Central Carbon Metabolism, Sodium-Motive Electron Transfer, and Ammonium Formation by the Vaginal Pathogen Prevotella bivia

Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.

Enzyme Catalysis Enhances Lateral Lipid Motility and Directional Particle Transport on Membranes

The dynamic interplay between the composition of lipid membranes and the behavior of membrane-bound enzymes is critical to the understanding of cellular function and viability, and the design of membrane-based biosensing platforms. While there is a significant body of knowledge on how lipid composition and dynamics affect membrane-bound enzymes, little is known about how enzyme catalysis influences the motility and lateral transport in lipid membranes. Using enzymes-attached lipids in supported bilayers (SLB), we show catalysis-induced enhanced lateral diffusion of lipids in the bilayer. Enhancing the membrane viscosity by increasing the cholesterol content in the bilayer suppresses the overall diffusion but not the relative diffusion enhancement of the enzyme-attached lipids. We also provide direct evidence of catalysis-induced membrane fluctuations leading to the enhanced diffusion of passive tracers resting on the SLB. Additionally, by using active enzyme patches, we demonstrate the directional transport of tracers on SLBs. These are first steps in understanding diffusion and transport in lipid membranes due to active, out-of-equilibrium processes that are the hallmark of living systems. In general, our study demonstrates how active enzymes can be used to control diffusion and transport in confined 2-D environments.

Molecular characterization and transcriptional regulation of two types of H+-pyrophosphatases in the scuticociliate parasite Philasterides dicentrarchi

Proton-translocating inorganic pyrophosphatases (H+-PPases) are an ancient family of membrane bound enzymes that couple pyrophosphate (PPi) hydrolysis to H+ translocation across membranes. In this study, we conducted a molecular characterization of two isoenzymes (PdVP1 and PdVP2) located in respectively the alveolar sacs and in the membranes of the intracellular vacuoles of a scuticociliate parasite (Philasterides dicentrarchi) of farmed turbot. We analyzed the genetic expression of the isoenzymes after administration of antiparasitic drugs and after infection in the host. PdVP1 and PdVP2 are encoded by two genes of 2485 and 3069 bp, which respectively contain 3 and 11 exons and express proteins of 746 and 810 aa of molecular mass 78.9 and 87.6 kDa. Topological predictions from isoenzyme sequences indicate the formation of thirteen transmembrane regions (TMRs) for PdVP1 and seventeen TMRs for PdVP2. Protein structure modelling indicated that both isoenzymes are homodimeric, with three Mg2+ binding sites and an additional K+ binding site in PdVP2. The levels of identity and similarity between the isoenzyme sequences are respectively 33.5 and 51.2%. The molecular weights of the native proteins are 158 kDa (PdVP1) and 178 kDa (PdVP2). The isoenzyme sequences are derived from paralogous genes that form a monophyletic grouping with other ciliate species. Genetic expression of the isoenzymes is closely related to the acidification of alveolar sacs (PdVP1) and intracellular vacuoles (PdVP2): antiparasitic drugs inhibit transcription, while infection increases transcription of both isoenzymes. The study findings show that P. dicentrarchi possesses two isoenzymes with H+-PPase activity which are located in acidophilic cell compartment membranes and which are activated during infection in the host and are sensitive to antiparasitic drugs. The findings open the way to using molecular modelling to design drugs for the treatment of scuticociliatosis.

(2-Aminobenzothiazole)-Methyl-1,1-Bisphosphonic Acids: Targeting Matrix Metalloproteinase 13 Inhibition to the Bone

