(41/17430) A genetic approach to trace neural circuits.
Mammalian nervous system function involves billions of neurons which are interconnected in a multitude of neural circuits. Here we describe a genetic approach to chart neural circuits. By using an olfactory-specific promoter, we selectively expressed barley lectin in sensory neurons in the olfactory epithelium and vomeronasal organ of transgenic mice. The lectin was transported through the axons of those neurons to the olfactory bulb, transferred to the bulb neurons with which they synapse, and transported through the axons of bulb neurons to the olfactory cortex. The lectin also was retrogradely transported from the bulb to neuromodulatory brain areas. No evidence could be obtained for adverse effects of the lectin on odorant receptor gene expression, sensory axon targeting in the bulb, or the generation or transmission of signals by olfactory sensory neurons. Transneuronal transfer was detected prenatally in the odor-sensing pathway, but only postnatally in the pheromone-sensing pathway, suggesting that odors, but not pheromones, may be sensed in utero. Our studies demonstrate that a plant lectin can serve as a transneuronal tracer when its expression is genetically targeted to a subset of neurons. This technology can potentially be applied to a variety of vertebrate and invertebrate neural systems and may be particularly valuable for mapping connections formed by small subsets of neurons and for studying the development of connectivity as it occurs in utero. (+info)
(42/17430) EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases.
A major class of plant disease resistance (R) genes encodes leucine-rich-repeat proteins that possess a nucleotide binding site and amino-terminal similarity to the cytoplasmic domains of the Drosophila Toll and human IL-1 receptors. In Arabidopsis thaliana, EDS1 is indispensable for the function of these R genes. The EDS1 gene was cloned by targeted transposon tagging and found to encode a protein that has similarity in its amino-terminal portion to the catalytic site of eukaryotic lipases. Thus, hydrolase activity, possibly on a lipid-based substrate, is anticipated to be central to EDS1 function. The predicted EDS1 carboxyl terminus has no significant sequence homologies, although analysis of eight defective eds1 alleles reveals it to be essential for EDS1 function. Two plant defense pathways have been defined previously that depend on salicylic acid, a phenolic compound, or jasmonic acid, a lipid-derived molecule. We examined the expression of EDS1 mRNA and marker mRNAs (PR1 and PDF1.2, respectively) for these two pathways in wild-type and eds1 mutant plants after different challenges. The results suggest that EDS1 functions upstream of salicylic acid-dependent PR1 mRNA accumulation and is not required for jasmonic acid-induced PDF1.2 mRNA expression. (+info)
(43/17430) Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes.
The shoot apical meristem and cotyledons of higher plants are established during embryogenesis in the apex. Redundant CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 as well as SHOOT MERISTEMLESS (STM) of Arabidopsis are required for shoot apical meristem formation and cotyledon separation. To elucidate how the apical region of the embryo is established, we investigated genetic interactions among CUC1, CUC2 and STM, as well as the expression patterns of CUC2 and STM mRNA. Expression of these genes marked the incipient shoot apical meristem as well as the boundaries of cotyledon primordia, consistent with their roles for shoot apical meristem formation and cotyledon separation. Genetic and expression analyses indicate that CUC1 and CUC2 are redundantly required for expression of STM to form the shoot apical meristem, and that STM is required for proper spatial expression of CUC2 to separate cotyledons. A model for pattern formation in the apical region of the Arabidopsis embryo is presented. (+info)
(44/17430) Role of P225 and the C136-C201 disulfide bond in tissue plasminogen activator.
The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu2+ was discovered. The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 microM. In an attempt to engineer Na+ binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu2+ to inhibit the enzyme. Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+ binding site of allosteric serine proteases. (+info)
(45/17430) Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems.
In higher plants, organogenesis occurs continuously from self-renewing apical meristems. Arabidopsis thaliana plants with loss-of-function mutations in the CLAVATA (CLV1, 2, and 3) genes have enlarged meristems and generate extra floral organs. Genetic analysis indicates that CLV1, which encodes a receptor kinase, acts with CLV3 to control the balance between meristem cell proliferation and differentiation. CLV3 encodes a small, predicted extracellular protein. CLV3 acts nonautonomously in meristems and is expressed at the meristem surface overlying the CLV1 domain. These proteins may act as a ligand-receptor pair in a signal transduction pathway, coordinating growth between adjacent meristematic regions. (+info)
(46/17430) High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2).
Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to beta-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12-beta-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the beta-oxidation pathway, a binding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1-CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosanoic acid (24:0) was negligible. (+info)
(47/17430) Rapid purification of membrane extrinsic F1-domain of chloroplast ATP synthase in monodisperse form suitable for 3D-crystallization.
A new chromatographic procedure for purification of the membrane extrinsic F1-domain of chloroplast ATP synthase is presented. The purification is achieved by a single anion exchange chromatography step. Determination of the enzyme-bound nucleotides reveals only 1 mole of ADP per complex. The purified enzyme shows a latent Ca(2+)-dependent ATPase activity of 1.0 mumol.mg-1 min-1 and a Mg(2+)-dependent activity of 4.4 mumol.mg-1 .min-1. Both activities are increased up to 8-10-fold after dithiothreitol activation. Analysis of the purified F1-complex by SDS/PAGE, silver staining and immunoblotting revealed that the preparation is uncontaminated by fragmented subunits or ribulose-1,5-bisphosphate carboxylase/oxygenase. Gel filtration experiments indicate that the preparation is homogenous and monodisperse. In order to determine the solubility minimum of the purified F1-complex the isoelectric point of the preparation was calculated from pH mapping on ion exchange columns. In agreement with calculations based on the amino acid sequence, a slightly acidic pI of 5.7 was found. Using ammonium sulphate as a precipitant the purified CF1-complex could be crystallized by MicroBatch. (+info)
(48/17430) The covalent attachment of polyamines to proteins in plant mitochondria.
Plant mitochondria from both potato and mung bean incorporated radioactivity into acid insoluble material when incubated with labelled polyamines (spermine, spermidine and putrescine). Extensive washing of mitochondrial precipitates with trichloroacetic acid and the excess of cold polyamine failed to remove bound radioactivity. Addition of nonradioactive polyamine stopped further incorporation of radioactivity but did not release radioactivity already bound. The radioactivity is incorporated into the membrane fraction. The labelling process has all the features of an enzymatic reaction: it is long lasting with distinctive kinetics peculiar to each polyamine, it is temperature dependent and is affected by N-ethylmaleimide. The latter inhibits the incorporation of putrescine but stimulates the incorporation of spermine and spermidine. Treatment of prelabelled mitochondria with pepsin releases bound radioactivity thus indicating protein to be the ligand for the attachment of polyamines. HPLC of mitochondrial hydrolysates revealed that the radioactivity bound to mitochondria is polyamines; traces of acetyl polyamines were also found in some samples. On autoradiograms of SDS/PAGE gels several radioactive bands of proteins were detected. Protein sequencing of labelled spots from a 2D gel gave a sequence which was 60% identical to catalase. We suggest that the attachment of polyamines to mitochondrial proteins occurs cotranslationally possibly via transglutaminases. (+info)