Modulation of mitochondrial K permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K⁺ current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K⁺ in the assay medium. The effects of p13 on ROS are mimicked by the K⁺ ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K⁺ vs. H⁺ currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K⁺ permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.     

Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17(+)] and from the same cell line transfected with the corresponding empty vector [GPR17(−)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)-IAM and GPR17(−)-IAM stationary phases. Frontal chromatography experiments on both GPR17(+)-IAM and GPR17(−)-IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)-IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants (K_d) of three ligands—the antagonist cangrelor (formerly AR-C69931MX, a P2Y₁₂/P2Y₁₃ antagonist), MRS2179 (a P2Y₁ receptor antagonist), and the agonist UDP—all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated K_d values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)-IAM in ranking affinity studies for the selection of new potential candidates.     

Transmembrane Signaling in the Maltose ABC Transporter MalFGK2-E: PERIPLASMIC MalF-P2 LOOP COMMUNICATES SUBSTRATE AVAILABILITY TO THE ATP-BOUND MalK DIMER*

ABC transporters are ubiquitous membrane proteins that translocate solutes across biological membranes at the expense of ATP. In prokaryotic ABC importers, the extracytoplasmic anchoring of the substrate-binding protein (receptor) is emerging as a key determinant for the structural rearrangements in the cytoplasmically exposed ATP-binding cassette domains and in the transmembrane gates during the nucleotide cycle. Here the molecular mechanism of such signaling events was addressed by electron paramagnetic resonance spectroscopy of spin-labeled ATP-binding cassette maltose transporter variants (MalFGK₂-E). A series of doubly spin-labeled mutants in the MalF-P2 domain involving positions 92, 205, 239, 252, and 273 and one triple mutant labeled at positions 205/252 in P2 and 83 in the Q-loop of MalK were assayed. The EPR data revealed that the substrate-binding protein MalE is bound to the transporter throughout the transport cycle. Concomitantly with the three conformations of the ATP-binding cassette MalK₂, three functionally relevant conformations are found also in the periplasmic MalF-P2 loop, strictly dependent on cytoplasmic nucleotide binding and periplasmic docking of liganded MalE to MalFG. The reciprocal communication across the membrane unveiled here gives first insights into the stimulatory effect of MalE on the ATPase activity, and it is suggested to be an important mechanistic feature of receptor-coupled ABC transporters.     

Prebiotic potential and gastrointestinal effects of immature wheat grain (IWG) biscuits

In this study the effects of immature wheat grains (IWG), a natural source of fructo-oligosaccharides (FOS), on intestinal microbiota and gastrointestinal function were evaluated. Ileal effluents from three ileostomates were collected for 8 h after IWG-enriched or Control biscuit administration, and then fermented in vitro with human faecal inoculum. On fermentation broths, microbial counts and SCFA were measured. Moreover, we investigated the effect of IWG biscuits on gastric emptying. Twelve healthy volunteers underwent ultrasound measurement of gastric emptying of a standard meal consumed 5 h after a breakfast composed by lactose-free milk and IWG or Control biscuits; hunger and satiety sensations were also evaluated in this period. Bifidobacteria and lactic acid bacteria counts were higher (P < 0.05) in IWG than in Control cultures, supporting the prebiotic effects of IWG, probably linked to its FOS content; moreover IWG fermentation produced more acetate. In vivo IWG biscuits delayed gastric emptying of the next meal (P < 0.05), decreased hunger (P < 0.001) and increased satiety (P < 0.005). Therefore in vitro results substantiate the potential of IWG as a prebiotic ingredient and in vivo data suggest an effect of IWG on gastric emptying and on persistent satiety. These preliminary data tend to support the use of IWG in formulating functional prebiotic products suitable to promote satiety.     

Cytochrome P-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3A-null (Cyp3a(-/-)) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a(-/-) DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a(-/-) DA. However, responses of RA-treated WT and Cyp3a(-/-) mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a(-/-) newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally.     

