Evidence of compositional differences between the extracellular and intracellular DNA of a granular sludge biofilm

Aims: Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Methods and Results: Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR‐based denaturing gradient‐gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Conclusions: Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. Significance and Impact of the Study: The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.     

The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background

The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B.

Methodology/Principal Findings

As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212–216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers.

Conclusions/Significance

Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B'' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation.     

Selective alteration at the growth-hormone- releasing-hormone nerve terminals during aging in GHRH-green fluorescent protein mice

Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.     

Wnt/β-catenin signaling is required for development of the exocrine pancreas

Background  

β-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of β-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that β-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of β-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of β-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of β-catenin disrupts pancreatic development. A Pdx1-cre mouse line was used to delete a floxed β-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis.     

Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis

Giardia intestinalis is a flagellated protozoan which causes enteric disease worldwide. Giardia trophozoites infect epithelial cells of the proximal small intestine and can cause acute or chronic diarrhea. The mechanism of epithelial injury in giardiasis remains unknown. A number of enteric pathogens, including protozoan parasites, are able to induce enterocyte apoptosis. The aim of this work was to assess whether G. intestinalis strain WB clone C6 is able to induce apoptosis in the human intestinal epithelial cell line HCT-8, and to investigate the role of caspases in this process. Results demonstrated that the parasite induces cell apoptosis, as confirmed by DNA fragmentation analysis, detection of active caspase-3 and degradation of the caspase-3 substrate PARP [poly(ADP-ribose) polymerase]. Furthermore, G. intestinalis infection induces activation of both the intrinsic and the extrinsic apoptotic pathways, down-regulation of the antiapoptotic protein Bcl-2 and up-regulation of the proapoptotic Bax, suggesting a possible role for caspase-dependent apoptosis in the pathogenesis of giardiasis.     

Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from [Citrus sinensis (L.) Osbeck

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of anthocyanins. In this study we showed that a strong reduction in DFR expression occurs in the non-red orange cultivar (Navel and Ovale) compared to that of the red orange (Tarocco) suggesting that the enzyme could be involved in the lack of production of anthocyanins. Therefore, we isolated and compared the cDNAs, the genomic clones, as well as the promoter regions of blood and blond orange dfrs. Our data revealed that the cDNA sequences of pigmented and non-pigmented orange DFRs were 100% homologous and contained a 1017 bp open reading frame which encodes a protein of 338 amino acid residues, corresponding to a molecular mass of 38010.76 Da, with a theoretical pI of 5.96. Moreover, we found that there were no significant differences in non-coding regions (introns and 5' upstream region) of dfr sequences. Southern blot analysis of genomic DNA indicated that dfr was present as a single copy gene in both cultivars. From these findings the low expression level of blond orange dfr, which might play a role in the phenotypic change from blood to blond orange, is thought to be the result of a likely mutation in a regulatory gene controlling the expression of dfr. In addition, here we reported the successful expression of orange DFR cDNAs leading to an active DFR enzyme which converts dihydroquercetin to leucoanthocyanidin, thus confirming the involvement of the isolated genes in the biosynthesis of anthocyanins. Moreover, as far as we know, this is the first report concerning the in vitro expression of DFR from fruit flesh whose biochemical properties might be very different from those of other plant organ DFRs.     

Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.     

Specific sequence motifs direct the oxygenation and chlorination of tryptophan by myeloperoxidase

Most studies of protein oxidation have typically focused on the reactivity of single amino acid side chains while ignoring the potential importance of adjacent sequences in directing the reaction pathway. We previously showed that hypochlorous acid (HOCl), a specific product of myeloperoxidase, inactivates matrilysin by modifying adjacent tryptophan and glycine (WG) residues in the catalytic domain. Here, we use model peptides that mimic the region of matrilysin involved in this reaction, VVWGTA, VVWATA, and the library VVWXTA, to determine whether specific sequence motifs are targeted for chlorination or oxygenation by myeloperoxidase. Our results demonstrate that HOCl generated by myeloperoxidase or activated neutrophils converts the peptide VVWGTA to a chlorinated product, WG+32(Cl). Tandem mass spectrometry in concert with high resolution 1H and two-dimensional NMR analysis revealed that the modification required cross-linking of the tryptophan to the amide of glycine followed by chlorination of the indole ring of tryptophan. In contrast, when glycine in the peptide was replaced with alanine, the major products were mono- and dioxygenated tryptophan residues. When the peptide library VVWXTA (where X represents all 20 common amino acids) was exposed to HOCl, only WG produced a high yield of the chloroindolenine derivative. However, when glycine was replaced by other amino acids, oxygenated tryptophan derivatives were the major products. Our observations indicate that WG may represent a specific sequence motif in proteins that is targeted for chlorination by myeloperoxidase.     

Transmembrane helix-helix interactions: comparative simulations of the glycophorin a dimer

The glycophorin helix dimer is a paradigm for the exploration of helix-helix interactions in integral membrane proteins. Two NMR structures of the dimer are known, one in a detergent micelle and one in a lipid bilayer. Multiple (4 x 50 ns) molecular dynamics simulations starting from each of the two NMR structures, with each structure in either a dodecyl phosphocholine (DPC) micelle or a dimyristoyl phosphatidylcholine (DMPC) bilayer, have been used to explore the conformational dynamics of the helix dimer. Analysis of the helix-helix interaction, mediated by the GxxxG sequence motif, suggests convergence of the simulations to a common model. This is closer to the NMR structure determined in a bilayer than to micelle structure. The stable dimer interface in the final simulation model is characterized by (i) Gly/Gly packing and (ii) Thr/Thr interhelix H-bonds. These results demonstrate the ability of extended molecular dynamics simulations in a lipid bilayer environment to refine membrane protein structures or models derived from experimental data obtained in protein/detergent micelles.