Matrix Metalloproteinases (MMPs) are a family of secreted and membrane-bound enzymes, of which 24 isoforms are known in humans. These enzymes degrade the proteins of the extracellular matrix and play a role of utmost importance in the physiological remodeling of all tissues. However, certain MMPs, such as MMP-2, -9, and -13, can be overexpressed in pathological states, including cancer and metastasis. Consequently, the development of MMP inhibitors (MMPIs) has been explored for a long time as a strategy to prevent and hinder metastatic growth, but the important side effects linked to promiscuous inhibition of MMPs prevented the clinical use of MMPIs. Therefore, several strategies were proposed to improve the therapeutic profile of this pharmaceutical class, including improved selectivity toward specific MMP isoforms and targeting of specific organs and tissues. Combining both approaches, we conducted the synthesis and preliminary biological evaluation of a series of (2-aminobenzothiazole)-methyl-1,1-bisphosphonic acids active as selective inhibitors of MMP-13 via in vitro and in silico studies, which could prove useful for the treatment of bone metastases thanks to the bone-targeting capabilities granted by the bisphosphonic acid group.

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral sequence recon¬struction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as AncFMO1 and AncFMO4, respectively. AncFMO1, sharing 89.5 % sequence identity with human FMO1, was successfully expressed as functional enzyme. It displayed typical FMO activity as demonstrated by oxygenating benzydamine, tamoxifen and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the FAD and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2’-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.

Taurine supplementation restores antioxidant status and hepatic membrane-bound enzymes in streptozotocin-induced diabetic rats

Introduction and Aim: Chronic hyperglycemia in diabetes causes cellular damage through increased lipid peroxidation and reduced levels of antioxidants. The activities of membrane-bound enzymes are affected by oxidative stress. Taurine, a sulfur containing amino acid is shown to have hypoglycemic activity, antioxidant property and membrane stabilization. The aim of the study is to check the effect of supplementation of taurine on lipid peroxidation, antioxidant status and hepatic membrane-bound enzymes in streptozotocin- induced diabetic rats. Materials and Methods: Thirty-two Wistar male albino rats of 19±1 weeks of age weighing 200-220 grams were randomly divided into four groups and each group consisted of eight animals. Group I (control) standard chow diet; Group II (chow diet with taurine); Group III (diabetes induced) and Group IV (diabetic receiving taurine). At the end of 45th day, all animals were sacrificed by cervical decapitation after overnight fasting. Blood and liver tissue samples were collected. The levels of glucose in plasma and lipid peroxidation, antioxidants and the activities of Na+/ K+, Ca2+ and Mg2+-ATPases in liver homogenate were analyzed. Results: Altered levels of antioxidants and activities of Na+/ K+, Ca2+ and Mg2+-ATPases were restored to normal by taurine supplementation in diabetic rats. Conclusion: The present study indicates that supplementation of taurine could protect liver plasma membrane against oxidative damage by acting as antioxidant and restoring the normal activities of Na+/ K+, Ca2+ and Mg2+-ATPases.

Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations

In nature, many enzymes are attached or inserted into the cell membrane, having hydrophobic subunits or lipid chains for this purpose. Their reconstitution on electrodes maintaining their natural structural characteristics allows for optimizing their electrocatalytic properties and stability. Different biomimetic strategies have been developed for modifying electrodes surfaces to accommodate membrane-bound enzymes, including the formation of self-assembled monolayers of hydrophobic compounds, lipid bilayers, or liposomes deposition. An overview of the different strategies used for the formation of biomimetic membranes, the reconstitution of membrane enzymes on electrodes, and their applications as biosensors is presented.

Is there a role for tau glutathione transferases in tetrapyrrole metabolism and retrograde signalling in plants?

In plants, tetrapyrrole biosynthesis occurs in chloroplasts, the reactions being catalysed by stromal and membrane-bound enzymes. The tetrapyrrole moiety is a backbone for chlorophylls and cofactors such as sirohaems, haems and phytochromobilins. Owing to this diversity, the potential cytotoxicity of some precursors and the associated synthesis costs, a tight control exists to adjust the demand and the fluxes for each molecule. After synthesis, haems and phytochromobilins are incorporated into proteins found in other subcellular compartments. However, there is only very limited information about the chaperones and membrane transporters involved in the trafficking of these molecules. After summarizing evidence indicating that glutathione transferases (GST) may be part of the transport and/or degradation processes of porphyrin derivatives, we provide experimental data indicating that tau glutathione transferases (GSTU) bind protoporphyrin IX and haem moieties and use structural modelling to identify possible residues responsible for their binding in the active site hydrophobic pocket. Finally, we discuss the possible roles associated with the binding, catalytic transformation (i.e. glutathione conjugation) and/or transport of tetrapyrroles by GSTUs, considering their subcellular localization and capacity to interact with ABC transporters. This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.

