Discovery of novel 5-(ethyl or hydroxymethyl) analogs of 2'-'up' fluoro (or hydroxyl) pyrimidine nucleosides as a new class of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium inhibitors

Discovery of novel antimycobacterial compounds that work on distinctive targets and by diverse mechanisms of action is urgently required for the treatment of mycobacterial infections due to the emerging global health threat of tuberculosis. We have identified a new class of 5-ethyl or hydroxy (or methoxy) methyl-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium bovis, Mycobacterium tuberculosis (H37Ra, H37Rv) and Mycobacterium avium. A series of 2'-'up' fluoro (or hydroxy) nucleosides (1, 2, 4-6, 9, 10, 13, 16, 18, 21, 24) was synthesized and evaluated for antimycobacterial activity. Among 2'-fluorinated compounds, 1-(3-bromo-2,3-dideoxy-2-fluoro-β-d-arabinofuranosyl)-5-ethyluracil (13) exhibited promising activity against M. bovis and Mtb alone, and showed synergism when combined with isoniazid. The most active compound emerging from these studies, 1-(β-d-arabinofuranosyl)-4-thio-5-hydroxymethyluracil (21) inhibited Mtb (H37Ra) (MIC(50)=0.5 μg/mL) and M. bovis (MIC(50)=0.5 μg/mL) at low concentrations, and was ten times more potent against Mtb (H37Ra) than cycloserine (MIC(50)=5.0 μg/mL), a second line drug. It also showed an additive effect when combined with isoniazid. Compound 21 retained sensitivity against a rifampicin-resistant (H37Rv) strain of Mtb (MIC(50)=1 μg/mL) at concentrations similar to that for a rifampicin-sensitive (H37Rv) strain, suggesting that it has no cross-resistance to a first-line anti-TB drug. In addition, the replication of M. avium was also inhibited by 21 (MIC(50)=10 μg/mL). No cellular toxicity of 13 or 21 was observed up to the highest concentration tested (CC(50)>100 μg/mL). These observations offer promise for a new drug treatment regimen to augment and complement the current chemotherapy of TB.     

Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum

Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 10(7) cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (K(m)=1.7 microM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target.     

Genome Scan of M. tuberculosis Infection and Disease in Ugandans

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is an enduring public health problem globally, particularly in sub-Saharan Africa. Several studies have suggested a role for host genetic susceptibility in increased risk for TB but results across studies have been equivocal. As part of a household contact study of Mtb infection and disease in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB, by studying three phenotypes. First, we analyzed culture confirmed TB disease compared to latent Mtb infection (LTBI) or lack of Mtb infection. Second, we analyzed resistance to Mtb infection in the face of continuous exposure, defined by a persistently negative tuberculin skin test (PTST-); this outcome was contrasted to LTBI. Third, we analyzed an intermediate phenotype, tumor necrosis factor-alpha (TNFα) expression in response to soluble Mtb ligands enriched with molecules secreted from Mtb (culture filtrate). We conducted a full microsatellite genome scan, using genotypes generated by the Center for Medical Genetics at Marshfield. Multipoint model-free linkage analysis was conducted using an extension of the Haseman-Elston regression model that includes half sibling pairs, and HIV status was included as a covariate in the model. The analysis included 803 individuals from 193 pedigrees, comprising 258 full sibling pairs and 175 half sibling pairs. Suggestive linkage (p<10⁻³) was observed on chromosomes 2q21-2q24 and 5p13-5q22 for PTST-, and on chromosome 7p22-7p21 for TB; these findings for PTST- are novel and the chromosome 7 region contains the IL6 gene. In addition, we replicated recent linkage findings on chromosome 20q13 for TB (p = 0.002). We also observed linkage at the nominal α = 0.05 threshold to a number of promising candidate genes, SLC11A1 (PTST- p = 0.02), IL-1 complex (TB p = 0.01), IL12BR2 (TNFα p = 0.006), IL12A (TB p = 0.02) and IFNGR2 (TNFα p = 0.002). These results confirm not only that genetic factors influence the interaction between humans and Mtb but more importantly that they differ according to the outcome of that interaction: exposure but no infection, infection without progression to disease, or progression of infection to disease. Many of the genetic factors for each of these stages are part of the innate immune system.     

Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway

Endocytosis of the nicotinic acetylcholine receptor (AChR) is a proposed major mechanism of neuromodulation at neuromuscular junctions and in the pathology of synapses in the central nervous system. We show that binding of the competitive antagonist alpha-bungarotoxin (alphaBTX) or antibody-mediated cross-linking induces the internalization of cell surface AChR to late endosomes when expressed heterologously in Chinese hamster ovary cells or endogenously in C2C12 myocytes. Internalization occurs via sequestration of AChR-alphaBTX complexes in narrow, tubular, surface-connected compartments, which are indicated by differential surface accessibility of fluorescently tagged alphaBTX-AChR complexes to small and large molecules and real-time total internal reflection fluorescence imaging. Internalization occurs in the absence of clathrin, caveolin, or dynamin but requires actin polymerization. alphaBTX binding triggers c-Src phosphorylation and subsequently activates the Rho guanosine triphosphatase Rac1. Consequently, inhibition of c-Src kinase activity, Rac1 activity, or actin polymerization inhibits internalization via this unusual endocytic mechanism. This pathway may regulate AChR levels at ligand-gated synapses and in pathological conditions such as the autoimmune disease myasthenia gravis.     

Processing of DNA replication and repair intermediates by the concerted action of RecQ helicases and Rad2 structure-specific nucleases

Processing of DNA replication and repair intermediates is a critical aspect of genome stability maintenance. The coordinated action of RecQ-like helicases with structure-specific nucleases such as Flap Endonuclease 1 plays an important role in the processing of certain DNA structures associated with the replication fork, DNA repair, or telomeres. We will summarize our current understanding of how and in what context these interactions take place, with a particular emphasis on the mechanisms of RecQ helicases in processing of key DNA replication and repair intermediates by their protein interactions with FEN-1 and related structure-specific nucleases.     

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg²⁺ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg²⁺ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg²⁺ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the γ-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.     

Involvement and interaction of various signaling compounds on the plant metabolic events during defense response,resistance to stress factors,formation of secondary metabolites and their molecular aspects

Plants are a nearly unlimited source of phytochemicals. The plants produce various secondary metabolites, which are useful in its interaction with the environment, various stress factors and development of resistance against pathogen attack. A wide array of external stimuli are capable of triggering changes in the plant cell which leads to a cascade of reactions, ultimately resulting in the formation and accumulation of secondary metabolites which helps the plant to overcome the stress factors. The biotic and abiotic elicitors can result in an enhancement of the secondary metabolite production. The stimuli are perceived by receptors, which then result in the activation of the secondary messengers. These then transmit the signals into the cell through the signal transduction pathways leading to gene expression and biochemical changes. There is interplay of the signaling molecules also which regulates the entire pathway. This review is oriented towards the factors, which influence signal transduction pathway(s) with special reference to polyamines, calcium, jasmonates, salicylates, nitric oxide and ethylene. The interplay of these components to elicit a defense response is discussed. Molecular aspects of disease resistance and regulation of plant secondary metabolism has also been presented.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.