GAPDH but not the FNR antisense plants. We propose that this reflects the occurrence of a previously undescribed regulatory pathway responding to the redox poise of the NADP(H) pool.     

Photosynthetic activity of diimidoester-modified cells,permeaplasts, and cell-free membrane fragments of the blue-green alga Anacystis nidulans

On treating the blue-green alga Anacystis nidulans with dimethylsuberimidate up to 70% of the free NH₂ of the photosynthetic membrane is amidinated, and presumably inter- and intramolecular cross-links are established in the membrane proteins. Amidination destroys the ability of A. nidulans to photoreduce HCO₃^? but leaves the photochemical activities of Photosystems II and I nearly intact. With added electron acceptors, photosynthetic O₂ evolution can be demonstrated both with permeable cells (permeaplasts) prepared by digestion of the cell wall of dimethylsuberimidate-reacted A. nidulans with lysozyme, as well as with heavy membrane particles (36 000 × g) prepared from dimethylsuberimidate-reacted cells.Permeaplasts prepared from dimethylsuberimidate-reacted cells resist damage in hypoosmotic medium, whereas those prepared from unreacted cells are induced to release C-phycocyanin. On the other hand, the former are inactivated more easily by heat stress than the latter. On this basis, it is concluded that cross-linking with dimethylsuberimidate confers functional instability to photosynthetic membranes.     

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c₂, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{3–5 μ}\text{s}$). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (1–2 ms) antimycin-sensitive phase of ferricytochrome c₂ reduction, attributed to a specific ubiquinone-10 molecule (Q_z), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Q_z (Q_zH₂) reduces the Rieske center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c₂ reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center.UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from g_y 1.90 to g_y 1.89, and shifts the E_m,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c₂, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of H⁺_II; these may reflect additional sites of action of the inhibitor.     

Antibodies against oxidation-specific epitopes and risk of acute myocardial infarction

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A mechanistic link between oxidative stress and membrane mediated amyloidogenesis revealed by infrared spectroscopy

The fully developed lesion of Alzheimer's disease is a dense plaque composed of fibrillar amyloid β-proteins (Aβ) with a characteristic and well-ordered β-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form β-sheet structure, it is important to understand factors that induced Aβ to adopt this conformation. In this review, we describe the application of polarized attenuated total internal reflection infrared FT-IR spectroscopy for characterizing the conformation, orientation, and rate of accumulation of Aβ on lipid membranes. We also describe the application and yield of linked analysis, whereby multiple spectra are fit simultaneously with component bands that are constrained to share common fitting parameters. Results have shown that membranes promote β-sheet formation under a variety of circumstances that may be significant to the pathogenesis of Alzheimer's disease.     

Examination of protein sequence homologies: IV. Twenty-seven bacterial ferredoxins

Summary Sequence homologies of 27 bacterial ferredoxins were examined using a computer program that quantitatively evaluates extent of similarity as a correlation coefficient. The results of a similarity search among the sequences demonstrated that the basal sequence consists of a pair of extremely similar segments of 26 amino acids connected by a three-amino acid group. The segment pairs, which would have arisen from gene duplication, are termed the first and second units. Because of the gene duplication, the connector sequence appears to have been introduced as a structurally important chain reversal. Each of the two units contains four cysteine residues, which are inserted one by one among seven, two, two, three, and eight amino acid alignments, respectively. The bacterial ferredoxins were categorized with regard to basal constitution as follows: group 1, in which both units closely conform to the basal structure; group 2, in which the second unit is modified in a characteristic manner among members; group 3, in which the first unit is modified in a characteristic manner, while the conforming second unit is accompanied by a long accessory sequence; group 4, in which there are modifications before and/or after the units, of which the respective central domains remain nearly intact; and group 5, where only the former of two Fe:S cluster ligation sets of four cysteines is estimated to remain intact, whereas the latter set is extremely modified. It is noteworthy that throughout all bacterial ferredosins, one of two cysteine sets never fails to be completely intact and, moreover, the connector of three amino acids also exists intact. Based on this grouping and on the correspondences among the groups, average correlation coefficients among all members were computed, and the respective evolutionary relationships were examined. The results supported the proposition that transposition had occurred in theAzotobacter-type ferredoxins of group 3.     

Purification and properties of ferredoxinBPH, a component of biphenyl 2,3-dioxygenase of Pseudomonas sp strain LB400

The ferredoxin component (ferredoxin_BPH) of biphenyl 2,3-dioxygenase was purified to homogeneity from crude cell extract of Pseudomonas sp strain LB400 using ion exchange, hydrophobic interaction and gel filtration column chromatography. The protein was a monomer with a molecular weight of 15000 and contained 2 gram-atoms each of iron and acid-labile sulfur. Ultraviolet-visible absorbance spectroscopy showed peaks at 325 nm and 460 nm with a broad shoulder around 575 nm. The spectrum was partially bleached in the visible region upon reduction by reductase_BPH with NADPH as the source of electrons. Electron paramagnetic resonance spectrometry showed no signals for the oxidized protein. Upon reduction with sodium dithionite, signals with g_x = 1.82, g_y = 1.92 and g_z = 2.02 were detected. These results indicate that the protein contains a Rieske-type (2Fe-2S) iron-sulfur center. Ferredoxin_BPH was required for the oxidation of biphenyl by the terminal oxygenase component of the enzyme and is probably involved in the transfer of reducing equivalents from reductase_BPH to the terminal oxygenase during catalysis. Received 01 November 1996/ Accepted in revised form 27 May 1997