Identification of Novel Imidazo[1,2-a]pyridine Inhibitors Targeting M. tuberculosis QcrB

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1–4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism ⁹³⁷ACC>⁹³⁷GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.     

Effect of Oxidizing Disinfectants (Chlorine, Monochloramine, and Ozone) on Helicobacter pylori

The susceptibility of Helicobacter pylori to disinfectants was compared to that of Escherichia coli. H. pylori is more resistant than E. coli to chlorine and ozone but not monochloramine. H. pylori may be able to tolerate disinfectants in distribution systems and, therefore, may be transmitted by a waterborne route.     

The Impact of Cyclone Fanele on a Tropical Dry Forest in Madagascar

Cyclones, which change tree communities and alter forest structure, are thought to have had a significant selective pressure on the flora and fauna of Madagascar. Very little information, however, is available on the actual impact of cyclones on Malagasy ecosystems. On 21 January 2009, Cyclone Fanele made landfall on the western coast of Madagascar with sustained winds of 185 km/h. We examined the immediate effects of the cyclone on tropical dry forest structure in the Kirindy Mitea National Park. In July and August 2009, we measured the height, diameter at breast height (dbh), and damage for 1361 trees in nine 25 × 25 m plots. We found that: (1) over 95 percent of trees experienced some sort of damage, including 8.8 percent mortality; (2) understory and emergent trees experienced significantly higher mortality than canopy trees; and (3) stem density was reduced 9.2±4.5 percent and biomass was reduced 13.4±8.1 percent after the cyclone. Dbh was the best predictor of trunk damage and mortality. This extensive alteration of forest structure will have a substantial short‐ and long‐term impact on the biotic communities of western Madagascar.     

Solution structure of mu-conotoxin PIIIA,a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels

Mu-conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of mu-conotoxins PIIIA and PIIIA-(2-22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2-22), and mu-conotoxins GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. (1)H NMR studies found that PIIIA adopts two conformations in solution due to cis/trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of mu-conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective mu-conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by mu-conotoxins.     

Tuning of the optical and electrochemical properties of the primary donor bacteriochlorophylls in the reaction centre from Rhodobacter sphaeroides: spectroscopy and structure

A series of mutations have been introduced at residue 168 of the L-subunit of the reaction centre from Rhodobacter sphaeroides. In the wild-type reaction centre, residue His L168 donates a strong hydrogen bond to the acetyl carbonyl group of one of the pair of bacteriochlorophylls (BChl) that constitutes the primary donor of electrons. Mutation of His L168 to Phe or Leu causes a large decrease in the mid-point redox potential of the primary electron donor, consistent with removal of this strong hydrogen bond. Mutations to Lys, Asp and Arg cause smaller decreases in redox potential, indicative of the presence of weak hydrogen bond and/or an electrostatic effect of the polar residue. A spectroscopic analysis of the mutant complexes suggests that replacement of the wild-type His residue causes a decrease in the strength of the coupling between the two primary donor bacteriochlorophylls. The X-ray crystal structure of the mutant in which His L168 has been replaced by Phe (HL168F) was determined to a resolution of 2.5 A, and the structural model of the HL168F mutant was compared with that of the wild-type complex. The mutation causes a shift in the position of the primary donor bacteriochlorophyll that is adjacent to residue L168, and also affects the conformation of the acetyl carbonyl group of this bacteriochlorophyll. This conformational change constitutes an approximately 27 degrees through-plane rotation, rather than the large into-plane rotation that has been widely discussed in the context of the HL168F mutation. The possible structural basis of the altered spectroscopic properties of the HL168F mutant reaction centre is discussed, as is the relevance of the X-ray crystal structure of the HL168F mutant to the possible structures of the remaining mutant complexes.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.     

Familial mediterranean fever (FMF) in Moroccan Jews: Demonstration of a founder effect by extended haplotype analysis

Familial Mediterranean fever (FMF) is an autosomal recessive disease causing attacks of fever and serositis. The FMF gene (designated “MEF”) is on 16p, with the gene order 16cen–D16S80–MEF–D16S94–D16S283–D16S291–16pter. Here we report the association of FMF susceptibility with alleles at D16S94, D16S283, and D16S291 among 31 non-Ashkenazi Jewish families (14 Moroccan, 17 non-Moroccan). We observed highly significant associations at D16S283 and D16S291 among the Moroccan families. For the non-Moroccans, only the allelic association at D16S94 approached statistical significance. Haplotype analysis showed that 18/25 Moroccan FMF chromosomes, versus 0/21 noncarrier chromosomes, bore a specific haplotype for D16S94–D16S283–D16S291. Among non-Moroccans this haplotype was present in 6/26 FMF chromosomes versus 1/28 controls. Both groups of families are largely descended from Jews who fled the Spanish Inquisition. The strong haplotype association seen among the Moroccans is most likely a founder effect, given the recent origin and genetic isolation of the Moroccan Jewish community. The lower haplotype frequency among non-Moroccan carriers may reflect differences both in history and in population genetics.     

