Inhibitors of HIV-1 attachment. Part 2: An initial survey of indole substitution patterns

The effects of introducing simple halogen, alkyl, and alkoxy substituents to the 4, 5, 6 and 7 positions of 1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione, an inhibitor of the interaction between HIV gp120 and host cell CD4 receptors, on activity in an HIV entry assay was examined. Small substituents at C-4 generally resulted in increased potency whilst substitution at C-7 was readily tolerated and uniformly produced more potent HIV entry inhibitors. Substituents deployed at C-6 and, particularly, C-5 generally produced a modest to marked weakening of potency compared to the prototype. Small alkyl substituents at N-1 exerted minimal effect on activity whilst increasing the size of the alkyl moiety led to progressively reduced inhibitory properties. These studies establish a basic understanding of the indole element of the HIV attachment inhibitor pharmacophore.     

Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

The assembly and maintenance of eukaryotic flagella are regulated by intraflagellar transport (IFT), the bidirectional traffic of IFT particles (recently renamed IFT trains) within the flagellum. We previously proposed the balance-point length control model, which predicted that the frequency of train transport should decrease as a function of flagellar length, thus modulating the length-dependent flagellar assembly rate. However, this model was challenged by the differential interference contrast microscopy observation that IFT frequency is length independent. Using total internal reflection fluorescence microscopy to quantify protein traffic during the regeneration of Chlamydomonas reinhardtii flagella, we determined that anterograde IFT trains in short flagella are composed of more kinesin-associated protein and IFT27 proteins than trains in long flagella. This length-dependent remodeling of train size is consistent with the kinetics of flagellar regeneration and supports a revised balance-point model of flagellar length control in which the size of anterograde IFT trains tunes the rate of flagellar assembly.     

Microbial Biogeography of Six Salt Lakes in Inner Mongolia,China, and a Salt Lake in Argentina