The effects of ATP and sodium chloride on the cytochrome c-cardiolipin interaction: the contrasting behavior of the horse heart and yeast proteins

In cells a portion of cytochrome c (cyt c) (15–20%) is tightly bound to cardiolipin (CL), one of the phospholipids constituting the mitochondrial membrane. The CL-bound protein, which has nonnative tertiary structure, altered heme pocket, and disrupted Fe(III)-M80 axial bond, is thought to play a role in the apoptotic process. This has attracted considerable interest in order to clarify the mechanisms governing the cyt c–CL interaction. Herein we have investigated the binding reaction of CL with the c-type cytochromes from horse heart and yeast. Although the two proteins possess a similar tertiary architecture, yeast cyt c displays lower stability and, contrary to the equine protein, it does not bind ATP and lacks pro-apoptotic activity. The study has been performed in the absence and in the presence of ATP and NaCl, two compounds that influence the (horse cyt c)-CL binding process and, thus, the pro-apoptotic activity of the protein. The two proteins behave differently: while CL interaction with horse cyt c is strongly influenced by the two effectors, no effect is observed for yeast cyt c. It is noteworthy that NaCl induces dissociation of the (horse cyt c)–CL complex but has no influence on that of yeast cyt c. The differences found for the two proteins highlight that specific structural factors, such as the different local structure conformation of the regions involved in the interactions with either CL or ATP, can significantly affect the behavior of cyt c in its reaction with liposomes and the subsequent pro-apoptotic action of the protein.     

The anandamide effect on NO/cGMP pathway in human platelets

In this study the effect of the endocannabinoid anandamide on platelet nitric oxide (NO)/cGMP pathway was investigated. Data report that anandamide in a dose-and time-dependent manner increased NO and cGMP levels and stimulated endothelial nitric oxide synthase (eNOS) activity. These parameters were significantly reduced by LY294002, selective inhibitor of PI3K and by MK2206, specific inhibitor of AKT. Moreover anandamide stimulated both eNOSser1177 and AKTser473 phosphorylation. Finally the anandamide effect on NO and cGMP levels, eNOS and AKT phosphorylation/activation were inhibited by SR141716, specific cannabinoid receptor 1 antagonist, supporting the involvement of anandamide binding to this receptor. Overall data of this report indicate that low concentrations of anandamide, through PI3K/AKT pathway activation, stimulates eNOS activity and increases NO levels in human platelets. In such way anandamide contributes to extend platelet survival.     

Cysteine co-oxidation process driven by native peptide folding: an example on HER2 receptor model system

Synthetic models of receptors that have relevant biological roles are valuable tools for studying receptors itself and the corresponding ligands. Their properties can be validated at first by their capacity to fold in solution under native-like conditions and to assume conformations structurally and functionally equivalent to those in the native receptor. In this context, a new strategy to prepare the two-fragments synthetic receptor model HER2-DIVMP, an independent structural and functional motif of HER2, has been developed and the folding properties have been investigated. The strategy is based on a one-step cysteine co-oxidation procedure in slightly alkaline aqueous buffers, whereby the two separate peptide chains are allowed to self-assemble in solution. Under these conditions, the two chains spontaneously form the expected heterodimer with the correct pattern of disulfide bridges. To gain insights on the folding mechanism, we investigated the folding of two scrambled variants of the constituent peptide chains.     

Safety and efficacy of lactoferrin versus ferrous sulphate in curing iron deficiency and iron deficiency anaemia in hereditary thrombophilia pregnant women: an interventional study

Objective Evaluate the safety and efficacy of bovine lactoferrin (bLf) versus the ferrous sulphate standard intervention in curing iron deficiency (ID) and ID anaemia (IDA) in pregnant women affected by hereditary thrombophilia (HT). Design Interventional study. Setting Secondary-level hospital for complicated pregnancies in Rome, Italy. Population 295 HT pregnant women (≥18 years) suffering from ID/IDA. Methods Women were enrolled in Arm A or B in accordance with their personal choice. In Arm A, 156 women received oral administration of 100 mg of bLf twice a day; in Arm B, 139 women received 520 mg of ferrous sulphate once a day. Therapies lasted until delivery. Main outcome measures Red blood cells, haemoglobin, total serum iron, serum ferritin (haematological parameters) were assayed before and every 30 days during therapy until delivery. Serum IL-6, key factor in inflammatory and iron homeostasis disorders, was detected at enrolment and after therapy at delivery. Possible maternal, foetal, and neonatal adverse effects were assessed. Results Haematological parameters were significantly higher in Arm A than in Arm B pregnant women (P ≤ 0.0001). Serum IL-6 significantly decreased in bLf-treated women and increased in ferrous sulphate-treated women. BLf did not exert any adverse effect. Adverse effects in 16.5 % of ferrous sulphate-treated women were recorded. Arm A women experienced no miscarriage compared to five miscarriages in Arm B women. Conclusions Differently from ferrous sulphate, bLf is safe and effective in curing ID/IDA associated with a consistent decrease of serum IL-6. The absence of miscarriage among bLf-treated women provided an unexpected benefit. Trial registration: ClinicalTrials.gov Identifier NCT01221844.