7-Alkyl-N(2)-substituted-3-deazaguanines. Synthesis, DNA polymerase III inhibition and antibacterial activity

Several 2-anilino- and 2-benzylamino-3-deaza-6-oxopurines [3-deazaguanines] and selected 8-methyl and 8-aza analogs have been synthesized. 7-Substituted N²-(3-ethyl-4-methylphenyl)-3-deazaguanines were potent and selective inhibitors of Gram+ bacterial DNA polymerase (pol) IIIC, and 7-substituted N²-(3,4-dichlorobenzyl)-3-deazaguanines were potent inhibitors of both pol IIIC and pol IIIE from Gram+ bacteria, but weakly inhibited pol IIIE from Gram− bacteria. Potent enzyme inhibitors in both classes inhibited the growth of Gram+ bacteria (MICs 2.5-10 μg/ml), and were inactive against the Gram− organism Escherichia coli. Several derivatives had moderate protective activity in Staphylococcus aureus-infected mice.     

Accelerated evolution associated with genome reduction in a free-living prokaryote

Background

Three complete genomes of Prochlorococcus species, the smallest and most abundant photosynthetic organism in the ocean, have recently been published. Comparative genome analyses reveal that genome shrinkage has occurred within this genus, associated with a sharp reduction in G+C content. As all examples of genome reduction characterized so far have been restricted to endosymbionts or pathogens, with a host-dependent lifestyle, the observed genome reduction in Prochlorococcus is the first documented example of such a process in a free-living organism.

Results

Our results clearly indicate that genome reduction has been accompanied by an increased rate of protein evolution in P. marinus SS120 that is even more pronounced in P. marinus MED4. This acceleration has affected every functional category of protein-coding genes. In contrast, the 16S rRNA gene seems to have evolved clock-like in this genus. We observed that MED4 and SS120 have lost several DNA-repair genes, the absence of which could be related to the mutational bias and the acceleration of amino-acid substitution.

Conclusions

We have examined the evolutionary mechanisms involved in this process, which are different from those known from host-dependent organisms. Indeed, most substitutions that have occurred in Prochlorococcus have to be selectively neutral, as the large size of populations imposes low genetic drift and strong purifying selection. We assume that the major driving force behind genome reduction within the Prochlorococcus radiation has been a selective process favoring the adaptation of this organism to its environment. A scenario is proposed for genome evolution in this genus.     

Study of nuclear basic proteins of Physarum polycephalum: Differential synthesis during plasmodial cell cycle and spherule germination

An electrophoresis system providing a good resolution of all basic proteins and their variants or modified forms extracted from Physarum polycephalum nuclei has been used to study the synthesis of the basic nuclear proteins of this organism during three developmental stages: the S-phase and the G2-phase of the plasmodial cell cycle and the premitotic stage of germinating spherules. Differences have been seen in the synthesis of the different classes of histones and HMG like proteins during these three stages.     

Effects of rho-fluorophenylalanine on the growth of Lactobacillus arabinosus

p-Fluorophenylalanine has been found to be inhibitory to Lactobacillus arabinosus 17-5. This inhibition has been shown to be competitive with phenylalanine but not with tyrosine, and is described by a low antibacterial index, which varies over a range of approximately 1–20, dependent upon the temporal stage of the culture for which the data are employed in calculation.The organism has been found also to adapt readily to the inhibitor. Evidence that this adjustment involves a mutation to a type with a selective advantage in media containing the inhibitor is presented.Under limiting conditions, which have been described, the organism is capable of greater growth in the presence of both metabolite and antimetabolite than in the presence of the same amount of metabolite alone.Discussion of the significance of the results has been presented.     

Mutants of Anabaena cylindrica altered in heterocyst spacing

Nitrosoguanidine induced mutants of Anabaena cylindrica have been obtained, which are altered in heterocyst spacing. In the wild type organism the pattern is composed of single intercalary heterocysts. The mutant patterns fall into several classes: those with only terminal heterocysts, with both terminal and intercalary heterocysts, with groups of heterocysts and those totally lacking heterocysts. The mutants are described in detail, and the various pattern modifications are interpreted in terms of a model we have proposed.     

