Incorporation of esterified soybean isoflavones with antioxidant activity into low density lipoprotein.

We have recently reported that dietary intake of soybean isoflavone phytoestrogens resulted in increased oxidation resistance of isolated low density lipoprotein (LDL). In order to explore the underlying mechanisms we designed two types of in vitro experiments. First, we prepared several different isoflavone fatty acid esters to increase their lipid solubility and studied their incorporation into LDL. Second, the oxidation resistance of the isoflavone-containing LDLs was investigated with Esterbauer's 'conjugated diene' method using Cu2+ as prooxidant. Unesterified daidzein and genistein as well as genistein stearic acid esters were incorporated into LDL to a relatively small extent (0.33 molecules per LDL particle, or less) and they did not significantly influence oxidation resistance. The oleic acid esters of isoflavones were incorporated more effectively, reaching a level of 2.19 molecules per LDL particle or more, and the 4',7-O-dioleates of daidzein and genistein exhibited prolongations of lag times by 46% (P<0.05) and 202% (P<0.01), respectively. A smaller but significant increase in lag time (20.5%, P<0.01) was caused by daidzein 7-mono-oleate. In summary, esterification of soybean isoflavones daidzein and genistein with fatty acids at different hydroxyl groups provided lipophilicity needed for incorporation into LDL. Some isoflavone oleic acid esters increased oxidation resistance of LDL following their incorporation.     

Norepinephrine-induced Ca2+ waves depend on InsP3 and ryanodine receptor activation in vascular myocytes

In rat portal veinmyocytes, Ca²⁺ signals can begenerated by inositol 1,4,5-trisphosphate(InsP₃)- and ryanodine-sensitive Ca²⁺ release channels, which arelocated on the same intracellular store. Using a laser scanningconfocal microscope associated with the patch-clamp technique, weshowed that propagated Ca²⁺ wavesevoked by norepinephrine (in the continuous presence of oxodipine) werecompletely blocked after internal application of ananti-InsP₃ receptor antibody.These propagated Ca²⁺ waves werealso reduced by ~50% and transformed in homogenous Ca²⁺ responses after applicationof an anti-ryanodine receptor antibody or ryanodine. All-or-noneCa²⁺ waves obtained withincreasing concentrations of norepinephrine were transformed in adose-response relationship with a Hill coefficient close to unity afterryanodine receptor inhibition. Similar effects of the ryanodinereceptor inhibition were observed on the norepinephrine- andACh-induced Ca²⁺ responses innon-voltage-clamped portal vein and duodenal myocytes and on thenorepinephrine-induced contraction. Taken together, these results showthat ryanodine-sensitive Ca²⁺release channels are responsible for the fast propagation of Ca²⁺ responses evoked by variousneurotransmitters producing InsP₃ in vascular and visceral myocytes.

     

Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes: application to viability assessment in waters

The CSE dye (Chemunex, Maisons-Alfort, France) was combined with an activity marker to improve bacterial activity assessment in natural waters. Its effectiveness to counterstain dead cells with permeabilised membranes was investigated on live and dead cells of a variety of strains from collections or isolated from the natural environment. Cells were killed by heat treatment. For all strains tested, the fluorescent dye showed an intense staining of killed cells having permeabilised membranes while no significant signal was detected when applied to live cells. Furthermore, the CSE dye had no toxicity on viable cells. Then, CSE was combined with the ChemChrome V6 dye (Chemunex) to assess the activity of bacterial cells in different waters. Both fluorescences were analysed simultaneously by solid-phase cytometry. The active cell counts were sometimes lower when both dyes were combined suggesting that CSE was able to counterstain cells having a residual esterase activity and compromised membranes. These cells were subtracted from the active cell counts determined with ChemChrome V6. In most samples, active cell counts were congruent with those determined by the direct viable count method.     

Focus: Rare Disease: Necrotizing Fasciitis: Association with Pregnancy-related Risk Factors Early in Life

Background: Pregnancy-related risk factors for necrotizing fasciitis are poorly understood. We investigated pregnancy-related characteristics associated with the long-term risk of developing necrotizing fasciitis, a rare life-threatening infectious disease. Methods: We analyzed a longitudinal cohort of 1,344,996 parous women in Quebec, Canada between 1989 and 2020. The main exposure measures included complications of pregnancy such as gestational diabetes, preterm delivery, metabolic disorder, and other maternal characteristics. We followed the women over time to identify future hospitalizations for necrotizing fasciitis up to three decades after delivery. We estimated adjusted hazard ratios (HR) and 95% confidence intervals (CI) for the association of pregnancy characteristics with risk of necrotizing fasciitis in time-varying Cox proportional hazards regression models. Results: A total of 420 women were hospitalized for necrotizing fasciitis during follow-up, including 83 (19.8%) with diabetes-related necrotizing fasciitis. The incidence of necrotizing fasciitis was elevated for women with gestational diabetes (2.9 per 100,000 person-years), preterm delivery (3.2 per 100,000 person-years), and metabolic disorders (5.4 per 100,000 person-years), compared with no pregnancy complication (1.1 per 100,000 person-years). Compared with no pregnancy complication, gestational diabetes was associated with 1.87 times the risk (95% CI 1.38-2.53), preterm delivery with 2.10 times the risk (95% CI 1.65-2.66), and metabolic disorder with 3.72 times the risk (95% CI 2.92-4.74) of developing necrotizing fasciitis over time. Pregnancy complications were more strongly associated with the risk of necrotizing fasciitis 5 years or more after delivery. Conclusions: Complications of pregnancy may be associated with the long-term risk of necrotizing fasciitis in women.     

