In vivo kinetics of a redox-regulated transcriptional switch

SoxR is a transcription activator governing a cellular response to superoxide and nitric oxide in Escherichia coli. SoxR protein is a homodimer, and each monomer has a redox-active [2Fe–2S] cluster. Oxidation and reduction of the [2Fe–2S] clusters can reversibly activate and inactivate SoxR transcriptional activity. Here, we use electron paramagnetic resonance spectroscopy to follow the redox-switching process of SoxR protein in vivo. SoxR [2Fe–2S] clusters were in the fully reduced state during normal aerobic growth, but were completely oxidized after only 2-min aerobic exposure of the cells to superoxide-generating agents such as paraquat. The oxidized SoxR [2Fe–2S] clusters were rapidly re-reduced in vivo once the oxidative stress was removed. The in vivo kinetics of SoxR [2Fe–2S] cluster oxidation and reduction exactly paralleled the increase and decrease of transcription of soxS, the target gene for SoxR. The kinetic analysis also revealed that an oxidative stress-linked decrease in soxS mRNA stability contributes to the rapid attainment of a new steady state after SoxR activation. Such a redox stress-related change in soxS mRNA stability may represent a new level of biological control.

Iron accumulation in Alzheimer disease is a source of redox-generated free radicals

Damage from free radicals has been demonstrated in susceptible neuronal populations in cases of Alzheimer disease. In this study, we investigated whether iron, a potent source of the highly reactive hydroxyl radical that is generated by the Fenton reaction with H2O2, might contribute to the source of radicals in Alzheimer disease. We found, using a modified histochemical technique that relies on the formation of mixed valence iron complexes, that redox-active iron is associated with the senile plaques and neurofibrillary tangles—the pathological hallmark lesions of this disease. This lesion-associated iron is able to participate in in situ oxidation and readily catalyzes an H2O2-dependent oxidation. Furthermore, removal of iron was completely effected using deferoxamine, after which iron could be rebound to the lesions. Characterization of the iron-binding site suggests that binding is dependent on available histidine residues and on protein conformation. Taken together, these findings indicate that iron accumulation could be an important contributor toward the oxidative damage of Alzheimer disease.

The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior

We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E. coli to environments where maximal energy is available for growth.

Recurrent excitatory postsynaptic potentials induced by synchronized fast cortical oscillations

Gamma frequency (about 20–70 Hz) oscillations occur during novel sensory stimulation, with tight synchrony over distances of at least 7 mm. Synchronization in the visual system has been proposed to reflect coactivation of different parts of the visual field by a single spatially extended object. We have shown that intracortical mechanisms, including spike doublet firing by interneurons, can account for tight long-range synchrony. Here we show that synchronous gamma oscillations in two sites also can cause long-lasting (>1 hr) potentiation of recurrent excitatory synapses. Synchronous oscillations lasting >400 ms in hippocampal area CA1 are associated with an increase in both excitatory postsynaptic potential (EPSP) amplitude and action potential afterhyperpolarization size. The resulting EPSPs stabilize and synchronize a prolonged beta frequency (about 10–25 Hz) oscillation. The changes in EPSP size are not expressed during non-oscillatory behavior but reappear during subsequent gamma-oscillatory events. We propose that oscillation-induced EPSPs serve as a substrate for memory, whose expression either enhances or blocks synchronization of spatially separated sites. This phenomenon thus provides a dynamical mechanism for storage and retrieval of stimulus-specific neuronal assemblies.

Calcium electrogenesis in distal apical dendrites of layer 5 pyramidal cells at a critical frequency of back-propagating action potentials

Action potentials in juvenile and adult rat layer-5 neocortical pyramidal neurons can be initiated at both axonal and distal sites of the apical dendrite. However, little is known about the interaction between these two initiation sites. Here, we report that layer 5 pyramidal neurons are very sensitive to a critical frequency of back-propagating action potentials varying between 60 and 200 Hz in different neurons. Bursts of four to five back-propagating action potentials above the critical frequency elicited large regenerative potentials in the distal dendritic initiation zone. The critical frequency had a very narrow range (10–20 Hz), and the dendritic regenerative activity led to further depolarization at the soma. The dendritic frequency sensitivity was suppressed by blockers of voltage-gated calcium channels, and also by synaptically mediated inhibition. Calcium-fluorescence imaging revealed that the site of largest transient increase in intracellular calcium above the critical frequency was located 400–700 μm from the soma at the site for initiation of calcium action potentials. Thus, the distal dendritic initiation zone can interact with the axonal initiation zone, even when inputs to the neuron are restricted to regions close to the soma, if the output of the neuron exceeds a critical frequency.