Genome-Wide RNAi Longevity Screens in Caenorhabditis elegans

Progress in aging research has identified genetic and environmental factors that regulate longevity across species. The nematode worm Caenorhabditis elegans is a genetically tractable model system that has been widely used to investigate the molecular mechanisms of aging, and the development of RNA interference (RNAi) technology has provided a powerful tool for performing large-scale genetic screens in this organism. Genome-wide screens have identified hundreds of genes that influence lifespan, many of which fall into distinct functional classes and pathways. The purpose of this review is to summarize the results of large-scale RNAi longevity screens in C. elegans, and to provide an in-depth comparison and analysis of their methodology and most significant findings.     

Assays optimized for detection and quantification of antibacterial activity in shark cell lysates under high salt conditions

To assess non-cellular innate immune mechanisms that play a role in the antimicrobial defense of an organism several assay systems have been devised to screen for such factors. Most assays, however, have been developed to measure activity against clinical isolates of medical importance. There is scant information on methods optimal for assaying material from sharks and other marine fish for antimicrobial activity particularly against salt tolerant organisms that are likely to be encountered in the marine environment. We have modified and optimized agar diffusion and broth dilution assays for detection and quantification of antibacterial activity of shark leukocyte lysates. By replacing marine agar, typically used for marine organisms, with artificial sea water complete medium (SCM) enriched with tryptone and yeast extract has resulted in an improved inhibition zone assay that uses Planococcus citreus, a salt-tolerant organism as the target organism. Antibacterial activity is correlated to the size of zone of no bacterial growth around wells containing bioactive test sample. An alternative broth based microdilution growth assay uses the 96 well format and the antibacterial effect of the sample on growth of P. citreus, the target organism, is measured spectrophotometrically as percent inhibition of bacterial growth when compared to the growth of P. citreus grown in medium alone that represents 100% growth. The assay can also be used to titrate antibacterial activity and express the level of growth inhibition as a titer.     

Cloning and Characterization of Two Pyruvate Decarboxylase Genes from Pichia stipitis CBS 6054

In Pichia stipitis, fermentative and pyruvate decarboxylase (PDC) activities increase with diminished oxygen rather than in response to fermentable sugars. To better characterize PDC expression and regulation, two genes for PDC (PsPDC1 and PsPDC2) were cloned and sequenced from P. stipitis CBS 6054. Aside from Saccharomyces cerevisiae, from which three PDC genes have been characterized, P. stipitis is the only organism from which multiple genes for PDC have been identified and characterized. PsPDC1 and PsPDC2 have diverged almost as far from one another as they have from the next most closely related known yeast gene. PsPDC1 contains an open reading frame of 1,791 nucleotides encoding 597 amino acids. PsPDC2 contains a reading frame of 1,710 nucleotides encoding 570 amino acids. An 81-nucleotide segment in the middle of the β domain of PsPDC1 codes for a unique segment of 27 amino acids, which may play a role in allosteric regulation. The 5′ regions of both P. stipitis genes include two putative TATA elements that make them similar to the PDC genes from S. cerevisiae, Kluyveromyces marxianus, and Hanseniaspora uvarum.     

Characterization of Pneumocystis carinii PHR1, a pH-Regulated Gene Important for Cell Wall Integrity

Pneumocystis carinii remains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region coding GSC-1, a gene mediating β-glucan cell wall synthesis in P. carinii. The predicted amino acid sequence of this new gene, termed P. carinii PHR1, exhibited 38% homology to Saccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology to Candida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking β-1,3- and β-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression of P. carinii PHR1 was examined. As in C. albicans, P. carinii PHR1 expression was repressed under acidic conditions but induced at neutral and more alkaline pH. PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties with P. carinii culture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression of P. carinii genes in related fungi to address functional correlates of P. carinii-encoded proteins. Therefore, the potential role of P. carinii PHR1 in cell wall integrity was examined by assessing its ability to rescue an S. cerevisiae gas1 mutant with absent endogenous Phr1p-like activity. Interestingly, P. carinii PHR1 DNA successfully restored proliferation of S. cerevisiae gas1 mutants under lethal conditions of cell wall stress. These results indicate that P. carinii PHR1 encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.     

