The 5-HT3 receptor is a neurotransmitter-gated ion channel. It is a member of the Cys-loop family of receptors, which also includes nicotinic acetylcholine, glycine and GABAA receptors. Each member of the family consists of an arrangement of five subunits surrounding a central ion-conducting pore. The 5-HT3 receptor binding site is composed of six loops from two adjacent subunits, and the critical ligand binding residues within these loops are well documented. There are a range of 5-HT3 receptor agonists and competitive antagonists, but it is the antagonists that dominate their clinical use. Studies have proposed a range of disease symptoms that might be amenable to 5-HT3 receptor selective compounds; however, so far only the treatment of emesis and irritable bowel syndrome have been fully realised. In this review, the authors look at the structure, function and distribution of 5-HT3 receptors and how this may influence their role in disease. The authors also describe the existing clinical applications of 5-HT3 antagonists and the future potential of these drugs.

We compared the activity of free D-Ser on the potentiation of cloned NMDA receptors with that of Gly by using a Xenopus oocyte expression system. The extracellular concentration of free D-Ser and Gly was further studied by means of microdialysis. The ED50 values of D-Ser were three to four times lower than those of Gly in any combination of epsilon 1, epsilon 2, epsilon 3, or epsilon 4 and zeta 1. Site-directed mutagenesis of zeta 1 subunits revealed that some aromatic residues necessary for the action of Gly affected the ED50 value of D-Ser. This result showed that the residues play crucial roles in the action of D-Ser. In vivo microdialysis of rodent brain revealed that the extracellular concentration of free D-Ser in the frontal cortex (6.5 microM) was high enough to saturate the Gly site on the NMDA receptor, but that in the cerebellum was not. These findings suggest that D-Ser is a candidate of the endogenous potentiator of the NMDA receptor in the rodent frontal cortex.

Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US$1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.

Heat-stable enterotoxin (ST) produced by porcine strains of enterotoxigenic (ENT+) Escherichia coli has been purified to apparent homogeneity by sequential ultrafiltration, acetone fractionation, preparative gel electrophoresis, diethylaminoethyl Bio-Gel A ion-exchange chromatography, and Bio-Gel P-10 gel filtration. The enterotoxin, purified more than 1,500-fold, exhibited a molecular weight of 4,400, as determined by both sodium dodecyl sulfate-gel electrophoresis and gel filtration. A molecular weight of 5,100, representing 47 residues, was calculated from amino acid analysis data. The amino acid content was distinctive, with an unusually high proportion of cystines and few hydrophobic amino acids. A single amino-terminal residue, glycine, was observed. Purified ST was stable to heating (100 degrees C, 30 min) and did not lose biological activity after treatment with Pronase, trypsin, proteinase K, deoxyribonuclease, ribonuclease, and phospholipase C. Periodic acid oxidation and several organic solvents (acetone, phenol, chloroform, and methanol) had no effect on the biological activity of ST. Further, purified ST was stable to acid treatment at pH 1.0 but lost biological activity at pH values greater than 9.0. Neither lipopolysaccharide nor lipid contamination was evident in purified preparations. A characteristic absorption spectrum was observed during the course of the purification, which shifted from a maximum at 260 nm in crude preparations to 270 nm for the purified toxin. Antiserum obtained from rabbits immunized with ST or ST coupled to bovine serum albumin neutralized the action of the enterotoxin in suckling mice; however, passive hemagglutination and hemolysis titer assays suggested that ST is a poor antigen.

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report ( Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81, 8374-8383 ), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable alpha-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.

hy8 long hypocotyl mutants of Arabidopsis defective in responsiveness to prolonged far-red light (the so-called "far-red high-irradiance response") are selectively deficient in functional phytochrome A. To define the molecular lesion in these mutants, we sequenced the phytochrome A gene (phyA) in lines carrying one or other of two classes of hy8 alleles. The hy8-1 and hy8-2 mutants that express no detectable phytochrome A each have a single nucleotide change that inserts a translational stop codon in the protein coding sequence. These results establish that phyA resides at the HY8 locus. The hy8-3 mutant that expresses wild-type levels of photochemically active phytochrome A has a glycine-to-glutamate missense mutation at residue 727 in the C-terminal domain of the phyA sequence. Quantitative fluence rate response analysis showed that the mutant phytochrome A molecule produced by hy8-3 exhibited no detectable regulatory activity above that of the phyA-protein-deficient hy8-2 mutant. This result indicates that glycine-727, which is invariant in all sequenced phytochromes, has a function important to the regulatory activity of phytochrome A but not to photoperception.

