Quantifying Interactions Within the NADP(H) Enzyme Network in Drosophila melanogaster

In this report, we use synthetic, activity-variant alleles in Drosophila melanogaster to quantify interactions across the enzyme network that reduces nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. We examine the effects of large-scale variation in isocitrate dehydrogenase (IDH) or glucose-6-phosphate dehydrogenase (G6PD) activity in a single genetic background and of smaller-scale variation in IDH, G6PD, and malic enzyme across 10 different genetic backgrounds. We find significant interactions among all three enzymes in adults; changes in the activity of any one source of a reduced cofactor generally result in changes in the other two, although the magnitude and directionality of change differs depending on the gene and the genetic background. Observed interactions are presumably through cellular mechanisms that maintain a homeostatic balance of NADPH/NADP, and the magnitude of change in response to modification of one source of reduced cofactor likely reflects the relative contribution of that enzyme to the cofactor pool. Our results suggest that malic enzyme makes the largest single contribution to the NADPH pool, consistent with the results from earlier experiments in larval D. melanogaster using naturally occurring alleles. The interactions between all three enzymes indicate functional interdependence and underscore the importance of examining enzymes as components of a network.IN traits determined by a network of gene products, the phenotype is a function of the alleles present and of the relative contributions of individual network member genes. Since selection is on phenotype, the total composite genotype, not just individual loci, determines the fitness of an organism. In establishing the connection between genotype and phenotype for such networks, the first challenge is to quantify the relative contribution of each member of the network to the endpoint phenotype. By addressing function on a network-wide basis, interactions and interconnections that may not be apparent in individual gene examinations can be determined (Proulx et al. 2005).In most organisms, reduction of the cofactor nicotinamide adenine dinucleotide phosphate, or NADP, to NADPH is primarily the function of four enzymes: cytosolic malic enzyme (MEN), cytosolic isocitrate dehydrogenase (IDH), and the two oxidative enzymes of the pentose shunt, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate (6PGD; recently reviewed in Ying 2008). In larval Drosophila melanogaster, MEN produces ∼30% of the available NADPH, IDH ∼20%, and G6PD and 6PGD the remaining ∼40% (Geer et al. 1979a,b). It is believed that these four enzymes interact to maintain the NADP/NADPH balance and supply of reducing power for lipogenesis and antioxidation (Geer et al. 1976, 1978, 1981; Wilton et al. 1982; Bentley et al. 1983; Geer and Laurie-Ahlberg 1984; Merritt et al. 2005; Pollak et al. 2007; Singh et al. 2007; Ying 2008). Dietary induction studies and observations of natural genetic variation have found connections between MEN activity and the activities of the pentose shunt enzymes to be generally straightforward and compensatory; reductions in one lead to increases in the other. The interactions involving IDH activity, however, have been found to be more complicated and at times counterintuitive; reductions in reducing power sometimes lead to decreases in IDH activity.In an earlier study (Merritt et al. 2005), we quantified the impact of genetic variation in Men activity on IDH and G6PD activities and triglyceride (a strong correlate with total lipid; Clark and Keith 1989) concentration. 6PGD was not independently assayed because earlier works suggest that G6PD and 6PGD activities are highly correlated, likely because of their coupled function in the pentose shunt (Wilton et al. 1982). We examined both naturally occurring Men alleles and synthetic alleles created by P-element excision and found significant associations between MEN activity and induction of the activities of both IDH and G6PD. The apparent interactions between MEN and IDH and G6PD across these 10 different third chromosome lines were quantified as mean elasticity coefficients: = −0.76 ± 0.236 and = −0.88 ± 0.208. Because MEN activity was reduced by 20%, both IDH and G6PD activity varied in a compensatory direction, increasing almost 1:1 with the decrease in MEN.The significant change in enzyme activity of two members of the NADPH network in response to our genetic reduction of the activity of a third strongly suggests that a physiological mechanism coregulates the three enzymes. Such functional interdependence would mean that individual members of the network do not act in isolation and should be examined collectively, not as isolated units. In this study, we characterize the effects of the independently varying activity levels of IDH, G6PD, and MEN on the activity of each other and triglyceride concentration in adult flies. We found significant responses to changes in all three enzymes, although the responses to genetic changes in IDH and G6PD were generally small; variation in MEN caused the greatest changes in the other enzymes.     

The basic domain of TRF2 directs binding to DNA junctions irrespective of the presence of TTAGGG repeats

The replication of long tracts of telomeric repeats may require specific factors to avoid fork regression (Fouché, N., Ozgür, S., Roy, D., and Griffith, J. (2006) Nucleic Acids Res., in press). Here we show that TRF2 binds to model replication forks and four-way junctions in vitro in a structure-specific but sequence-independent manner. A synthetic peptide encompassing the TRF2 basic domain also binds to DNA four-way junctions, whereas the TRF2 truncation mutant (TRF2(DeltaB)) and a mutant basic domain peptide do not. In the absence of the basic domain, the ability of TRF2 to localize to model telomere ends and facilitate t-loop formation in vitro is diminished. We propose that TRF2 plays a key role during telomere replication in binding chickenfoot intermediates of telomere replication fork regression. Junction-specific binding would also allow TRF2 to stabilize a strand invasion structure that is thought to exist at the strand invasion site of the t-loop.     

