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.

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor–urotensin–sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of ≈1 μM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29–R30 and R30–A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.

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.

Complementary DNA Cloning and Characterization of Ferredoxin Localized in Bundle-Sheath Cells of Maize Leaves1

In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells. A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe. The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry. RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively. The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds. Fd I and Fd II had similar redox potentials of −423 and −406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I. Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase. Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I. These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.

Desorption/ionization on silicon (DIOS): A diverse mass spectrometry platform for protein characterization

Since the advent of matrix-assisted laser desorption/ionization and electrospray ionization, mass spectrometry has played an increasingly important role in protein functional characterization, identification, and structural analysis. Expanding this role, desorption/ionization on silicon (DIOS) is a new approach that allows for the analysis of proteins and related small molecules. Despite the absence of matrix, DIOS-MS yields little or no fragmentation and is relatively tolerant of moderate amounts of contaminants commonly found in biological samples. Here, functional assays were performed on an esterase, a glycosidase, a lipase, as well as exo- and endoproteases by using enzyme-specific substrates. Enzyme activity also was monitored in the presence of inhibitors, successfully demonstrating the ability of DIOS to be used as an inhibitor screen. Because DIOS is a matrix-free desorption technique, it also can be used as a platform for multiple analyses to be performed on the same protein. This unique advantage was demonstrated with acetylcholine esterase for qualitative and quantitative characterization and also by its subsequent identification directly from the DIOS platform.

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.

Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.

Computation of Surface Electrical Potentials of Plant Cell Membranes : Correspondence to Published Zeta Potentials from Diverse Plant Sources

A Gouy-Chapman-Stern model has been developed for the computation of surface electrical potential (ψ0) of plant cell membranes in response to ionic solutes. The present model is a modification of an earlier version developed to compute the sorption of ions by wheat (Triticum aestivum L. cv Scout 66) root plasma membranes. A single set of model parameters generates values for ψ0 that correlate highly with published ζ potentials of protoplasts and plasma membrane vesicles from diverse plant sources. The model assumes ion binding to a negatively charged site (R− = 0.3074 μmol m−2) and to a neutral site (P0 = 2.4 μmol m−2) according to the reactions R− + IΖ ⇌ RIΖ−1 and P0 + IΖ ⇌ PIΖ, where IΖ represents an ion of charge Ζ. Binding constants for the negative site are 21,500 m−1 for H+, 20,000 m−1 for Al3+, 2,200 m−1 for La3+, 30 m−1 for Ca2+ and Mg2+, and 1 m−1 for Na+ and K+. Binding constants for the neutral site are 1/180 the value for binding to the negative site. Ion activities at the membrane surface, computed on the basis of ψ0, appear to determine many aspects of plant-mineral interactions, including mineral nutrition and the induction and alleviation of mineral toxicities, according to previous and ongoing studies. A computer program with instructions for the computation of ψ0, ion binding, ion concentrations, and ion activities at membrane surfaces may be requested from the authors.

Root Growth and Oxygen Relations at Low Water Potentials. Impact of Oxygen Availability in Polyethylene Glycol Solutions1

Polyethylene glycol (PEG), which is often used to impose low water potentials (ψw) in solution culture, decreases O2 movement by increasing solution viscosity. We investigated whether this property causes O2 deficiency that affects the elongation or metabolism of maize (Zea mays L.) primary roots. Seedlings grown in vigorously aerated PEG solutions at ambient solution O2 partial pressure (pO2) had decreased steady-state root elongation rates, increased root-tip alanine concentrations, and decreased root-tip proline concentrations relative to seedlings grown in PEG solutions of above-ambient pO2 (alanine and proline accumulation are responses to hypoxia and low ψw, respectively). Measurements of root pO2 were made using an O2 microsensor to ensure that increased solution pO2 did not increase root pO2 above physiological levels. In oxygenated PEG solutions that gave maximal root elongation rates, root pO2 was similar to or less than (depending on depth in the tissue) pO2 of roots growing in vermiculite at the same ψw. Even without PEG, high solution pO2 was necessary to raise root pO2 to the levels found in vermiculite-grown roots. Vermiculite was used for comparison because it has large air spaces that allow free movement of O2 to the root surface. The results show that supplemental oxygenation is required to avoid hypoxia in PEG solutions. Also, the data suggest that the O2 demand of the root elongation zone may be greater at low relative to high ψw, compounding the effect of PEG on O2 supply. Under O2-sufficient conditions root elongation was substantially less sensitive to the low ψw imposed by PEG than that imposed by dry vermiculite.

Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.

Ice as a matrix for IR-matrix-assisted laser desorption/ionization: mass spectra from a protein single crystal.

Lasers emitting in the ultraviolet wavelength range of 260-360 nm are almost exclusively used for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of macromolecules. Reports about the use of lasers emitting in the infrared first appeared in 1990/1991. In contrast to MALDI in the ultraviolet, a very limited number of reports on IR-MALDI have since been published. Several matrices have been identified for infrared MALDI yielding spectra of a quality comparable to those obtained in the ultraviolet. Water (ice) was recognized early as a potential matrix because of its strong O-H stretching mode near 3 microm. Interest in water as matrix derives primarily from the fact that it is the major constituent of most biological tissues. If functional as matrix, it might allow the in situ analysis of macromolecular constituents in frozen cell sections without extraction or exchanging the water. We present results that show that IR-MALDI of lyophilized proteins, air dried protein solutions, or protein crystals up to a molecular mass of 30 kDa is possible without the addition of any separate matrix. Samples must be frozen to retain a sufficient fraction of the water of hydration in the vacuum. The limited current sensitivity, requiring at least 10 pmol of protein for a successful analysis needs to be further improved.