VANILLIC ACID INHIBITS LUNG CARCINOGENESIS BY MODULATES GLYCOPROTEIN ABNORMALITIES, MEMBRANE-BOUND ENZYMES, AND INFLAMMATORY MARKERS

Objective: The current plan was accompanied to explicate the possible protective role of vanillic acid (VA), on modification in lipid peroxidation, inflammatory cytokines, membrane-bound enzymes, and glycoconjugates during B(a)P induced lung cancer in Swiss albino mice. Methods: Benzo(a)pyrene was administered orally (50 mg/kg b. wt) to induce lung cancer in Swiss albino mice. lipid peroxidation, serum marker enzymes, inflammatory cytokines, membrane-bound ATPases and protein-bound carbohydrate components (Hexose, hexosamine, sialic acid and fucose) and Mast cells and PAS staining were carried out. Results: Lung cancer possessing animals exhibited increased levels of lipid peroxidation, ADA, AHH, γ-GT, 5’-NT, LDH, cytokines such as TNF-α and IL-1β, protein-bound carbohydrate components (protein-hexose, hexosamine, sialic acid, and fucose) also diminished activity of membrane-bound ATPases (Na+/K+ATPases, Ca2+ATPases, and Mg2+ATPase). Treatment with VA significantly ameliorated all these activities. Conclusion: Overall, the present study evidence to the VA has effective anti-inflammatory in addition to free radical scavenging activity for the duration of lung carcinogenesis in Swiss albino mice.

Myocardial potency of Caesalpinia bonducella Linn. on doxorubicin induced myocardial infarction in albino rats

Objective Caesalpinia bonducella L. is well known and extremely valuable herb in ayurvedic system of medicine. The present study is aimed to design the evaluation of aqueous extract of Caesalpinia bonducella L. on doxorubicin induced myocardial infarction in wistar strains of albino rats of both sex. Materials and methods The experimental animals are divided in to 5 groups of 6 animals each. Group I (Normal Control), Group II (Negative Control, 2.5 mg/kgbw of Doxorubicin i.p.), Group III (2.5 mg/kgbw of Doxorubicin i.p.) + AECB (150 mg/kgbw), Group IV (2.5 mg/kgbw of Doxorubicin i.p.) + AECB (300 mg/kgbw), Group V (2.5 mg/kgbw of Doxorubicin i.p.) + standard drug (Propranolol 5 mg/kgbw). Doxorubicin induced myocardial infarction was confirmed by disturbances in levels of cardiac markers (Lactate Dehydrogenase, Troponin-T, Creatine Kinase-MB Isoenzyme, Creatine Phosphokinase), nucleic acid contents (DNA and RNA), Challenged levels of Membrane bound enzymes such as Na+/K + ATPase, Ca2 + ATPase and Mg2 + ATPase, Decreased tissue protein and altered lipid profile markers. Results Doxorubicin induced rats significantly showed increase in the activities of LDH, CK-MB, CPK, Troponin-T, nucleic acids, membrane bound enzymes, lipid profiles and decrease in the serum HDL. Treatment with AECB simultaneously at two different doses such as 150 mg/kg bw, 300 mg/kg bw prevented the leakage of myocardium markers and altered the levels of Protein, DNA, RNA and membrane bound enzymes. The AECB prevented the altered variations in Cholesterol, Triacylglycerols, Phospholipids and Free Fatty Acids. This extract also brought back the levels of Lipoproteins like HDL, LDL and VLDL which were varied in disease control animals. Conclusion The present study concludes that AECB is effective in controlling the cardiac markers and lipid levels which could be due to its ability to maintain the membrane stability and repair the myocardial damage.