Chemical complementation identifies a proton acceptor for redox-active tyrosine D in photosystem II

Through the use of site-directed mutagenesis and chemical rescue, we have identified the proton acceptor for redox-active tyrosine D in photosystem II (PSII). Effects of chemical rescue on the tyrosyl radical were monitored by EPR spectroscopy. We also have acquired the Fourier–transform infrared (FT-IR) spectrum associated with the oxidation of tyrosine D and concomitant protonation of the acceptor. Mutant and isotopically labeled PSII samples are used to assign vibrational lines in the 3,600–3,100 cm−1 region to N-H modes of His-189 in the D2 polypeptide. When His-189 in D2 is changed to a leucine (HL189D2) in PSII, dramatic alterations of both EPR and FT-IR spectra are observed. When imidazole is introduced into HL189D2 samples, results from both EPR and FT-IR spectroscopy argue that imidazole is functionally reconstituted into an accessible pocket and that imidazole acts as a chemical mimic for His-189. Small perturbations of EPR and FT-IR spectra are consistent with access to this pocket in wild-type PSII, as well. Structures of the analogous site in bacterial reaction centers suggest that an accessible pocket, large enough to contain imidazole, is bordered by tyrosine D and His-189 in the D2 polypeptide. These data provide evidence that His-189 in the D2 polypeptide of PSII acts as a proton acceptor for redox-active tyrosine D and that proton transfer to the imidazole ring facilitates the efficient oxidation/reduction of tyrosine D.

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.

Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Cα), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators.

Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell

We have discovered that intracellular redox state appears to be a necessary and sufficient modulator of the balance between self-renewal and differentiation in dividing oligodendrocyte-type-2 astrocyte progenitor cells. The intracellular redox state of freshly isolated progenitors allows prospective isolation of cells with different self-renewal characteristics. Redox state is itself modulated by cell-extrinsic signaling molecules that alter the balance between self-renewal and differentiation: growth factors that promote self-renewal cause progenitors to become more reduced, while signaling molecules that promote differentiation cause progenitors to become more oxidized. Moreover, pharmacological antagonists of the redox effects of these cell-extrinsic signaling molecules antagonize their effects on self-renewal and differentiation, indicating that cell-extrinsic signaling molecules that modulate this balance converge on redox modulation as a critical component of their effector mechanism.

Plastid redox state and sugars: Interactive regulators of nuclear-encoded photosynthetic gene expression

Feedback regulation of photosynthesis by carbon metabolites has long been recognized, but the underlying cellular mechanisms that control this process remain unclear. By using an Arabidopsis cell culture, we show that a block in photosynthetic electron flux prevents the increase in transcript levels of chlorophyll a/b-binding protein and the small subunit of Rubisco that typically occurs when intracellular sugar levels are depleted. In contrast, the expression of the nitrate reductase gene, which is induced by sugars, is not affected. These findings were confirmed in planta by using Arabidopsis carrying the firefly luciferase reporter gene fused to the plastocyanin and chlorophyll a/b-binding protein 2 gene promoters. Transcription from both promoters increases on carbohydrate depletion. Blocking photosynthetic electron transport with 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea prevents this increase in transcription. We conclude that plastid-derived redox signaling can override the sugar-regulated expression of nuclear-encoded photosynthetic genes. In the sugar-response mutant, sucrose uncoupled 6 (sun6), plastocyanin-firefly luciferase transcription actually increases in response to exogenous sucrose rather than decreasing as in the wild type. Interestingly, plastid-derived redox signals do not influence this defective pattern of sugar-regulated gene expression in the sun6 mutant. A model, which invokes a positive inducer originating from the photosynthetic electron transport chain, is proposed to explain the nature of the plastid-derived signal.

