Rise and fall of vector infectivity during sequential strain displacements by mosquito-borne dengue virus.

Each of the four serotypes of mosquito-borne dengue virus (DENV-1-4) comprises multiple, genetically distinct strains. Competitive displacement between strains within a serotype is a common feature of DENV epidemiology and can trigger outbreaks of dengue disease. We investigated the mechanisms underlying two sequential displacements by DENV-3 strains in Sri Lanka that each coincided with abrupt increases in dengue haemorrhagic fever (DHF) incidence. First, the post-DHF strain displaced the pre-DHF strain in the 1980s. We have previously shown that post-DHF is more infectious than pre-DHF for the major DENV vector, Aedes aegypti. Then, the ultra-DHF strain evolved in situ from post-DHF and displaced its ancestor in the 2000s. We predicted that ultra-DHF would be more infectious for Ae. aegypti than post-DHF but found that ultra-DHF infected a significantly lower percentage of mosquitoes than post-DHF. We therefore hypothesized that ultra-DHF had effected displacement by disseminating in Ae. aegypti more rapidly than post-DHF, but this was not borne out by a time course of mosquito infection. To elucidate the mechanisms that shape these virus-vector interactions, we tested the impact of RNA interference (RNAi), the principal mosquito defence against DENV, on replication of each of the three DENV strains. Replication of all strains was similar in mosquito cells with dysfunctional RNAi, but in cells with functional RNAi, replication of pre-DHF was significantly suppressed relative to the other two strains. Thus, differences in susceptibility to RNAi may account for the differences in mosquito infectivity between pre-DHF and post-DHF, but other mechanisms underlie the difference between post-DHF and ultra-DHF.

Identification of a putative chromosomal replication origin from Helicobacter pylori and its interaction with the initiator protein DnaA.

The key elements of the initiation of Helicobacter pylori chromosome replication, DnaA protein and putative oriC region, have been characterized. The gene arrangement in the H.pylori dnaA region differs from that found in many other eubacterial dnaA regions (rnpA-rmpH-dnaA-dnaN-recF-gyrB). Helicobacter pylori dnaA is flanked by two open reading frames with unknown function, while dnaN-gyrB and rnpA-rmpH loci are separated from the dnaA gene by 600 and 90 kb, respectively. We show that the dnaA gene encoding initiator protein DnaA is expressed in H.pylori cells. The H.pylori DnaA protein, like other DnaA proteins, can be divided into four domains. Here we demonstrate that the C-terminal domain of H.pylori DnaA protein is responsible for DNA binding. Using in silico and in vitro studies, the putative oriC region containing five DnaA boxes has been located upstream of the dnaA gene. DNase I and gel retardation analyses show that the C-terminal domain of H.pylori DnaA protein specifically binds each of five DnaA boxes.

Functions of the DnaA protein of Escherichia coli in replication and transcription.

The function of DnaA protein as a replisome organizer in the initiation of DNA replication is reviewed. A model is presented showing the construction of two basic types of DnaA-dependent replication origin. New data demonstrate that the dnaA box-DnaA protein complex is a transcription terminator. Only one orientation of the dnaA box results in termination of transcription. Mutation of the dnaA box within the dnaA reading frame shows that DnaA-mediated transcription termination has a role in the autoregulation of the dnaA gene.

Complexes at the replication origin of Bacillus subtilis with homologous and heterologous DnaA protein.

The initial steps in the formation of the initiation complex at oriC of Bacillus subtilis were analyzed with special emphasis on the exchangeability of B. subtilis DnaA protein by DnaA of Escherichia coli. The DNA binding domain of B. subtilis DnaA protein was localized in the 93 C-terminal amino acids. Formation of the "initial complex", as analyzed by electron microscopy, was indistinguishable with B. subtilis DnaA protein or with E. coli DnaA. Similarly, both proteins were able to form loops by interaction of DnaA proteins bound to the DnaA box regions upstream and downstream of the dnaA gene in B. subtilis oriC. The region of local unwinding in the "open complex" was precisely defined. It is located at one side of a region of helical instability, a DNA unwinding element (DUE). Unwinding in oriC could only be catalyzed by the homologous DnaA protein.

Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA.

