Long-term potentiation and dual-component quantal signaling in the dentate gyrus

Long-term potentiation (LTP) of excitatory transmission is an important candidate cellular mechanism for the storage of memories in the mammalian brain. The subcellular phenomena that underlie the persistent increase in synaptic strength, however, are incompletely understood. A potentially powerful method to detect a presynaptic increase in glutamate release is to examine the effect of LTP induction on the rate at which the use-dependent blocker MK-801 attenuates successive N-methyl-d-aspartic acid (NMDA) receptor-mediated synaptic signals. This method, however, has given apparently contradictory results when applied in hippocampal CA1. The inconsistency could be explained if NMDA receptors were opened by glutamate not only released from local presynaptic terminals, but also diffusing from synapses on neighboring cells where LTP was not induced. Here we examine the effect of pairing-induced LTP on the MK-801 blocking rate in two afferent inputs to dentate granule cells. LTP in the medial perforant path is associated with a significant increase in the MK-801 blocking rate, implying a presynaptic increase in glutamate release probability. An enhanced MK-801 blocking rate is not seen, however, in the lateral perforant path. This result still could be compatible with a presynaptic contribution to LTP in the lateral perforant path if intersynaptic cross-talk occurred. In support of this hypothesis, we show that NMDA receptors consistently sense more quanta of glutamate than do α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. In the medial perforant path, in contrast, there is no significant difference in the number of quanta mediated by the two receptors. These results support a presynaptic contribution to LTP and imply that differences in intersynaptic cross-talk can complicate the interpretation of experiments designed to detect changes in transmitter release.

P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase

Protein tyrosine phosphatases (PTPs) have long been thought to play a role in tumor suppression due to their ability to antagonize the growth promoting protein tyrosine kinases. Recently, a candidate tumor suppressor from 10q23, termed P-TEN, was isolated, and sequence homology was demonstrated with members of the PTP family, as well as the cytoskeletal protein tensin. Here we show that recombinant P-TEN dephosphorylated protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibited a high degree of substrate specificity, showing selectivity for extremely acidic substrates in vitro. Furthermore, we demonstrate that mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan–Zonana syndrome, resulted in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is necessary for its ability to function as a tumor suppressor.

Dual roles for DNA sequence identity and the mismatch repair system in the regulation of mitotic crossing-over in yeast

Sequence divergence acts as a potent barrier to homologous recombination; much of this barrier derives from an antirecombination activity exerted by mismatch repair proteins. An inverted repeat assay system with recombination substrates ranging in identity from 74% to 100% has been used to define the relationship between sequence divergence and the rate of mitotic crossing-over in yeast. To elucidate the role of the mismatch repair machinery in regulating recombination between mismatched substrates, we performed experiments in both wild-type and mismatch repair defective strains. We find that a single mismatch is sufficient to inhibit recombination between otherwise identical sequences, and that this inhibition is dependent on the mismatch repair system. Additional mismatches have a cumulative negative effect on the recombination rate. With sequence divergence of up to approximately 10%, the inhibitory effect of mismatches results mainly from antirecombination activity of the mismatch repair system. With greater levels of divergence, recombination is inefficient even in the absence of mismatch repair activity. In both wild-type and mismatch repair defective strains, an approximate log-linear relationship is observed between the recombination rate and the level of sequence divergence.

Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1α and its dual block by ovarian steroids

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1α (IL-1α) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1α and repress MMP-1 production by IL-1α-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1α and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1α is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1α, thereby exerting an effective control via a “double-block” mechanism.

Intracellular gene transfer in action: Dual transcription and multiple silencings of nuclear and mitochondrial cox2 genes in legumes

The respiratory gene cox2, normally present in the mitochondrion, was previously shown to have been functionally transferred to the nucleus during flowering plant evolution, possibly during the diversification of legumes. To search for novel intermediate stages in the process of intracellular gene transfer and to assess the evolutionary timing and frequency of cox2 transfer, activation, and inactivation, we examined nuclear and mitochondrial (mt) cox2 presence and expression in over 25 legume genera and mt cox2 presence in 392 genera. Transfer and activation of cox2 appear to have occurred during recent legume evolution, more recently than previously inferred. Many intermediate stages of the gene transfer process are represented by cox2 genes in the studied legumes. Nine legumes contain intact copies of both nuclear and mt cox2, although transcripts could not be detected for some of these genes. Both cox2 genes are transcribed in seven legumes that are phylogenetically interspersed with species displaying only nuclear or mt cox2 expression. Inactivation of cox2 in each genome has taken place multiple times and in a variety of ways, including loss of detectable transcripts or transcript editing and partial to complete gene loss. Phylogenetic evidence shows about the same number (3–5) of separate inactivations of nuclear and mt cox2, suggesting that there is no selective advantage for a mt vs. nuclear location of cox2 in plants. The current distribution of cox2 presence and expression between the nucleus and mitochondrion in the studied legumes is probably the result of chance mutations silencing either cox2 gene.

Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1

The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. The structural basis for cooperative binding of NFAT and AP-1 to these sites, and indeed for the cooperative binding of transcription factors to composite regulatory elements in general, is not well understood. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein–protein interactions with NFAT in the ternary NFAT/AP-1/DNA complex. In a model of the ternary complex, the segment of NFAT nearest AP-1 is the Rel insert region (RIR), a feature that is notable for its hypervariability in size and in sequence amongst members of the Rel transcription factor family. Here we have used mutational analysis to study the role of the NFAT RIR in binding to DNA and AP-1. Parallel yeast one-hybrid screening assays in combination with alanine-scanning mutagenesis led to the identification of four amino acid residues in the RIR of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, and Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, and Asn-541). These results were confirmed and extended through in vitro binding assays. We thus conclude that the NFAT RIR plays an essential dual role in DNA recognition and cooperative binding to AP-1 family transcription factors.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.

Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets

A definite diagnosis of prion diseases such as Creutzfeldt–Jakob disease (CJD) relies on the detection of pathological prion protein (PrPSc). However, no test for PrPSc in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule detection, we developed a highly sensitive detection method for PrPSc. Pathological prion protein aggregates were labeled by specific antibody probes tagged with fluorescent dyes, resulting in intensely fluorescent targets, which were measured by dual-color fluorescence intensity distribution analysis in a confocal scanning setup. In a diagnostic model system, PrPSc aggregates were detected down to a concentration of 2 pM PrPSc, corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sensitive than Western blot analysis. A PrPSc-specific signal could also be detected in a number of CSF samples from patients with CJD but not in control samples, providing the basis for a rapid and specific test for CJD and other prion diseases. Furthermore, this method could be adapted to the sensitive detection of other disease-associated amyloid aggregates such as in Alzheimer's disease.

The dual functions of Fas ligand in the regulation of peripheral CD8+ and CD4+ T cells

Although Fas ligand (FasL) is well characterized for its capacity to deliver a death signal through its receptor Fas, recent work demonstrates that FasL also can receive signals facilitating antigen (Ag)-specific proliferation of CD8+ T cells. The fact that the gld mutation differentially influences the proliferative capacity of CD8+ and CD4+ T cells presented the intriguing possibility that a single molecule may play opposing roles in these two subpopulations. The present study focuses on how these positive and negative regulatory roles are balanced. We show that naive CD4+ T cells are responsive to FasL-mediated costimulation on encounter with Ag when Fas-mediated death is prevented. Thus, the machinery responsible for transducing the FasL positive reverse signal operates in both CD4+ and CD8+ T cells. Instead, differential control of FasL expression distinguishes the role of FasL in these two T cell subpopulations. FasL costimulation occurs immediately on T cell receptor ligation and correlates with the up-regulation of FasL expression on CD8+ and naive CD4+ T cells, both of which are sensitive to the FasL costimulatory signal. Conversely, FasL-initiated death occurs late in an immune response when high levels of FasL expression are maintained on CD4+ T cells that are sensitive to Fas-mediated death, but not on CD8+ T cells that are relatively insensitive to this signal. This careful orchestration of FasL expression during times of susceptibility to costimulation and conversely, to death, endows FasL with the capacity to both positively and negatively regulate the peripheral T cell compartment.

Dual pathways for regulation of root branching by nitrate

Root development is extremely sensitive to variations in nutrient supply, but the mechanisms are poorly understood. We have investigated the processes by which nitrate (NO3−), depending on its availability and distribution, can have both positive and negative effects on the development and growth of lateral roots. When Arabidopsis roots were exposed to a locally concentrated supply of NO3− there was no increase in lateral root numbers within the NO3−-rich zone, but there was a localized 2-fold increase in the mean rate of lateral root elongation, which was attributable to a corresponding increase in the rate of cell production in the lateral root meristem. Localized applications of other N sources did not stimulate lateral root elongation, consistent with previous evidence that the NO3− ion is acting as a signal rather than a nutrient. The axr4 auxin-resistant mutant was insensitive to the stimulatory effect of NO3−, suggesting an overlap between the NO3− and auxin response pathways. High rates of NO3− supply to the roots had a systemic inhibitory effect on lateral root development that acted specifically at the stage when the laterals had just emerged from the primary root, apparently delaying final activation of the lateral root meristem. A nitrate reductase-deficient mutant showed increased sensitivity to this systemic inhibitory effect, suggesting that tissue NO3− levels may play a role in generating the inhibitory signal. We present a model in which root branching is modulated by opposing signals from the plant’s internal N status and the external supply of NO3−.

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease EcoRI.

A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5′-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.