Infection of legume roots with Rhizobium species results in the development of a root nodule structure in which the bacteria form an intracellular symbiosis with the plant. We report here that the infection of soybean (Glycine max L.) roots with Rhizobium japonicum results in the synthesis by the plant of at least 18-20 polypeptides other than leghemoglobin during the development of root nodules. Identification of these "nodule-specific" host polypeptides (referred to as nodulins) was accomplished by two-dimensional gel analysis of the immunoprecipitates formed by a "nodule-specific" antiserum with in vitro translation products of root-nodule polysomes that are free of bacteroidal contaminations. Nodulins account for 7-11% of the total 35S-methionine-labeled protein synthesized in the host cell cytoplasm, and the majority of them are of 12,000-20,000 molecular weight. These proteins are absent from the uninfected roots, bacteroids and free-living Rhizobium, and appear to be coded for the plant genes that may be obligatory for the development of symbiosis in the legume root nodules. Analysis of nodulins in ineffective (unable to fix nitrogen) nodules developed due to Rhizobium strains SM5 and 61A24 showed that their synthesis is reduced and their expression differentially influenced by mutations in rhizobia. Two polypeptides of bacterial origin were also found to be cross-reactive with the "nodule-specific" antiserum, suggesting that they are secreted by Rhizobium into the host cell cytoplasm during symbiotic nitrogen fixation.

The vitamins folic acid, B12 and B6 and B2 are the source of coenzymes which participate in one carbon metabolism. In this metabolism, a carbon unit from serine or glycine is transferred to tetrahydrofolate (THF) to form methylene-THF. This is either used as such for the synthesis of thymidine, which is incorporated into DNA, oxidized to formyl-THF which is used for the synthesis of purines, which are building blocks of RNA and DNA, or it is reduced to methyl-THF which used to methylate homocysteine to form methionine, a reaction which is catalyzed by a B12-containing methyltransferase. Much of the methionine which is formed is converted to S-adenosylmethionine (SAM), a universal donor of methyl groups, including DNA, RNA, hormones, neurotransmitters, membrane lipids, proteins and others. Because of these functions, interest in recent years has been growing particularly in the area of aging and the possibility that certain diseases that afflict the aging population, loss of cognitive function, Alzheimer's disease, cardiovascular disease, cancer and others, may be in part explained by inadequate intake or inadequate status of these vitamins. Homocysteine, a product of methionine metabolism as well as a precursor of methionine synthesis, was shown recently to be a risk factor for cardiovascular disease, stroke and thrombosis when its concentration in plasma is slightly elevated. There are now data which show association between elevated plasma homocysteine levels and loss of neurocognitive function and Alzheimer's disease. These associations could be due to a neurotoxic effect of homocysteine or to decreased availability of SAM which results in hypomethylation in the brain tissue. Hypomethylation is also thought to exacerbate depressive tendency in people, and for (colorectal) cancer DNA hypomethylation is thought to be the link between the observed relationship between inadequate folate status and cancer. There are many factors that contribute to the fact that the status of these vitamins in the elderly is inadequate. These factors are in part physiological such as the achlorhydria which affects vitamin B12 absorption and in part socioeconomic and habitual. We need more studies to confirm that these vitamins have important functions in the etiology of these diseases. We also need to establish if these diseases can be prevented or diminished by proper nutrition starting at a younger age.