Prevalence of known mutations in the MEFV gene in a population screening with high rate of carriers

The Familial Mediterranean Fever (FMF) shows an autosomal recessive pattern of inheritance and affects certain ethnic groups. Disease is caused by mutations in MEFV gene and more than 180 mutations have been defined in affected individuals. Current study aimed to determine the frequency-type of the mutations for MEFV gene in Sivas??middle Anatolian city. The cohort was composed of 3340 patients. MEFV gene mutations were studied by multiplex PCR based reverse hybridization stripAssay method. Patients?? clinical features were; family history: 68%, erysipelas-like erythema: 17.6%, fever: 89.9%, abdominal pain: 84.2%, peritonitis: 90.2%, arthritis: 33%, pleuritis: 14.2%, parental consanguinity: 21.2%. Current results revealed that M694V is the most frequent mutation (43.12%), followed by E148Q (20.18), M680I(G/C) (15.00%) and V726A (11.32%). The study population has a high rate of carriers and the E148Q mutation frequency was found to be highest when compared to the other regions of Turkey and other Mediterranean groups.     

Quorum quenching is an antivirulence strategy employed by endophytic bacteria

Bacteria predominantly use quorum sensing to regulate a plethora of physiological activities such as cell-cell crosstalk, mutualism, virulence, competence, biofilm formation, and antibiotic resistance. In this study, we investigated how certain potent endophytic bacteria harbored in Cannabis sativa L. plants use quorum quenching as an antivirulence strategy to disrupt the cell-to-cell quorum sensing signals in the biosensor strain, Chromobacterium violaceum. We used a combination of high-performance liquid chromatography high-resolution mass spectrometry (HPLC-ESI-HRMSⁿ) and matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) to first quantify and visualize the spatial distribution of the quorum sensing molecules in the biosensor strain, C. violaceum. We then showed, both quantitatively and visually in high spatial resolution, how selected endophytic bacteria of C. sativa can selectively and differentially quench the quorum sensing molecules of C. violaceum. This study provides fundamental insights into the antivirulence strategies used by endophytes in order to survive in their ecological niches. Such defense mechanisms are evolved in order to thwart the plethora of pathogens invading associated host plants in a manner that prevents the pathogens from developing resistance against the plant/endophyte bioactive secondary metabolites. This work also provides evidence towards utilizing endophytes as tools for biological control of bacterial phytopathogens. In continuation, such insights would even afford new concepts and strategies in the future for combating drug resistant bacteria by quorum-inhibiting clinical therapies.     

Role of exonic variation in chemokine receptor genes on AIDS: CCRL2 F167Y association with pneumocystis pneumonia

Chromosome 3p21–22 harbors two clusters of chemokine receptor genes, several of which serve as major or minor coreceptors of HIV-1. Although the genetic association of CCR5 and CCR2 variants with HIV-1 pathogenesis is well known, the role of variation in other nearby chemokine receptor genes remain unresolved. We genotyped exonic single nucleotide polymorphisms (SNPs) in chemokine receptor genes: CCR3, CCRL2, and CXCR6 (at 3p21) and CCR8 and CX3CR1 (at 3p22), the majority of which were non-synonymous. The individual SNPs were tested for their effects on disease progression and outcomes in five treatment-naïve HIV-1/AIDS natural history cohorts. In addition to the known CCR5 and CCR2 associations, significant associations were identified for CCR3, CCR8, and CCRL2 on progression to AIDS. A multivariate survival analysis pointed to a previously undetected association of a non-conservative amino acid change F167Y in CCRL2 with AIDS progression: 167F is associated with accelerated progression to AIDS (RH = 1.90, P = 0.002, corrected). Further analysis indicated that CCRL2-167F was specifically associated with more rapid development of pneumocystis pneumonia (PCP) (RH = 2.84, 95% CI 1.28–6.31) among four major AIDS–defining conditions. Considering the newly defined role of CCRL2 in lung dendritic cell trafficking, this atypical chemokine receptor may affect PCP through immune regulation and inducing inflammation.     

Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes

Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid-protein nanodomains, termed "rafts". In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids ("raft lipids", e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GM1 exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena.     

Novel ribonucleotide discrimination in the RNA polymerase-like two-barrel catalytic core of Family D DNA polymerases

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2′ modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.     

Pneumonia due to Enterobacter cancerogenus infection

Enterobacter cancerogenus (formerly known as CDC Enteric Group 19; synonym with Enterobacter taylorae) has rarely been associated with human infections, and little is known regarding the epidemiology and clinical significance of this organism. We describe a community-acquired pneumonia case in a 44-year-old female due to E. cancerogenus. Identification and antimicrobial susceptibility of the microorganism was performed by the automatized VITEK 2 Compact system (bioMerieux, France). The clinical case suggests that E. cancerogenus is a potentially pathogenic microorganism in determined circumstances; underlying diseases such as bronchial asthma, empiric antibiotic treatment, wounds, diagnostic, or therapeutic instruments.