Photoheterotrophic growth of Physcomitrella patens

Physcomitrella patens is a model bryophyte representing an early land plant in the green plant lineage. This organism possesses many advantages as a model organism. Its genome has been sequenced, its predominant life cycle stage is the haploid gametophyte, it is readily transformable and it can integrate transformed DNA into its genome by homologous recombination. One limitation for the use of P. patens in photosynthesis research is its reported inability to grow photoheterotrophically, in the presence of sucrose and the Photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which prevents linear photosynthetic electron transport. In this communication we describe the facile isolation of a P. patens strain which can grow photoheterotrophically. Additionally, we have examined a number of photosynthetic parameters for this strain grown under photoautotrophic, mixotrophic (in the presence of sucrose) and photoheterotrophic conditions, as well as the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-inhibited state. The ability to grow P. patens photoheterotrophically should significantly facilitate its use in photosynthetic studies.     

Caenorhabditis elegans as a model system for studying non-cell-autonomous mechanisms in protein-misfolding diseases

Caenorhabditis elegans has a number of distinct advantages that are useful for understanding the basis for cellular and organismal dysfunction underlying age-associated diseases of protein misfolding. Although protein aggregation, a key feature of human neurodegenerative diseases, has been typically explored in vivo at the single-cell level using cells in culture, there is now increasing evidence that proteotoxicity has a non-cell-autonomous component and is communicated between cells and tissues in a multicellular organism. These discoveries have opened up new avenues for the use of C. elegans as an ideal animal model system to study non-cell-autonomous proteotoxicity, prion-like propagation of aggregation-prone proteins, and the organismal regulation of stress responses and proteostasis. This Review focuses on recent evidence that C. elegans has mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism. These findings emphasize the potential of C. elegans to provide insights into non-cell-autonomous proteotoxic mechanisms underlying age-related protein-misfolding diseases.KEY WORDS: Caenorhabditis elegans, Cell non-autonomous proteotoxicity, Prion-like spreading     

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway

We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis.In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26.Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3^? infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads.Detailed examination of 8^? lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well.All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.     

Effect of long-term starvation in salty microcosm on biofilm formation and motility in Pseudomonas aeruginosa

The development of antibiotic resistance in the opportunistic pathogen Pseudomonas aeruginosa is a major cause of the pathogen’s morbidity and is strongly correlated with the biofilm formation. Motility and adherence capacity in long-term stressed cells have not been extensively analyzed even though P. aeruginosa considered a model organism for the study of biofilm formation. In this investigation, P. aeruginosa ATCC 27853 strain has been stored for 12 months in LB broth with 0.5 M NaCl. Several experiments demonstrated that the strain recovery from the salty microcosm had the ability to increase the biofilm formation and to reduce motility comparing with that of the original strain. To identify genes involved in the regulation of biofilm and/or in stress response by the recovered P. aeruginosa, differential display “DDRT-PCR” technique was used. The genes speD and ccoN2, coding, respectively, for an S-adenosylmethionine decarboxylase and Cbb3-type cytochrome oxidase, were identified in recovered strain of P. aeruginosa ATCC 27853 as two differentially expressed gene fragments. A comparison of the biofilm produced by the wild-type strain PA14 and the transposon insertion mutant for speD gene suggested that spermidine has a potential role in the adaptive response in P. aeruginosa incubated in long-term stress conditions.     

Pyrazole-based arylalkyne Cathepsin S inhibitors. Part III: Modification of P4 region

Novel classes of tetrahydropyrido-pyrazole thioether amines and arylalkynes that display potency against human Cathepsin S have been previously reported. Here, key pharmacophoric elements of these two classes are merged, and SAR investigations of the P4 region are described, in conjunction with re-optimization of the P5 and P1/P1′/P3 regions. Identification of meta-substituted arylalkynes with good potency and improved solubility is described.     