NMR studies of the phosphorylation motif of the HIV-1 protein Vpu bound to the F-box protein beta-TrCP

A protein-protein association regulated by phosphorylation of serine is examined by NMR studies. Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at two sites, Ser52 and Ser56, on the motif DSGXXS is required for the interaction of Vpu with the ubiquitin ligase SCF-betaTrCP which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. To elucidate the basis of beta-TrCP recognition, the bound conformation of the P-Vpu(41-62) peptide was determined by using NMR and MD. The TRNOE intensities provided distance constraints which were used in simulated annealing. The beta-TrCP-bound structure of P-Vpu was found to be similar to the structure of the free peptide in solution and to the structure recognized by its antibody. Residues 50-57 formed a bend while the phosphate groups are pointing away. The binding fragment was studied by STD-NMR spectroscopy. The phosphorylated motif DpS(52)GNEpS(56) was found to make intimate contact with beta-TrCP, and pSer52 displays the strongest binding effect. It is suggested that Ser phosphorylation allows protein-protein association by electrostatic stabilization: an obvious negative binding region of Vpu was recognizable by positive residues (Arg and Lys) of the WD domain of beta-TrCP. The Ile46 residue was also found essential for interaction with the beta-TrCP protein. Leu45 and Ile46 side chains lie in close proximity to a hydrophobic pocket of the WD domain.     

The dispanins: a novel gene family of ancient origin that contains 14 human members

The Interferon induced transmembrane proteins (IFITM) are a family of transmembrane proteins that is known to inhibit cell invasion of viruses such as HIV-1 and influenza. We show that the IFITM genes are a subfamily in a larger family of transmembrane (TM) proteins that we call Dispanins, which refers to a common 2TM structure. We mined the Dispanins in 36 eukaryotic species, covering all major eukaryotic groups, and investigated their evolutionary history using Bayesian and maximum likelihood approaches to infer a phylogenetic tree. We identified ten human genes that together with the known IFITM genes form the Dispanin family. We show that the Dispanins first emerged in eukaryotes in a common ancestor of choanoflagellates and metazoa, and that the family later expanded in vertebrates where it forms four subfamilies (A-D). Interestingly, we also find that the family is found in several different phyla of bacteria and propose that it was horizontally transferred to eukaryotes from bacteria in the common ancestor of choanoflagellates and metazoa. The bacterial and eukaryotic sequences have a considerably conserved protein structure. In conclusion, we introduce a novel family, the Dispanins, together with a nomenclature based on the evolutionary origin.     

Top down analysis ceramide-induced mitochondrial dysfunctions: role of mitochondrial swelling

Mitochondrial role in ceramide-induced apoptosis pathway remains unclear. Direct effects of ceramide on mitochondria (cytochrome c release, respiratory chain inhibition, oxygen radicals production...) have been reported [1, 2] and we previously showed that addition of ceramide to intact cells or isolated mitochondria triggers mitochondrial swelling which appeared to be insensitive to cyclosporin A (CsA) [3, 4]. The purpose of this work was to determine to which extent this CsA-insensitive mitochondrial swelling, therefore distinct from permeability transition, participates to ceramide-induced apoptosis. To achieve this, we applied Top-Down analysis of integrated mitochondrial function [5], in order to better understand ceramide-induced mitochondrial dysfunctions.     

Metabolite Proofreading in Carnosine and Homocarnosine Synthesis: MOLECULAR IDENTIFICATION OF PM20D2 AS β-ALANYL-LYSINE DIPEPTIDASE*

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-alanyl-histidine) and homocarnosine (γ-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a “metabolite repair” enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic β-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with β-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on β-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from β-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some “classic dipeptides” like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was ∼100–200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.     

SAR inspired by aldehyde oxidase (AO) metabolism: Discovery of novel,CNS penetrant tricyclic M4 PAMs

This letter describes progress towards an M₄ PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M₄ PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.     

Proteomic Profiling of the Outer Membrane Fraction of the Obligate Intracellular Bacterial Pathogen Ehrlichia ruminantium

The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed previously, but information is limited about OMPs in this organism and about their role in the protective immune response. Identification of OMPs is also essential for understanding Ehrlichia’s OM architecture, and how the bacterium interacts with the host cell environment. First, we developed an OMP extraction method using the ionic detergent sarkosyl, which enriched the OM fraction. Second, proteins were separated via one-dimensional electrophoresis, and digested peptides were analyzed via nano-liquid chromatographic separation coupled with mass spectrometry (LC-MALDI-TOF/TOF). Of 46 unique proteins identified in the OM fraction, 18 (39%) were OMPs, including 8 proteins involved in cell structure and biogenesis, 4 in transport/virulence, 1 porin, and 5 proteins of unknown function. These experimental data were compared to the predicted subcellular localization of the entire E. ruminantium proteome, using three different algorithms. This work represents the most complete proteome characterization of the OM fraction in Ehrlichia spp. The study indicates that suitable subcellular fractionation experiments combined with straightforward computational analysis approaches are powerful for determining the predominant subcellular localization of the experimentally observed proteins. We identified proteins potentially involved in E. ruminantium pathogenesis, which are good novel targets for candidate vaccines. Thus, combining bioinformatics and proteomics, we discovered new OMPs for E. ruminantium that are valuable data for those investigating new vaccines against this organism. In summary, we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.