Identification of the RecR Toprim domain as the binding site for both RecF and RecO. A role of RecR in RecFOR assembly at double-stranded DNA-single-stranded DNA junctions

The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4:2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-stranded DNA (ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR.     

Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.     

Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.     

Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration and neuritogenesis

Enteric nervous system (ENS) development requires complex interactions between migrating neural-crest-derived cells and the intestinal microenvironment. Although some molecules influencing ENS development are known, many aspects remain poorly understood. To identify additional molecules critical for ENS development, we used DNA microarray, quantitative real-time PCR and in situ hybridization to compare gene expression in E14 and P0 aganglionic or wild type mouse intestine. Eighty-three genes were identified with at least two-fold higher expression in wild type than aganglionic bowel. ENS expression was verified for 39 of 42 selected genes by in situ hybridization. Additionally, nine identified genes had higher levels in aganglionic bowel than in WT animals suggesting that intestinal innervation may influence gene expression in adjacent cells. Strikingly, many synaptic function genes were expressed at E14, a time when the ENS is not needed for survival. To test for developmental roles for these genes, we used pharmacologic inhibitors of Snap25 or vesicle-associated membrane protein (VAMP)/synaptobrevin and found reduced neural-crest-derived cell migration and decreased neurite extension from ENS precursors. These results provide an extensive set of ENS biomarkers, demonstrate a role for SNARE proteins in ENS development and highlight additional candidate genes that could modify Hirschsprung's disease penetrance.     

Tuatara (Sphenodon) genomics: BAC library construction, sequence survey, and application to the DMRT gene family

The tuatara (Sphenodon punctatus) is of "extraordinary biological interest" as the most distinctive surviving reptilian lineage (Rhyncocephalia) in the world. To provide a genomic resource for an understanding of genome evolution in reptiles, and as part of a larger project to produce genomic resources for various reptiles (evogen.jgi.doe.gov/second_levels/BACs/our_libraries.html), a large-insert bacterial artificial chromosome (BAC) library from a male tuatara was constructed. The library consists of 215 424 individual clones whose average insert size was empirically determined to be 145 kb, yielding a genomic coverage of approximately 6.3x. A BAC-end sequencing analysis of 121 420 bp of sequence revealed a genomic GC content of 46.8%, among the highest observed thus far for vertebrates, and identified several short interspersed repetitive elements (mammalian interspersed repeat-type repeats) and long interspersed repetitive elements, including chicken repeat 1 element. Finally, as a quality control measure the arrayed library was screened with probes corresponding to 2 conserved noncoding regions of the candidate sex-determining gene DMRT1 and the DM domain of the related DMRT2 gene. A deep coverage contig spanning nearly 300 kb was generated, supporting the deep coverage and utility of the library for exploring tuatara genomics.     

Opposing effects of NaCl on reversibility and thermal stability of halophilic beta-lactamase from a moderate halophile, Chromohalobacter sp. 560

Beta-lactamase from a moderately halophilic organism is expected to show salt-dependent stability. Here we examined the temperature-dependence of stability at different salt concentrations using circular dichroism (CD) and enzyme activity. NaCl showed opposing effects on melting temperature and reversibility of the thermal melting. Increasing NaCl concentration greatly increased the melting temperature from, e.g., 41 degrees C in the absence of NaCl to 61 degrees C in 3 M NaCl. Conversely, reversibility decreased from 92% to 0% in the corresponding NaCl solutions. When beta-lactamase was heated at different temperatures and NaCl concentrations, the activity recovery followed the reversibility, not the melting temperature. Heating beta-lactamase at 63 degrees C, slightly above the onset temperature of melting in 2 M NaCl and far above the melting in 0.2 M NaCl, showed a much greater recovery of activity in 0.2 M NaCl than in 2 M NaCl, again consistent with the reversibility of melting.     

Leptin facilitates learning and memory performance and enhances hippocampal CA1 long-term potentiation and CaMK II phosphorylation in rats

Leptin, an adipocytokine encoded by an obesity gene and expressed in adipose tissue, affects feeding behavior, thermogenesis, and neuroendocrine status via leptin receptors distributed in the brain, especially in the hypothalamus. Leptin may also modulate the synaptic plasticity and behavioral performance related to learning and memory since: leptin receptors are found in the hippocampus, and both leptin and its receptor share structural and functional similarities with the interleukin-6 family of cytokines that modulate long-term potentiation (LTP) in the hippocampus. We therefore examined the effect of leptin on (1) behavioral performance in emotional and spatial learning tasks, (2) LTP at Schaffer collateral-CA1 synapses, (3) presynaptic and postsynaptic activities in hippocampal CA1 neurons, (4) the intracellular Ca(2+) concentration ([Ca(2+)](i)) in CA1 neurons, and (5) the activity of Ca(2+)/calmodulin protein kinase II (CaMK II) in the hippocampal CA1 tissue that exhibits LTP. Intravenous injection of 5 and/or 50mug/kg, but not of 500mug/kg leptin, facilitated behavioral performance in passive avoidance and Morris water-maze tasks. Bath application of 10(-12)M leptin in slice experiments enhanced LTP and increased the presynaptic transmitter release, whereas 10(-10)M leptin suppressed LTP and reduced the postsynaptic receptor sensitivity to N-methyl-d-aspartic acid. The increase in the [Ca(2+)](i) induced by 10(-10)M leptin was two times greater than that induced by 10(-12)M leptin. In addition, the facilitation (10(-12)M) and suppression (10(-10)M) of LTP by leptin was closely associated with an increase and decrease in Ca(2+)-independent activity of CaMK II. Our results show that leptin not only affects hypothalamic functions (such as feeding, thermogenesis, and neuroendocrine status), but also modulates higher nervous functions, such as the behavioral performance related to learning and memory and hippocampal synaptic plasticity.     

René Couteaux (1909-1999) and the morphological identification of synapses

During a historical research, we realized that René Couteaux (1909-1999) was the first histologist who stained the postsynaptic structure of the neuromuscular junction. By means of Janus Green B dye, he revealed the membranous 'subneural apparatus' related to the 'synaptic gutter'. Hence, he justified the use of the physiological term 'synapse' in histology. A few years later, Couteaux localized acetylcholinesterase activity on this subneural apparatus with a histo-enzymatic method. The histochemical staining confirmed his histological observations that the subneural apparatus of the muscle cells was distinct from the nerve terminal. Thus Couteaux not only supported Cajal's neuron theory, but also gave decisive morphological basis to the concept of the synapse, thought to be up to that time a physiological entity. These observations were performed several years before the first electron microscopic data obtained in the U.S.A. When the electron microscopy became available in Europe, Couteaux studied the ultrastructural details of the myoneural synapse. He found evidence for the presynaptic 'active zone' as a restricted zone of the thickened presynaptic membrane flanked on both sides with synaptic vesicles, some of which were observed to be opening into the synaptic cleft by exocytosis. Furthermore, the synaptic vesicles were shown to be displayed in two rows along the linearly prolonged active zone which is situated just above the 'trench-like' junctional folds of the muscle membrane. The discontinuity of the synaptic structure was confirmed by the three-dimensional observation of the intraneuronal network of neurofibrils with the first prototype of high-voltage electron microscope.