The chromosomal replication origin oriC and the gene encoding the replication initiator protein DnaA from Thermus thermophilus have been identified and cloned into an Escherichia coli vector system. The replication origin is composed of 13 characteristically arranged DnaA boxes, binding sites for the DnaA protein, and an AT-rich stretch, followed by the dnaN gene. The dnaA gene is located upstream of the origin and expresses a typical DnaA protein that follows the division into four domains, as with other members of the DnaA protein family. Here, we report the purification of Thermus-DnaA (Tth-DnaA) and characterize the interaction of the purified protein with the replication origin, with regard to the binding kinetics and stoichiometry of this interaction. Using gel retardation assays, surface plasmon resonance (SPR) and electron microscopy, we show that, unlike the E. coli DnaA, Tth-DnaA does not recognize a single DnaA box, instead a cluster of three tandemly repeated DnaA boxes is the minimal requirement for specific binding. The highest binding affinities are observed with full-length oriC or six clustered, tandemly repeated DnaA boxes. Furthermore, high-affinity DNA-binding of Tth-DnaA is dependent on the presence of ATP. The Thermus DnaA/oriC interaction will be compared with oriC complex formation generated by other DnaA proteins.

Architecture of the Streptomyces lividans DnaA protein-replication origin complexes.

The Streptomyces oriC region contains two clusters of 19 DnaA boxes separated by a spacer (134 bp). The Streptomyces DnaA protein consists, like all other DnaA proteins, of four domains: domain III and the carboxyterminal part (domain IV) are responsible for binding of ATP and DNA, respectively. Binding of the DnaA protein to the entire oriC region analysed by electron microscopy showed that the DnaA protein forms separate complexes at each of the clusters of DnaA boxes, but not at the spacer separating them. In vivo mutational analysis revealed that the number of DnaA boxes and the presence of the spacer linking both groups of DnaA boxes seem to be important for a functional Streptomyces origin. We suggest that the arrangement of DnaA boxes allows the DNA-bound DnaA protein to induce bending and looping of the oriC region. As it was shown by electrophoretic mobility shift assay and "one hybrid system", two domains, I and III, facilitate interactions between DnaA molecules. We postulate that domain I and domain III could be involved in cooperativity at distant and at closely spaced DnaA boxes, respectively. The long domain II extends the range over which N termini (domain I) of DNA-bound DnaA protein can form dimers. Thus, interactions between DnaA molecules may bring two clusters of DnaA boxes separated by the spacer into functional contact by loop formation. Removal of the spacer region or deletion of domains I and II resulted, respectively, in nucleoprotein complexes which are not fully developed, or huge nucleoprotein aggregates.

Identification and characterization of the dnaA upstream region of Thermus thermophilus.

The gene order in the dnaA region of Thermus thermophilus was determined. Previously, we showed that the putative oriC of T. thermophilus is located in the dnaA-dnaN intergenic region. In the 4 kb region upstream of the dnaA gene four ORFs were found, all orientated in the same direction which is opposite to that of dnaA. The ORFs were identified as T. thermophilus homologs of gidA, gidB, soj and spo0J of Bacillus subtilis. The gene order spo0J-soj-gidB-gidA-dnaA-dnaN resembles that of B. subtilis, Pseudomonas putida, Coxiella burnetii, Streptomyces coelicolor, Mycobacterium leprae, and Mycobacterium tuberculosis. We identified the transcriptional start point of the dnaA gene. The -10 region shows significant homology to the Escherichia coli -10 consensus sequence. The putative -35 region shows homology neither to the E. coli -35 consensus sequence nor to known -35 sequences of T. thermophilus. There are no DnaA boxes in the promoter region, and consequently dnaA transcription is not repressed by DnaA protein in vitro, i.e. the dnaA gene of T. thermophilus is not autoregulated.

Termination of the Escherichia coli asnC transcript. The DnaA protein/dnaA box complex blocks transcribing RNA polymerase.

Genes clockwise of oriC, the Escherichia coli replication origin (oriC-mioC-asnC), show anticlockwise transcription. The intergenic region between mioC and asnC contains both a terminator and a consensus DnaA-protein-binding site (dnaA box). We analysed termination in this region using galK expression to monitor for transcription. About 50% of the asnC transcripts were not terminated, and about 25% terminated at the asnC terminator. We found that the DnaA protein/dnaA box complex acts as a terminator of transcription for about 25% of the transcripts. Its efficiency could be increased by raising the level of DnaA protein, or it could be inactivated by deletion in the dnaA box or by thermal denaturation of the DnaA protein.