A type VI secretion system (T6SS) was recently shown to be required for full virulence of Vibrio cholerae O37 serogroup strain V52. In this study, we systematically mutagenized each individual gene in T6SS locus and characterized their functions based on expression and secretion of the hemolysin co-regulated protein (Hcp), virulence towards amoebae of Dictyostelium discoideum and killing of Escherichia coli bacterial cells. We group the 17 proteins characterized in the T6SS locus into four categories: twelve (VipA, VipB, VCA0109-VCA0115, ClpV, VCA0119, and VasK) are essential for Hcp secretion and bacterial virulence, and thus likely function as structural components of the apparatus; two (VasH and VCA0122) are regulators that are required for T6SS gene expression and virulence; another two, VCA0121 and valine-glycine repeat protein G 3 (VgrG-3), are not essential for Hcp expression, secretion or bacterial virulence, and their functions are unknown; the last group is represented by VCA0118, which is not required for Hcp expression or secretion but still plays a role in both amoebae and bacterial killing and may therefore be an effector protein. We also showed that the clpV gene product is required for Dictyostelium virulence but is less important for killing E. coli. In addition, one vgrG gene (vgrG-2) outside of the T6SS gene cluster was required for bacterial killing but another (vgrG-1) was not. However, a bacterial killing defect was observed when vgrG-1 and vgrG-3 were both deleted. Several genes encoded in the same putative operon as vgrG-1 and vgrG-2 also contribute to virulence toward Dictyostelium but have a smaller effect on bacterial killing. Our results provide new insights into the functional requirements of V. cholerae's T6SS in the context of secretion as well as killing of bacterial and eukaryotic phagocytic cells.

Six peptidase activities have been distinguished electrophoretically in cell extracts of Salmonella typhimurium with the aid of a histochemical stain. The activities can also be partially separated by chromatography on diethylaminoethyl-cellulose. These peptidases show overlapping substrate specificities. Mutants (pepN) of the parent strain leu-485 lacking one of these enzymes (peptidase N) were obtained by screening for colonies that do not hydrolyze the chromogenic substrate l-alanyl-beta-naphthylamide. The absence of this broad-specificity peptidase in leu-485 pepN(-) mutants allowed the selection of mutants unable to use l-leucyl-l-alaninamide as a leucine source. These mutants (leu-485 pepN(-)pepA(-)) lack a broad-specificity peptidase (peptidase A) similar to aminopeptidase I previously described in Escherichia coli. Mutants (pepD) lacking a dipeptidase (peptidase D) have been isolated from a leu-485 pepN(-)pepA(-) parent by penicillin selection for mutants unable to use l-leucyl-l-glycine as a leucine source. Mutants (pepB) lacking a fourth peptidase (peptidase B) have been isolated from a leu-485 pepN(-)pepA(-)pepD(-) strain by penicillin selection for failure to utilize l-leucyl-l-leucine as a source of leucine. Single recombinants were obtained by transduction for each of the peptidases missing in a leu-485 pepN(-)pepA(-)pepD(-)pepB(-) strain. The growth response of these recombinants to leucine peptides shows that all of these peptidases can function in the catabolism of peptides and that they display overlapping substrate specificities in vivo.

Glycine betaine, which functions as an osmoprotectant, is accumulated to high intracellular concentrations in Escherichia coli at high osmolarity. We demonstrate the presence of a high-affinity, binding protein dependent transport system for glycine betaine, which is encoded by the proU region. We show the osmotically regulated synthesis of a 32 kDa periplasmic protein that is a glycine betaine binding protein with a KD of 1.4 microM. ProU-mediated glycine betaine transport is osmotically stimulated at the level of gene expression. The osmolarity of the medium also regulates the activity of the transport system, while binding of glycine betaine to its binding protein is independent of the osmolarity. We also find a second glycine betaine transport system that is dependent on proP and exhibits a lower substrate affinity. Like ProU, this system is regulated at two levels: both gene expression and the activity of the transport system are osmotically stimulated. Using lambda plac Mu-generated lacZ operon and gene fusions, we find that expression of the proU region is osmotically regulated at the level of transcription. We cloned a part of the proU region together with the phi(proU-lacZ)hyb2 gene fusion into a multicopy plasmid and show that the DNA sequences required in cis for osmotic regulation are present on the plasmid.