Stimulable and Spontaneous Bioluminescence in the Marine Dinoflagellates, Pyrodinium bahamense, Gonyaulax polyedra, and Pyrocystis lunula

P. bahamense, G. polyedra, and P. lunula exhibit interspecies differences in stimulable and spontaneous bioluminescence. For each species the total number of photons that can be emitted upon mechanical stimulation is a constant, regardless of the time during scotophase at which stimulation occurs. Ratios of stimulable bioluminescence per organism during scotophase and photophase are as high as 950:1 for laboratory cultures and have been observed as high as 4000: 1 for natural populations of P. bahamense. Spontaneous emission in darkness shows flashing as well as low-level continuous emission. Natural populations of P. bahamense, placed in darkness during natural photophase, exhibit a dual character to their stimulable bioluminescence. Mechanical stimulation techniques are described for rapid and reproducible stimulation of bioluminescence.     

Bis-alkylamine quindolone derivatives as new antimalarial leads

Quindolone derivatives, designed to target the malaria parasite digestive vacuole and heme detoxification pathway, have been synthesized by reaction with 2-chloro-N,N-diethylethanamine. This reaction gave N,O-, N,N- and O-alkylated products containing one or two basic side-chains. The compounds were evaluated for antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum W2 strain and for cytotoxicity in HepG2 A16 hepatic cells. By incorporating alkylamine side chains and chlorine atoms in the quindolone nucleus we transformed the inactive tetracyclic parent quindolones into moderate or highly active and selective antimalarial compounds. The most active and selective compound, 5c, showed an IC₅₀ = 51 nM for P. falciparum and a selectivity ratio of 98.     

Gene-specific signatures of elevated non-synonymous substitution rates correlate poorly across the Plasmodium genus

Background

Comparative genome analyses of parasites allow large scale investigation of selective pressures shaping their evolution. An acute limitation to such analysis of Plasmodium falciparum is that there is only very partial low-coverage genome sequence of the most closely related species, the chimpanzee parasite P. reichenowi. However, if orthologous genes have been under similar selective pressures throughout the Plasmodium genus then positive selection on the P. falciparum lineage might be predicted to some extent by analysis of other lineages.

Principal Findings

Here, three independent pairs of closely related species in different sub-generic clades (P. falciparum and P. reichenowi; P. vivax and P. knowlesi; P. yoelii and P. berghei) were compared for a set of 43 candidate ligand genes considered likely to be under positive directional selection and a set of 102 control genes for which there was no selective hypothesis. The ratios of non-synonymous to synonymous substitutions (dN/dS) were significantly elevated in the candidate ligand genes compared to control genes in each of the three clades. However, the rank order correlation of dN/dS ratios for individual candidate genes was very low, less than the correlation for the control genes.

Significance

The inability to predict positive selection on a gene in one lineage by identifying elevated dN/dS ratios in the orthologue within another lineage needs to be noted, as it reflects that adaptive mutations are generally rare events that lead to fixation in individual lineages. Thus it is essential to complete the genome sequences of particular species of phylogenetic importance, such as P. reichenowi.     

Cloning and Characterization of a P2X Receptor Expressed in the Central Nervous System of Lymnaea stagnalis