DnaA proteins of Escherichia coli and Bacillus subtilis: coordinate actions with single-stranded DNA-binding protein and interspecies inhibition during open complex formation at the replication origins.

DnaA-mediated unwinding of the AT-rich region in the replication origins of Escherichia coli and Bacillus subtilis was analysed in vitro with and without single-stranded DNA-binding protein (SSB). In the presence of SSB, the unwound region was larger by a defined number of base pairs. Although the overall structure of the origins is very different, the size and structure of the unwound region were similar. The unwinding reaction at oriC of one organism was inhibited by DnaA protein of the other bacterium. Similarly, hybrid DnaA proteins with swapped DNA-binding domains were inactive and inhibitory to 'open complex' formation at both origins. We suggest that the inhibition is due to inactive mixed complexes.

The replication origin region of Escherichia coli: nucleotide sequence and functional units.

The minichromosome pCM959 contains the DNA segment from bp -677 (left) to bp + 3335 (right) of the Escherichia coli replication origin, oriC. The nucleotide sequence of this plasmid was determined. The coding regions for proteins were identified, and the possible function of those proteins is discussed. Within oriC two extended systems of dyad symmetry were found, and their possible significance is considered.

Evidence for muscarinic acetylcholine receptor subtypes in the pigeon telencephalon.

At least five subtypes of muscarinic acetylcholine receptors are expressed in various mammalian tissue preparations. The following experiment, through the use of direct binding assays (using tritiated quinuclidinyl benzilate), competitive binding assays (using tritiated quinuclidinyl benzilate and unlabeled pirenzepine or AF-DX 116), and autoradiographic techniques, examined whether two of these five putative muscarinic acetylcholine receptor subtypes can be found in avian brain. Accordingly, autoradiographic mapping of pirenzepine-sensitive (M1-like) and AF-DX 116-sensitive (M2-like) muscarinic acetylcholine receptor subtypes in the pigeon telencephalon was conducted. Although both ligands bound throughout the brain, most telencephalic regions, including the archistriatum, the neostriatum, and basal ganglia structures like lobus paraolfactorius, nucleus accumbens, and paleostriatum, showed a higher density of M1-like sites. The exception to this finding was the nucleus basalis which appeared as a region where M2-like sites predominated. Moreover, the telencephalic region with the largest ratio of M1-like to M2-like sites was the lateral portion of the parahippocampus; a characteristic shared with the mammalian dentate gyrus. The findings reported here are generally consistent with previous reports of mammalian M1/M2 receptor distributions.

Functions of histone-like proteins in the initiation of DNA replication at oriC of Escherichia coli.

Using methidiumpropyl-EDTA (MPE) footprinting we found one specific binding site for FIS protein in the E coli replication origin, oriC. We mutagenized the binding sites for FIS and IHF in oriC and analyzed the effect of the mutations on protein binding and oriC function. The replication efficiency of oriC plasmids paralleled the ability of the mutated DNA fragments to bind IHF or FIS. We conclude that these histone-like proteins function in cis in the initiation of DNA replication at oriC.

The Escherichia coli SeqA protein binds specifically to two sites in fully and hemimethylated oriC and has the capacity to inhibit DNA replication and affect chromosome topology.

The SeqA protein was identified as a factor that prevents reinitiation of newly replicated, hemimethylated origins. SeqA also seems to inhibit initiation of fully methylated origins, thus contributing to the regulation of chromosomal replication. The SeqA protein was found to bind to two sites in the left part of the origin, near the AT-rich region where strand separation takes place during initiation of replication. The same binding sites seemed to be preferred irrespective of whether the origin was in the newly replicated (hemimethylated) state or not. In addition to binding specifically to groups of GATC sites, the SeqA protein was capable of interacting non-specifically with negatively supercoiled DNA, restraining the supercoils in a fashion similar to that seen with histone-like protein HU. The restraint of supercoils by SeqA was, in contrast to that of HU, cooperative.

Functional domains of DnaA proteins.

Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

Bacterial replication initiator DnaA. Rules for DnaA binding and roles of DnaA in origin unwinding and helicase loading.

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

Histochemical staining in lymphocyte suspensions complexed with beta-galactosidase as an antigen. Unspecific staining of spleen cells by indigo.

Spleen cells from mice immunized with E. coli beta-galactosidase were incubated with the enzyme and with 5-bromo-4-chloroindolyl-beta-galactosidase as a histochemical substrate. Some cells are stainable in this reaction, however, not all of them carry specific receptors for beta-galactosidases. Some spleen cells can take up dye and thereby become unspecifically stained. This system is therefore not suitable for the detection of antigen binding to cells.

Alternative-splicing of serotonin receptor isoforms in the pharynx and muscle of the parasitic nematode, Ascaris suum.

Pharyngeal pumping is essential for nematode feeding and survival and is dramatically stimulated by serotonin (5-HT). In the present study, a cDNA pool was prepared from poly A + RNA isolated from pharynxes dissected from the parasitic nematode, Ascaris suum, and was used as a template for RT-PCR with degenerate primers designed from sequences conserved in 5-HT receptors from a variety of sources. A putative 5-HT receptor cDNA (AS1) was identified which exhibited most identity to the 5-HT2 family of receptors. AS1 was 1925 nucleotides, did not appear to be trans-spliced and contained a 3' untranslated region of 127 nucleotides with a polyadenylation signal (ATTAAA) and a short poly A+ tail. The coding region predicted a protein of 532 amino acids with a molecular weight of 60 176. When AS1 was transiently expressed in COS-7 cells, isolated membranes exhibited the high affinity, saturable binding of [125I]LSD. More importantly, [125I]LSD binding was inhibited by 5-HT, but not other biogenic amines, supporting the identification of AS1 as a 5-HT receptor. Additional cDNAs identical, in part, to AS1 were also identified. AS1deltaIV lacked a predicted 42 amino acids at the carboxy terminus of the third intracellular loop, while AS2 and AS3 contained different COOH-termini, regions implicated in G-protein coupling in other heptahelical receptors. A portion of the gene (5htn) encoding AS1 also was cloned and sequenced. This genomic fragment was about 10 kb, contained the entire AS1 open reading frame and included eight exons and seven introns. From this analysis, it appears that these different AS cDNAs were generated by alternative-splicing, AS1deltaIV from the deletion of exon IV, and AS2 and AS3 from the use of alternative sites within exon VII as 5' splice acceptor sites for exon VIII. Using RT-PCR and primers specific for each of the isoforms, AS1 -3 appeared to be expressed in pharynx, while only AS1 and AS2 were present in body wall muscle. More importantly, the deletion of exon IV appeared to be associated exclusively with AS1 in pharynx and AS2 in muscle.

Inhibition and inactivation of presynaptic cholinergic markers using redox-reactive choline analogs.

Inhibition and inactivation of two presynaptic cholinergic "markers", choline acetyltransferase and high affinity choline transporter, has been investigated using inhibitors designed with a redox-reactive catechol tethered to a quaternary ammonium group. Two quaternary ammonium alkyl-substituted catechols, 3[(trimethylammonio)methyl]catechol (TMC, 1) and N,N-dimethylepinephrine (catecholine, 2) were shown to bind weakly and noncompetitively to bovine choline acetyltransferase yet inactivated the enzyme in a time course consistent with the involvement of early intermediates in the spontaneous oxidation of these catechols. Both agents also inhibited high-affinity choline uptake. The time course of TMC and catecholine spontaneous oxidation-dependent inactivation of high affinity choline uptake sites was slower than, if it occurred at all, the spontaneous degradation of measurable choline transport in synaptosomes. When compared with inhibition of uptake of other neurotransmitters, it was shown that catecholine demonstrated more selectivity than TMC toward inhibition of choline transport. Km (microM) and Vmax (pmol/min per mg of protein) were measured for high affinity transport of choline, dopamine, and serotonin and were observed to be Km = 2.04 +/- 0.31, Vmax = 22 +/- 1; Km = 1.4, Vmax = 53; and Km = 0.15, Vmax = 23, respectively, in good agreement with published literature values. Ki's (mM) for catecholine and TMC, calculated from experimentally determined IC50's, were for catecholine 0.13 +/- 0.06, 0.53 +/- 0.09, and 0.39 +/- 0.10, and for TMC 0.06 +/- 0.03, 0.09 +/- 0.03, and 0.09 +/- 0.08, for choline, dopamine, and serotonin transport, respectively. In vivo studies using catecholine suggest that this compound impairs learning ability associated with long-term memory. Thus, catecholine represents a lead compound in a potential series of redox-reactive choline analogs, which may become useful irreversible antagonists of the critical cholinergic macromolecular targets underlying cholinergic hypofunction in disorders such as Alzheimer's disease.