We examined the regulation of glutamate transporter protein expression after stimulation with selective metabotropic glutamate receptor (mGluR) agonists in cultured human glial cells. mGluR3 and mGluR5 are expressed in human astrocytes and in human glioma cells in vivo as well as in vitro, as shown by either RT-PCR or western blot analysis. The selective group I agonist (S)-3,5-dihydroxyphenylglycine produced a significant down-regulation of both GLAST and GLT-1 protein expression in astrocytes cultured in the presence of growth factors. This condition mimics the morphology of reactive glial cells in vivo including an increased expression of mGluR5 protein (observed in pathological conditions). In contrast, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine, a selective agonist of group II metabotropic glutamate receptors, positively modulates the expression of GLAST and GLT-1 proteins. A similar opposite effect of (S)-3,5-dihydroxyphenylglycine and (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine was observed for the expression of EAAT3 protein in U373 glioblastoma cell line. Selective group I and II antagonists prevented these effects. Pharmacological inhibition of mitogen-activated protein kinase and phosphatidylinositol-3-K pathways reduces the induction of GLT-1 observed in response to the group II metabotropic glutamate receptor agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine. Thus, mGluR3 and mGluR5 can critically and differentially modulate the expression of glutamate transporters and may represent interesting pharmacological targets to regulate the extracellular levels of glutamate in pathological conditions.

1. Activation and desensitization of N-methyl-D-aspartate (NMDA) receptors were studied in large outside-out patches excised from cultured embryonic neurones dissociated from mouse forebrain. The patches were exposed to rapid changes of NMDA or L-glutamate concentrations in the presence of glycine at concentrations (10-20 microM) saturating the modulatory site of the NMDA receptor. 2. Immediately after formation of the patch the responses to NMDA and L-glutamate showed a slow and small desensitization, even with high concentrations of agonist. During the following hour, the peak response either decreased or remained relatively stable, but in all cases the desensitization increased and accelerated until it stabilized. In this 'stabilized' state, the desensitization produced by high concentrations of NMDA (1 mM) or L-glutamate (300 microM) had an exponential time course, with a time constant of about 30 ms. The ratio of the peak over the steady-state current was in the order of 40 for NMDA and about 30 for L-glutamate. 3. Concentration-response curves were built for the peak and the plateau responses, for NMDA and for L-glutamate. The comparison of these curves indicated that (i) the EC50 of the peak (K(app) was always higher than the EC50 of the plateau (Kss); (ii) the two EC50 values for NMDA (K(app) and Kss) were higher than those for L-glutamate; (iii) the Hill coefficient was close to 1.4 for each of the four curves. 4. The application of NMDA or L-glutamate at a low concentration for 3 s periods reduced the response to a subsequent application of the same agonist at a saturating concentration. The IC50 of this 'predesensitization', termed Kpre, was lower than the EC50 of the steady-state response, Kss. 5. The onset rates of desensitization increased with the concentration of agonist. The EC50 of this relation was close to the value of K(app). 6. The decay of the currents at the end of a 3 s application of agonist was usually well described by the sum of two exponentials both of which were faster for NMDA than for L-glutamate. 7. The recovery from desensitization after a long (3 s) pulse of agonist was approximately exponential, with a time constant of about 0.5 s for NMDA and about 3.5 s for L-glutamate.(ABSTRACT TRUNCATED AT 400 WORDS)

Over the last 20 years, glutamatergic models of schizophrenia have become increasingly accepted as etiopathological models of schizophrenia, based on the observation that phencyclidine (PCP) induces a schizophrenia-like psychosis by blocking neurotransmission at N-methyl-D-aspartate (NMDA)-type glutamate receptors. This article reviews developments in two key predictions of the model: first, that neurocognitive deficits in schizophrenia should follow the pattern of deficit predicted based on underlying NMDAR dysfunction and, second, that agents that stimulate NMDAR function should be therapeutically beneficial. As opposed to dopamine receptors, NMDAR are widely distributed throughout the brain, including subcortical as well as cortical brain regions, and sensory as well as association cortex. Studies over the past 20 years have documented severe sensory dysfunction in schizophrenia using behavioral, neurophysiological, and functional brain imaging approaches, including impaired generation of key sensory-related potentials such as mismatch negativity and visual P1 potentials. Similar deficits are observed in humans following administration of NMDAR antagonists such as ketamine in either humans or animal models. Sensory dysfunction, in turn, predicts impairments in higher order cognitive functions such as auditory or visual emotion recognition. Treatment studies have been performed with compounds acting directly at the NMDAR glycine site, such as glycine, D-serine, or D-cycloserine, and, more recently, with high-affinity glycine transport inhibitors such as RG1678 (Roche). More limited studies have been performed with compounds targeting the redox site. Overall, these compounds have been found to induce significant beneficial effects on persistent symptoms, suggesting novel approaches for treatment and prevention of schizophrenia.