Proline Accumulation in Maize (Zea mays L.) Primary Roots at Low Water Potentials. II. Metabolic Source of Increased Proline Deposition in the Elongation Zone1

The proline (Pro) concentration increases greatly in the growing region of maize (Zea mays L.) primary roots at low water potentials (ψw), largely as a result of an increased net rate of Pro deposition. Labeled glutamate (Glu), ornithine (Orn), or Pro was supplied specifically to the root tip of intact seedlings in solution culture at high and low ψw to assess the relative importance of Pro synthesis, catabolism, utilization, and transport in root-tip Pro deposition. Labeling with [3H]Glu indicated that Pro synthesis from Glu did not increase substantially at low ψw and accounted for only a small fraction of the Pro deposition. Labeling with [14C]Orn showed that Pro synthesis from Orn also could not be a substantial contributor to Pro deposition. Labeling with [3H]Pro indicated that neither Pro catabolism nor utilization in the root tip was decreased at low ψw. Pro catabolism occurred at least as rapidly as Pro synthesis from Glu. There was, however, an increase in Pro uptake at low ψw, which suggests increased Pro transport. Taken together, the data indicate that increased transport of Pro to the root tip serves as the source of low-ψw-induced Pro accumulation. The possible significance of Pro catabolism in sustaining root growth at low ψw is also discussed.

Root Xylem Embolisms and Refilling. Relation to Water Potentials of Soil, Roots, and Leaves, and Osmotic Potentials of Root Xylem Sap1

Embolism and refilling of vessels was monitored directly by cryomicroscopy of field-grown corn (Zea mays L.) roots. To test the reliability of an earlier study showing embolism refilling in roots at negative leaf water potentials, embolisms were counted, and root water potentials (Ψroot) and osmotic potentials of exuded xylem sap from the same roots were measured by isopiestic psychrometry. All vessels were full at dawn (Ψroot −0.1 MPa). Embolisms were first seen in late metaxylem vessels at 8 am. Embolized late metaxylem vessels peaked at 50% at 10 am (Ψroot −0.1 MPa), fell to 44% by 12 pm (Ψroot −0.23 MPa), then dropped steadily to zero by early evening (Ψroot −0.28 MPa). Transpiration was highest (8.5 μg cm−2 s−1) between 12 and 2 pm when the percentage of vessels embolized was falling. Embolized vessels were refilled by liquid moving through their lateral walls. Xylem sap was very low in solutes. The mechanism of vessel refilling, when Ψroot is negative, requires further investigation. Daily embolism and refilling in roots of well-watered plants is a normal occurrence and may be a component of an important hydraulic signaling mechanism between roots and shoots.

Event-related brain potentials in the study of visual selective attention

Event-related brain potentials (ERPs) provide high-resolution measures of the time course of neuronal activity patterns associated with perceptual and cognitive processes. New techniques for ERP source analysis and comparisons with data from blood-flow neuroimaging studies enable improved localization of cortical activity during visual selective attention. ERP modulations during spatial attention point toward a mechanism of gain control over information flow in extrastriate visual cortical pathways, starting about 80 ms after stimulus onset. Paying attention to nonspatial features such as color, motion, or shape is manifested by qualitatively different ERP patterns in multiple cortical areas that begin with latencies of 100–150 ms. The processing of nonspatial features seems to be contingent upon the prior selection of location, consistent with early selection theories of attention and with the hypothesis that spatial attention is “special.”

Potentials of the National Corn Genome Initiative

The present paper summarizes future needs in information and tools, technology, infrastructure, training, funding, and bioinformatics, to provide the genomic knowledge and tools for breeding and biotechnological goals in maize. The National Corn Genome Initiative (NCGA) has developed through actions taken by the National Corn Growers Association (NCGA) and participation in a planning process by institutions, companies, and organizations. At the web address for the NCGI, http://www.inverizon.com/ncgi, are detailed analyses of goals and costs, impact and value, and strategy and approaches. The NCGI has also produced an informative and perceptive video suitable for public groups or schools, about agricultural contributions to life and the place of maize in these contributions. High potential can be expected, from cross-application of knowledge obtained in maize and other cereals. Development of information and tools for all crops, whether monocots or dicots, will be gained through an initiative, and each crop will be positioned to advance with cost-effective parallels, especially for expressed sequences, markers, and physical mapping.