We used cultivation-independent methods to investigate the prokaryotic biogeography of the water column in six salt lakes in Inner Mongolia, China, and a salt lake in Argentina. These lakes had different salt compositions and pH values and were at variable geographic distances, on both local and intercontinental scales, which allowed us to explore the microbial community composition within the context of both contemporary environmental conditions and geographic distance. Fourteen 16S rRNA gene clone libraries were constructed, and over 200 16S rRNA gene sequences were obtained. These sequences were used to construct biotic similarity matrices, which were used in combination with environmental similarity matrices and a distance matrix in the Mantel test to discover which factors significantly influenced biotic similarity. We showed that archaeal biogeography was influenced by contemporary environmental factors alone (Na⁺, CO₃²⁻, and HCO₃⁻ ion concentrations; pH; and temperature). Bacterial biogeography was influenced both by contemporary environmental factors (Na⁺, Mg²⁺, and HCO₃⁻ ion concentrations and pH) and by geographic distance.Biogeography aims to explain spatial patterns of diversity in the context of evolutionary events such as speciation, dispersal, extinction, and species interactions (42). Macroecologists have long studied the biogeography of higher plants and animals in various habitats (9, 13). In contrast, there is very little information available on the biogeography of prokaryotes. This stemmed from the difficulty of assessing microbial communities by cultivation methods, which only sampled 0.1 to 10% of the microbial community (30). However, with the advent of cultivation-independent sequencing techniques, microbial communities of many environments have been characterized, including soil (43), the Arctic and Antarctic Oceans (5), and the Sargasso Sea (61). This in turn facilitated prokaryotic biogeography studies in a number of environments on scales ranging from 20,000 km to 0.002 km (42).A study of the biogeography of soil bacteria across the Americas showed that differences were largely attributed to soil pH, with higher diversity observed in neutral soils (20). Bacterial communities in an estuary in Massachusetts were found to vary with the salinity gradient (14). Such studies demonstrated that environmental parameters influenced biogeographical patterns in microbial diversity. Further studies demonstrated that biogeography of hot spring cyanobacteria, hyperthermophilic archaea, and Pseudomonas strains was influenced by geographic distance, which led to isolation of disparate populations and subsequent genetic divergence (12, 51, 63). The apparent allopatric speciation demonstrated in these studies therefore contested the idea that prokaryotes were not affected by limits to dispersal due to their small size, abundance, and metabolic plasticity (i.e., “everything is everywhere” [see below]) (21).A simple framework was suggested to distinguish between the effects of evolutionary events and contemporary environmental conditions on the spatial variation of microbial diversity (42). At the center of this framework were four hypotheses. The null hypothesis stated that microorganisms were distributed randomly over space. Upon rejection of the null hypothesis, the second hypothesis stated that spatial variation reflected the influence of contemporary environmental variation. It assumed that geographic distance did not affect diversity due to the wide dispersal of microorganisms. This hypothesis represents the famously quoted “everything is everywhere; the milieu selects” by Baas-Becking (4, 6). The third hypothesis stated that variation was shaped by evolutionary events (geographic distance) that limited dispersal and that past environmental conditions led to genetic divergence between different microbial assemblages. The fourth hypothesis stated that the biogeography of microorganisms was determined by both contemporary environmental conditions and past evolutionary events (geographic distance). It is important to note here the possibility that evolutionary events can be represented by geographic distances. (For more details on this framework, see reference 42.)Many studies have been carried out on salt lakes and salterns around the world (28), but few have tried to explain variations in microbial community composition. Those that did identified salinity, altitude, redox and ionic concentration, pO₂, and seasonal events as relevant factors (7, 11, 16, 17, 34, 35, 38, 65). To our knowledge, only two studies have looked at the effect of intercontinental geographic distances on microbial community composition in salt lakes. Foti and colleagues looked specifically at the biogeography of Thioalkalivibrio in soda lakes across Mongolia, Kenya, California, Egypt, and Siberia and found that these bacteria showed a tendency for endemism; hence, geographic distance was a significant factor in influencing community composition (22). A further study looked at the biogeography of Salinibacter ruber strains from salterns in the Mediterranean, Atlantic, and Peruvian regions using a metabolomic approach. Geographically distinct strains were distinguished by characteristic metabolites (58).We examined the prokaryotic community composition in several salt lakes using ribosomal DNA methods. Six of the salt lakes in this study were situated on the Inner Mongolian steppe, northwest of Beijing, which had an average elevation of 1,000 to 2,000 m above sea level. The lakes were mostly several hundred kilometers apart (0.147 to 395.2 km) and were in different climate and vegetation zones: from typical grassland steppe in the north and east to desert steppe bordering the Gobi desert in the south and west (70). The lakes were Bagaejinnor, Chagannor, Ejinnor, Erliannor, Shangmatala, and an unnamed lake near Xilinhot. Lakes Ejinnor and Erliannor were extensively developed into salterns. Salar Guayatayoc Lake was situated in the same basin as the Salinas Grandes in the Argentine Altiplano at an elevation of 3,432 m, north-west of the city Salta, ∼18,000 km from the other lakes. All salt lakes were athalassohaline, located in arid climates, and subjected to high solar radiation and wide ranges of temperature. The lakes had different salt compositions and allowed us to explore the microbial community composition within the context of both contemporary environmental conditions and geographic distance.Here we describe the microbial diversity of six salt lakes in Inner Mongolia and one salt lake in Argentina. Using the framework previously described, we present evidence that biogeography of Archaea in these salt lakes was significantly influenced (P < 0.05) by environmental factors (Na⁺, CO₃²⁻, and HCO₃⁻ ion concentrations, pH, and temperature), but not geographic distance, consistent with the previously stated hypothesis 2. We also show that the biogeography of Bacteria was significantly influenced (P < 0.05) by both environmental factors (Na⁺, Mg²⁺, and HCO₃⁻ ion concentrations and pH) and geographic distance, consistent with the previously stated hypothesis 4.     