Clostridium difficile spores must germinate in vivo to become actively growing bacteria in order to produce the toxins that are necessary for disease. C. difficile spores germinate in vitro in response to certain bile acids and glycine. In other sporulating bacteria, proteins embedded within the inner membrane of the spore sense the presence of germinants and trigger the release of Ca⁺⁺-dipicolinic acid (Ca⁺⁺-DPA) from the spore core and subsequent hydrolysis of the spore cortex, a specialized peptidoglycan. Based upon homology searches of known germinant receptors from other spore-forming bacteria, C. difficile likely uses unique mechanisms to recognize germinants. Here, we identify the germination-specific protease, CspC, as the C. difficile bile acid germinant receptor and show that bile acid-mediated germination is important for establishing C. difficile disease in the hamster model of infection. These results highlight the importance of bile acids in triggering in vivo germination and provide the first description of a C. difficile spore germinant receptor. Blocking the interaction of bile acids with the C. difficile spore may represent an attractive target for novel therapeutics.

The two amino acids GABA and glycine mediate fast inhibitory neurotransmission in different CNS areas and serve pivotal roles in the spinal sensory processing. Under healthy conditions, they limit the excitability of spinal terminals of primary sensory nerve fibers and of intrinsic dorsal horn neurons through pre- and postsynaptic mechanisms, and thereby facilitate the spatial and temporal discrimination of sensory stimuli. Removal of fast inhibition not only reduces the fidelity of normal sensory processing but also provokes symptoms very much reminiscent of pathological and chronic pain syndromes. This review summarizes our knowledge of the molecular bases of spinal inhibitory neurotransmission and its organization in dorsal horn sensory circuits. Particular emphasis is placed on the role and mechanisms of spinal inhibitory malfunction in inflammatory and neuropathic chronic pain syndromes.

Four small ubiquitin-related modifier (SUMO) genes have been identified in humans. However, little is known about the basic biology of SUMO-4. Here, we report that SUMO-4 differs from SUMO-1, -2, and -3 in that the maturation process of SUMO-4 to active form containing C-terminal di-glycine residues is inhibited by a unique proline residue located at position 90 (Pro-90). Although, both the hydrolase and isopeptidase activities of SUMO peptidases are significantly diminished by Pro-90 as compared to Gln-90 (glutamine) in mutated SUMO genes, only the defective hydrolase activity appears to be biologically relevant. Native SUMO-4, thus, appears to be unable to form covalent isopeptide bonds with substrates. A biological role of SUMO-4, through non-covalent interactions is proposed.

Class 2 aminoacyl-tRNA synthetases, which include the enzymes for alanine, aspartic acid, asparagine, glycine, histidine, lysine, phenylalanine, proline, serine and threonine, are characterised by three distinct sequence motifs 1,2 and 3 (reference 1). The structural and evolutionary relatedness of these ten enzymes are examined using alignments of primary sequences from prokaryotic and eukaryotic sources and the known three dimensional structure of seryl-tRNA synthetase from E. coli. It is shown that motif 1 forms part of the dimer interface of seryl-tRNA synthetase and motifs 2 and 3 part of the putative active site. It is further shown that the seven alpha 2 dimeric synthetases can be subdivided into class 2a (proline, threonine, histidine and serine) and class 2b (aspartic acid, asparagine and lysine), each subclass sharing several important characteristic sequence motifs in addition to those characteristic of class 2 enzymes in general. The alpha 2 beta 2 tetrameric enzymes (for glycine and phenylalanine) show certain special features in common as well as some of the class 2b motifs. In the alanyl-tRNA synthetase only motif 3 and possibly motif 2 can be identified. The sequence alignments suggest that the catalytic domain of other class 2 synthetases should resemble the antiparallel domain found in seryl-tRNA synthetase. Predictions are made about the sequence location of certain important helices and beta-strands in this domain as well as suggestions concerning which residues are important in ATP and amino acid binding. Strong homologies are found in the N-terminal extensions of class 2b synthetases and in the C-terminal extensions of class 2a synthetases suggesting that these putative tRNA binding domains have been added at a later stage in evolution to the catalytic domain.