P2X receptors are membrane ion channels gated by extracellular ATP. Mammals possess seven distinct P2X subtypes (P2X1-7) that have important functions in a wide array of physiological processes including roles in the central nervous system (CNS) where they have been linked to modulation of neurotransmitter release. We report here the cloning and functional characterization of a P2X receptor from the mollusc Lymnaea stagnalis. This model organism has a relatively simple CNS consisting of large readily identifiable neurones, a feature which together with a well characterized neuronal circuitry for important physiological processes such as feeding and respiration makes it an attractive potential model to examine P2X function. Using CODEHOP PCR we identified a single P2X receptor (LymP2X) in Lymnaea CNS which was subsequently cloned by RT-PCR. When heterologously expressed in Xenopus oocytes, LymP2X exhibited ATP evoked inward currents (EC₅₀ 6.2 µM) which decayed during the continued presence of agonist. UTP and ADP did not activate the receptor whereas αβmeATP was a weak agonist. BzATP was a partial agonist with an EC₅₀ of 2.4 µM and a maximal response 33% smaller than that of ATP. The general P2 receptor antagonists PPADS and suramin both inhibited LymP2X currents with IC₅₀ values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn²⁺ and Cu²⁺ ions exhibited a biphasic effect, potentiating currents up to 100 µM and inhibiting at higher concentrations. Quantitative RT-PCR and in situ hybridization detected expression of LymP2X mRNA in neurones of all CNS ganglia suggesting this ion channel may have widespread roles in Lymnaea CNS function.     

Chromosomal Copy Number Variation,Selection and Uneven Rates of Recombination Reveal Cryptic Genome Diversity Linked to Pathogenicity

Pathogenic fungi constitute a growing threat to both plant and animal species on a global scale. Despite a clonal mode of reproduction dominating the population genetic structure of many fungi, putatively asexual species are known to adapt rapidly when confronted by efforts to control their growth and transmission. However, the mechanisms by which adaptive diversity is generated across a clonal background are often poorly understood. We sequenced a global panel of the emergent amphibian pathogen, Batrachochytrium dendrobatidis (Bd), to high depth and characterized rapidly changing features of its genome that we believe hold the key to the worldwide success of this organism. Our analyses show three processes that contribute to the generation of de novo diversity. Firstly, we show that the majority of wild isolates manifest chromosomal copy number variation that changes over short timescales. Secondly, we show that cryptic recombination occurs within all lineages of Bd, leading to large regions of the genome being in linkage equilibrium, and is preferentially associated with classes of genes of known importance for virulence in other pathosystems. Finally, we show that these classes of genes are under directional selection, and that this has predominantly targeted the Global Panzootic Lineage (BdGPL). Our analyses show that Bd manifests an unusually dynamic genome that may have been shaped by its association with the amphibian host. The rates of variation that we document likely explain the high levels of phenotypic variability that have been reported for Bd, and suggests that the dynamic genome of this pathogen has contributed to its success across multiple biomes and host-species.     

Novel drug design for Chagas disease via targeting Trypanosoma cruzi tubulin: Homology modeling and binding pocket prediction on Trypanosoma cruzi tubulin polymerization inhibition by naphthoquinone derivatives

Chagas disease, also called American trypanosomiasis, is a parasitic disease caused by Trypanosoma cruzi (T. cruzi). Recent findings have underscored the abundance of the causative organism, (T. cruzi), especially in the southern tier states of the US and the risk burden for the rural farming communities there. Due to a lack of safe and effective drugs, there is an urgent need for novel therapeutic options for treating Chagas disease. We report here our first scientific effort to pursue a novel drug design for treating Chagas disease via the targeting of T. cruzi tubulin. First, the anti T. cruzi tubulin activities of five naphthoquinone derivatives were determined and correlated to their anti-trypanosomal activities. The correlation between the ligand activities against the T. cruzi organism and their tubulin inhibitory activities was very strong with a Pearson’s r value of 0.88 (P value <0.05), indicating that this class of compounds could inhibit the activity of the trypanosome organism via T. cruzi tubulin polymerization inhibition. Subsequent molecular modeling studies were carried out to understand the mechanisms of the anti-tubulin activities, wherein, the homology model of T. cruzi tubulin dimer was generated and the putative binding site of naphthoquinone derivatives was predicted. The correlation coefficient for ligand anti-tubulin activities and their binding energies at the putative pocket was found to be r = 0.79, a high correlation efficiency that was not replicated in contiguous candidate pockets. The homology model of T. cruzi tubulin and the identification of its putative binding site lay a solid ground for further structure based drug design, including molecular docking and pharmacophore analysis. This study presents a new opportunity for designing potent and selective drugs for Chagas disease.