Regional differences in the binding of selective muscarinic receptor antagonists in rat brain: comparison with minimum-energy conformations.

The binding of selective muscarinic receptor antagonists to regions of rat brain was examined through quantitative autoradiographic techniques. 5,11-Dihydro-11-[(4-methyl-1-piperazinyl)acetyl]-6H- pyrido[2,3-b][1,4]benzodiazepin-6-one [pirenzepine (compound I)] and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido[2,3-b][1,4]benzodiazepin-6-one [AF-DX 116 (compound II)] were chosen on the basis of their selectivity for M1 and M2 muscarinic receptors, respectively, and similarities in chemical structure. Pirenzepine displayed a higher potency than II for inhibition of [3H]-l-quinuclidinyl benzilate ([3H]-l-QNB) binding to rat brain sections. Scatchard analyses of binding to brain sections revealed heterogeneous binding profiles for both antagonists, suggesting the presence of multiple receptor binding sites. Quantitative autoradiographic techniques were utilized in regional analyses of antagonist binding. Pirenzepine displayed the highest affinity for hippocampal, striatal, and amygdaloid muscarinic receptors (IC50 values less than 0.4 microM), with a slightly lower affinity for cortical receptors (IC50 values between 0.4 and 0.8 microM). Pirenzepine displayed the lowest affinity for thalamic and brainstem regions with IC50 values generally greater than 1.0 microM. In contrast, II bound with higher affinity to muscarinic receptors in brainstem, cerebellar, and hypothalamic nuclei (IC50 values less than 0.5 microM) than to receptors in thalamic nuclei (IC50 values between 0.5 and 2.0 microM). Binding sites with the lowest affinity for II were found in cortical, striatal, and hippocampal regions (IC50 values greater than 2.0 microM). The binding profiles of the two selective muscarinic antagonists reveal the complexity and diversity of muscarinic receptor subtypes throughout the brain. The data provide a basis for identifying muscarinic receptor subtypes (as defined through cloning procedures) with selective ligands. Minimum-energy conformations of pirenzepine and II were calculated by using the program MacroModel (version 2.0). Pirenzepine displayed three energy minima, differing in the relative position of the piperazine ring with respect to the tricyclic system. In contrast, the (diethylamino)methyl substituent on the piperidine ring conferred a much larger set of minimum-energy conformations on II. It is suggested that the greater conformational flexibility of the side chain allows II to achieve a conformation inaccessible to pirenzepine, which allows it to bind preferentially to M2 receptors.

Design, synthesis, and neurochemical evaluation of 5-(3-alkyl-1,2,4- oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidines as M1 muscarinic receptor agonists.

A series of 5-(3-alkyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidines+ ++ (7a-h) was synthesized for biological evaluation as selective agonists for M1 receptors coupled to phosphoinositide (PI) metabolism in the central nervous system. Each ligand bound with high affinity to muscarinic receptors from rat brain as measured by inhibition of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) binding. 5-(3-Methyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine+ ++ trifluoroacetate (CDD-0098-J;7a) displayed high affinity (IC50 = 2.7 +/- 0.69 microM) and efficacy at muscarinic receptors coupled to PI metabolism in the rat cortex and hippocampus. Increasing the length of the alkyl substituent increased affinity for muscarinic receptors yet decreased activity in PI turnover assays. The hippocampal PI response of 7a was blocked by lower concentrations of pirenzepine (8) or by higher concentrations of either AF-DX 116 (9) or p-fluorohexahydrosiladifenidol (10), suggesting that at low concentrations 7a selectively stimulates PI turnover through M1 receptors.