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.

OBJECTIVE

Both K(ATP) channel opening drugs and ischaemic preconditioning have been suggested to protect the ischaemic heart by acting on K(ATP) channels in the inner mitochondrial membrane, uncoupling the proton gradient and partially dissipating the mitochondrial membrane potential. The aim of these studies was to use low concentrations of FCCP, a mitochondrial protonophore, to bypass the mitochondrial K(ATP) channel and partially uncouple the mitochondria and establish whether this activates protective pathways within the rat heart analogous to K(ATP) channel openers or preconditioning.

METHODS

Isolated, Langendorff-perfused rat hearts were subjected to 25 min global zero-flow ischaemia and functional recovery assessed. Hearts were pretreated with FCCP (30-300 nM) in the presence or absence of glibenclamide (1 microM), 5-hydroxydecanoate (5-HD: 100 microM), N-acetyl cysteine (4 mM), or N-2-mercaptopropionyl glycine (1 mM). The metabolic consequences of FCCP perfusion in isolated hearts were studied using (31)P NMR, and reactive oxygen species (ROS) production was measured using DCF fluorescence in isolated rat ventricular myocytes.

RESULTS

FCCP exerted a dose-dependent cardioprotective effect, with 100 nM FCCP being the optimal concentration. This effect could not be blocked by glibenclamide or 5-HD, but was completely attenuated by N-acetyl cysteine and N-2-mercaptopropionyl glycine. Perfusion with FCCP (100 nM) did not deplete bulk ATP during the pretreatment period but significantly depleted phosphocreatine. In ventricular myocytes, FCCP caused an antioxidant-sensitive increase in ROS production but diazoxide was without effect.

CONCLUSIONS

In the isolated rat heart, partial mitochondrial uncoupling with low-dose FCCP significantly improves post-ischaemic functional recovery via a ROS-dependent pathway. This cardioprotection is not mediated via the depletion of cellular ATP or mitochondrial K(ATP) channel activation.

The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding approximately 40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate-binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT.

Synthetic peptides containing the arginine-glycine-aspartate (RGD) motif have been used extensively as inhibitors of integrin-ligand interactions in studies of cell adhesion, migration, growth and differentiation, because the RGD motif is an integrin-recognition motif found in many ligands. Here we report that RGD-containing peptides are able to directly induce apoptosis without any requirement for integrin-mediated cell clustering or signals. We show that RGD-containing peptides enter cells and directly induce autoprocessing and enzymatic activity of procaspase-3, a pro-apoptotic protein. Using the breast carcinoma cell line MCF-7, which has a functional deletion of the caspase-3 gene, we confirm that caspase-3 is required for RGD-mediated cell death. In addition to an RGD motif, pro-caspase-3 also contains a potential RGD-binding motif, aspartate-aspartate-methionine (DDM), near the site of processing to produce the p12 and p17 subunits. On the basis of the ability of RGD-DDX interactions to trigger integrin activation, we suggest that RGD peptides induce apoptosis by triggering conformational changes that promote pro-caspase-3 autoprocessing and activation. These findings provide an alternative molecular explanation for the potent proapoptotic properties of RGD peptides in models of angiogenesis, inflammation and cancer metastasis.

Duration is a biologically important feature of sound. Some neurons in the inferior colliculus of the big brown bat, Eptesicus fuscus, are tuned to sound duration, but it is unclear at what level the tuning originates or what neural mechanisms are responsible for it. The application of antagonists of the inhibitory neurotransmitters gamma-aminobutyric acid or glycine to neurons in the inferior colliculus eliminated duration tuning. Whole-cell patch-clamp recordings of synaptic currents suggested that inhibition produces a temporal frame within which excitation can occur. A model is proposed in which duration tuning arises when an early, sustained inhibitory input interacts with a delayed, transient excitatory input.