Quantitative Method To Determine Sporicidal Decontamination of Building Surfaces by Gaseous Fumigants,and Issues Related to Laboratory-Scale Studies

Chlorine dioxide gas and vaporous hydrogen peroxide sterilant have been used in the cleanup of building interiors contaminated with spores of Bacillus anthracis. A systematic study, in collaboration with the U.S. Environmental Protection Agency, was jointly undertaken by the U.S. Army-Edgewood Chemical Biological Center to determine the sporicidal efficacies of these two fumigants on six building structural materials: carpet, ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. Critical issues related to high-throughput sample processing and spore recovery from porous and nonporous surfaces included (i) the extraction of spores from complex building materials, (ii) the effects of titer challenge levels on fumigant efficacy, and (iii) the impact of bioburden inclusion on spore recovery from surfaces and spore inactivation. Small pieces (1.3 by 1.3 cm of carpet, ceiling tile, wallboard, I-beam steel, and pinewood and 2.5 by 1.3 cm for cinder block) of the materials were inoculated with an aliquot of 50 μl containing the target number (1 × 10⁶, 1 × 10⁷, or 1 × 10⁸) of avirulent spores of B. anthracis NNR1Δ1. The aliquot was dried overnight in a biosafety cabinet, and the spores were extracted by a combination of a 10-min sonication and a 2-min vortexing using 0.5% buffered peptone water as the recovery medium. No statistically significant drop in the kill efficacies of the fumigants was observed when the spore challenge level was increased from 6 log units to 8 log units, even though a general trend toward inhibition of fumigant efficacy was evident. The organic burden (0 to 5%) in the spore inoculum resulted in a statistically significant drop in spore recovery (at the 2 or 5% level). The effect on spore killing was a function of the organic bioburden amount and the material type. In summary, a high-throughput quantitative method was developed for determining the efficacies of fumigants, and the spore recoveries from five porous materials and one nonporous material ranged between 20 and 80%.Biological terrorism has become a major concern in the United States since the anthrax spore-tainted letters in the fall of 2001 resulted in contamination and closure of the U.S. Postal Service Curseen-Morris Processing and Distribution Center (Brentwood Post Office), the Hart Senate Office Building, and the American Media Inc. office building in Boca Raton, FL. The contamination of infrastructure posed an unprecedented challenge of decontaminating over 20,000,000 cubic feet (∼1 million sq. ft.) of combined building interior space (6). The incident required concerted action from the government of the United States and the private sector to develop technologies for building interior cleanup. A number of liquid (29) and gaseous (3) products were granted crisis exemptions under the Federal Insecticide, Fungicide, and Rodenticide Act by the U.S. Environmental Protection Agency (EPA) for use as sterilants against Bacillus anthracis spores, but their application and efficacy in the context of large three-dimensional spaces and complex building material surfaces were not fully understood. No products were (or currently are) registered for use in such applications, involving large volumes and complex (porous and nonporous) structural building materials.In early 2005, a systematic study of laboratory-scale decontamination of five porous surfaces (carpet, ceiling tile, cinder block, painted wallboard, and unpainted wood) and one nonporous surface (painted I-beam steel) was initiated by the U.S. EPA in collaboration with the U.S. Army Edgewood Chemical Biological Center (ECBC). The overall objective of this collaborative study was to systematically investigate the abilities of fumigants to effectively decontaminate building materials contaminated with anthrax spores. This unprecedented systematic investigation involved the determination of efficacy (or log reduction in the number of viable spores) as a function of fumigant technology, technology operating parameters (e.g., fumigant concentration and exposure time), environmental conditions (temperature and relative humidity [RH]), and building material types. The magnitude and scope of this study required that new methods be developed to incorporate the use of complex materials in sporicidal efficacy testing and the processing of an unprecedented number of complex samples.Current standardized sporicidal test methods include the Association of Official Analytical Chemists (AOAC International) sporicidal activity of disinfectant test (AOAC Official Method 966.04) (4) and the American Society for Testing and Materials (ASTM) 2414-05 (3) and quantitative carrier test (QCT) (2). All of these methods are based on testing hard-surface carrier-based spores, which are submerged in a disinfectant for a desired contact time, followed by the addition of a neutralizer and enumeration of viable spores recovered from the carrier. Almost all standard test methods for liquid disinfectants use small coupons, e.g., 5- by 5-mm squares or 1-cm discs, on which 1 million to 10 million (6 to 7 log) spores are inoculated. While AOAC Official Method 966.04 is qualitative, the other two test methods are quantitative and provide log reduction estimates. Currently, demonstration of a >6-log-unit inactivation of B. anthracis or an appropriate surrogate spore (e.g., Bacillus subtilis) using a quantitative test method, such as QCT, which is also known as ASTM 2197-02, or the three-step method (TSM), also known as ASTM 2414-05, by a decontaminant is a requirement for product registration as a sporicidal agent against spores of B. anthracis Ames (18).Key information on three critical issues was lacking at the start of this study. First, optimal spore extraction protocols that could be scaled to process over 200 samples per run (or day) were lacking. Second, the appropriate spore challenge level for fumigation studies was unknown, even though a range between 5 and 8 log spores/coupon has been used in a number of recent disinfection studies (12, 13, 14, 16, and 17). Finally, it was not known if protein serum (an organic burden is included in standard procedures, such as AOAC Official Method 966.04) should be included in the testing performed with the fumigants. The specific objectives of this study, therefore, were to (i) develop scalable coupon-processing/spore extraction protocols from six building materials that would result in recovery of >20% of the spores inoculated per coupon, (ii) investigate the effects of three spore challenge levels on spore extraction and the efficacy of chlorine dioxide (CD) gas and vaporous hydrogen peroxide (VHP), and, finally, (iii) investigate the effect of organic burden inclusion on spore recovery and sterilization using CD gas.     