Two tumor models of curative adoptive chemoimmunotherapy using tumor-infiltrated spleen cells with potent antitumor cytotoxicity stimulated by antigen-sharing tumors

Previously we have established curative protocols for adoptive chemoimmunotherapy (ACIT) of mice bearing different plasmacytomas that are known to bear cross-reacting antigens: (a) the cure of mice bearing an early-stage, nonpalpable MOPC-315 tumor by a very low dose of cyclophosphamide (10 mg/kg) and cultured MOPC-315-tumor-infiltrated (TI) spleen cells (25×10⁶) and (b) the cure of mice bearing a late-stage, relatively drug-resistant, highly metastatic RPC-5 tumor with cyclophosphamide (100 mg/kg) and cultured RPC-5 TI spleen cells (25×10⁶–50×10⁶). In both models, the spleen cells were obtained from mice bearing a late-stage tumor and were cultured for 5 days in the presence of polyethyleneglycol 6000 and autochthonous tumor cells as a source of tumor antigen. Here we show that RPC-5 tumor cells could substitute for MOPC-315 tumor cells in the 5-day culture of MOPC-315 TI spleen cells so that they became curative in ACIT for mice bearing an early-stage MOPC-315 tumor. Similarly, MOPC-315 tumor cells could substitute for RPC-5 tumor cells in the 5-day culture of RPC-5 TI spleen cells so that they became curative in ACIT of mice bearing a late-stage RPC-5 tumor. In addition, RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were effective in curing all mice bearing an early-stage MOPC-315 tumor by ACIT. However, MOPC-315 TI spleen cells whether cultured with MOPC-315 or RPC-5 tumor cells, were much less effective than cultured RPC-5 TI spleen cells in curing mice bearing a late-stage RPC-5 tumor by ACIT (although the survival of these mice was extended significantly). Interestingly, whereas RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were as effective as MOPC-315 TI spleen cells cultured under the same conditions in lysing MOPC-315 tumor cells in vitro, MOPC-315 TI spleen cells that had been cultured with either MOPC-315 or RPC-5 tumor cells exerted a much weaker in vitro cytotoxic T lymphocyte activity against RPC-5 tumor cells than did RPC-5 TI spleen cells that had been cultured under the same conditions.Work was supported by research grant CA-30088 from the National Cancer Institute and IM-435 from the American Cancer Society. M. B. M. was supported by Career Development Award CA-01350 from the National Cancer InstituteThis work is in partial fulfillment of the requirements for the Ph. D. degree     

Trends in wood density and structure are linked to prevention of xylem implosion by negative pressure

Wood density (D_t), an excellent predictor of mechanical properties, is typically viewed in relation to support against gravity, wind, snow, and other environmental forces. In contrast, we show the surprising extent to which variation in D_t and wood structure is linked to support against implosion by negative pressure in the xylem pipeline. The more drought-tolerant the plant, the more negative the xylem pressure can become without cavitation, and the greater the internal load on the xylem conduit walls. Accordingly, D_t was correlated with cavitation resistance. This trend was consistent with the maintenance of a safety factor from implosion by negative pressure: conduit wall span (b) and thickness (t) scaled so that (t/b)² was proportional to cavitation resistance as required to avoid wall collapse. Unexpectedly, trends in D_t may be as much or more related to support of the xylem pipeline as to support of the plant.     

A localized zone of increased conductance progresses over the surface of the sea urchin egg during fertilization

Although activation of a sea urchin egg by sperm leads to three phases of membrane conductance increase in the egg, the mechanism by which the sperm causes these conductance changes is not known. We used the loose patch clamp technique to localize the conductance changes in voltage clamped eggs. A patch of the egg's membrane was isolated from the bath by pressing the loose patch clamp pipette against the egg surface. Sperm added to the bath attached to the surface of the egg in a region other than at the isolated membrane patch. During phase 1 of the activation current, no changes of the membrane conductance were detected. At the time of, and subsequent to the onset of phase 2, large currents recorded between the interior of the patch pipette and the bath were attributed to changes of the seal resistance between the surface of the egg and the pipette. A local change of membrane conductance was observed during phase 2 despite the changes of seal resistance. During phase 2, the large amplitude and short duration of the local membrane conductance increase relative to the membrane, conductance increase for the whole egg during phase 2 indicated that the conductance increase occurred over the entire surface of the egg, but not simultaneously. The time when the peak conductance for the membrane patch occurred, relative to the time of onset for phase 2 in the whole egg, depended on the distance, measured in a straight line, between the site of sperm attachment and the tip of the pipette. These data indicate that the localized conductance increase progressed over the surface of the egg from the site of sperm attachment to the opposite pole of the egg. It is proposed that the local conductance increase, the cortical reaction, and the change of seal resistance are all evoked by a common cytoplasmic message that progresses throughout the cytoplasm of the egg from the site of sperm attachment to the opposite pole of the egg.