Occurrence, biosynthesis and functions of N-acylphosphatidylethanolamines (NAPE): not just precursors of N-acylethanolamines (NAE)

N-acylphosphatidylethanolamine (NAPE) is a minor phospholipid resulting from the transfer of an acyl chain from an acyl donor to the primary amine of the ethanolamine moiety of phosphatidylethanolamine (PE). Occurring in plant and animal kingdoms as well as in prokaryotic cells, it is synthesized in higher amounts in membranes during cellular stresses and tissue damage, and it is widely thought to be the precursor of the lipid mediator, N-acylethanolamine (NAE), which modulates the endocannabinoid signaling pathway and therefore regulates various physiological processes. However, recent studies have shown that NAPE is also a bioactive molecule that is involved in several physiological functions. The present paper reviews the occurrence of NAPE in animals and plants and focuses on the various properties of NAPE observed in vitro and in vivo. The different metabolic pathways promoting the synthesis and degradation of NAPE are also discussed and the differences between animals and plants are underlined.     

Soil Bacterial Consortia and Previous Exposure Enhance the Biodegradation of Sulfonamides from Pig Manure

Persistence or degradation of synthetic antibiotics in soil is crucial in assessing their environmental risks. Microbial catabolic activity in a sandy loamy soil with pig manure using 12C- and 14C-labelled sulfamethazine (SMZ) respirometry showed that SMZ was not readily degradable. But after 100 days, degradation in sulfadiazine-exposed manure was 9.2%, far greater than soil and organic manure (0.5% and 0.11%, respectively, p < 0.05). Abiotic degradation was not detected suggesting microbial catabolism as main degradation mechanism. Terminal restriction fragment length polymorphism showed biodiversity increases within 1 day of SMZ spiking and especially after 200 days, although some species plummeted. A clone library from the treatment with highest degradation showed that most bacteria belonged to α, β and γ classes of Proteobacteria, Firmicutes, Bacteroidetes and Acidobacteria. Proteobacteria (α, β and γ), Firmicutes and Bacteroidetes which were the most abundant classes on day 1 also decreased most following prolonged exposure. From the matrix showing the highest degradation rate, 17 SMZ-resistant isolates biodegraded low levels of 14C-labelled SMZ when each species was incubated separately (0.2-1.5%) but biodegradation was enhanced when the four isolates with the highest biodegradation were incubated in a consortium (Bacillus licheniformis, Pseudomonas putida, Alcaligenes sp. and Aquamicrobium defluvium as per 16S rRNA gene sequencing), removing up to 7.8% of SMZ after 20 days. One of these species (B. licheniformis) was a known livestock and occasional human pathogen. Despite an environmental role of these species in sulfonamide bioremediation, the possibility of horizontal transfer of pathogenicity and resistance genes should caution against an indiscriminate use of these species as sulfonamide degraders.     

Comparative analysis of the first complete Enterococcus faecium genome

Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infections in health care facilities around the globe. In particular, infections caused by vancomycin-resistant Enterococcus faecium are becoming increasingly common. Comparative and functional genomic studies of E. faecium isolates have so far been limited owing to the lack of a fully assembled E. faecium genome sequence. Here we address this issue and report the complete 3.0-Mb genome sequence of the multilocus sequence type 17 vancomycin-resistant Enterococcus faecium strain Aus0004, isolated from the bloodstream of a patient in Melbourne, Australia, in 1998. The genome comprises a 2.9-Mb circular chromosome and three circular plasmids. The chromosome harbors putative E. faecium virulence factors such as enterococcal surface protein, hemolysin, and collagen-binding adhesin. Aus0004 has a very large accessory genome (38%) that includes three prophage and two genomic islands absent among 22 other E. faecium genomes. One of the prophage was present as inverted 50-kb repeats that appear to have facilitated a 683-kb chromosomal inversion across the replication terminus, resulting in a striking replichore imbalance. Other distinctive features include 76 insertion sequence elements and a single chromosomal copy of Tn1549 containing the vanB vancomycin resistance element. A complete E. faecium genome will be a useful resource to assist our understanding of this emerging nosocomial pathogen.     

Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization

We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.     

Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E_m) at pH 7.0 of approximately +210, −340, and −400 mV for the His/Met, His/Cys⁻, and active site His/CysS⁻-ligated heme, respectively. Exposing SoxAX to S₂O₄²⁻, a substrate analog with E_m ∼−450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E_m ∼−1140 mV), allows cyanide to displace the cysteine persulfide (CysS⁻) ligand to the active site heme. This provides the first evidence for the dissociation of CysS⁻ that has been proposed as a key event in SoxAX catalysis.     

Protein carbonylation and adipocyte mitochondrial function

Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte.     

The γ3 chain of laminin is widely but differentially expressed in murine basement membranes: expression and functional studies

Laminins are heterotrimeric extracellular glycoproteins found in, but not confined to, basement membranes (BMs). They are important components in formation of the molecular networks of BMs as well as in cell polarity, cell differentiation and tissue morphogenesis. Each laminin is composed by an α, a β and a γ chain. Previous studies have shown that the γ3 chain is partnered with either the β1 chain (in placenta) or β2 chain (in the CNS) (Libby et al., 2000). Several studies, including our own, suggested that the γ3 chain is expressed in both apical and basal compartments (Koch et al., 1999; Gersdorff et al., 2005; Yan and Cheng, 2006). This study investigates the expression pattern of the γ3 chain in mouse. We developed three new γ3-reactive antibodies, and we show that the γ3 chain is present in BMs. The distribution pattern is considerably more restricted than that of the γ1 chain and within any tissue there is differential deposition into BM compartments. This is particularly true in the retina and brain, where γ3 is uniquely expressed in a subset of the vascular basement membranes and the pial surface. We used conventional genetic ablation techniques to remove the γ3 chain in mice; unlike other laminin null mice (α5, β2, γ1 nulls), these mice live a normal lifespan and have only minor abnormalities, the most striking of which are ectopic granule cells in the cerebellum and an apparent increase in capillary branching in the outer retina. These data support the suggestion that the γ3 chain is deposited in BMs and contributes some unique properties to their function, particularly in the nervous system.