Isolation and characterization of 11 microsatellite loci from Oropsylla hirsuta, a vector of sylvatic plague

The flea (Oropsylla hirsuta) is an important vector of the plague bacterium, Yersinia pestis, in black-tailed prairie dog (Cynomys ludovicianus) colonies. We developed 11 anonymous microsatellite primers for O. hirsuta using a subtractive hybridization procedure. All primers were polymorphic exhibiting 4-12 alleles.     

Heterozygous Mutation of Opa1 in Drosophila Shortens Lifespan Mediated through Increased Reactive Oxygen Species Production

Optic atrophy 1 (OPA1) is a dynamin-like GTPase located in the inner mitochondrial membrane and mutations in OPA1 are associated with autosomal dominant optic atrophy (DOA). OPA1 plays important roles in mitochondrial fusion, cristae remodeling and apoptosis. Our previous study showed that dOpa1 mutation caused elevated reactive oxygen species (ROS) production and resulted in damage and death of the cone and pigment cells in Drosophila eyes. Since ROS-induced oxidative damage to the cells is one of the primary causes of aging, in this study, we examined the effects of heterozygous dOpa1 mutation on the lifespan. We found that heterozygous dOpa1 mutation caused shortened lifespan, increased susceptibility to oxidative stress and elevated production of ROS in the whole Drosophila. Antioxidant treatment partially restored lifespan in the male dOpa1 mutants, but had no effects in the females. Heterozygous dOpa1 mutation caused an impairment of respiratory chain complex activities, especially complexes II and III, and reversible decreased aconitase activity. Heterozygous dOpa1 mutation is also associated with irregular and dysmorphic mitochondria in the muscle. Our results, for the first time, demonstrate the important role of OPA1 in aging and lifespan, which is most likely mediated through augmented ROS production.     

Calcium Homeostasis and Cone Signaling Are Regulated by Interactions between Calcium Stores and Plasma Membrane Ion Channels

Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca²⁺ entry (SOCE) to Ca²⁺ homeostasis and its role in regulation of neurotransmission at cone synapses. Mn²⁺ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca²⁺ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca²⁺ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca²⁺ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca²⁺ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca²⁺ channels. Exposure to MRS 1845 resulted in ∼40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca²⁺ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca²⁺ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse.     

A xylanase produced by the rumen anaerobic protozoan Polyplastron multivesiculatum shows close sequence similarity to family 11 xylanases from gram-positive bacteria

We report for the first time the cloning and characterisation of a protozoal enzyme involved in plant cell wall polysaccharide degradation. A cDNA library was constructed from the ruminal protozoan Polyplastron multivesiculatum and a stable clone expressing xylanase activity was isolated. The encoded enzyme belongs to the glycoside hydrolase family 11, and phylogenetic analysis indicates a closer relationship with catalytic domains from Gram-positive bacteria than the other fibrolytic eukaryotes from the rumen, the anaerobic fungi.