FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

The proliferative effect of synthetic N-POMC(1-28) peptides in rat adrenal cortex: A possible role for cyclin E

Modified synthetic N-POMC(1-28) without disulfide bridges has been shown to act as an adrenal mitogen. Cyclins and their inhibitors are the major cell cycle controls, but in the adrenal cortex the effect of ACTH and N-POMC on the expression of these proteins remains unclear. In this work, we evaluate the effect of different synthetic N-POMC peptides on the S-phase of the cell cycle. In addition, we examine the cyclin E expression in rat adrenal cortex. Rats treated with dexamethasone were injected with ACTH and/or synthetic modified N-POMC and/or synthetic N-POMC with disulfide bridges. DNA synthesis was determined by BrdU incorporation and protein expression was analyzed by immunoblotting and immunohistochemistry. The results showed that similarly to modified N-POMC without disulfide bridges, administration of synthetic N-POMC with disulfide bridges and the combination of ACTH and N-POMC promoted an increase of BrdU-positive nuclei in adrenal cortex. However, the proliferative effect of N-POMC was comparable to that of ACTH only in the zona glomerulosa. An increase in cyclin E expression was observed 6 h after N-POMC treatment in the outer fraction of the adrenal cortex, in agreement with immunohistochemical findings in the zona glomerulosa. In summary, the effect of synthetic N-POMC with disulfide bridges was similar to modified synthetic N-POMC, increasing proliferation in the adrenal cortex, confirming previous evidence that disulfide bridges are not essential to the N-POMC mitogenic effect. Moreover, cyclin E appears to be involved in the N-POMC- and ACTH-stimulated proliferation in the zona glomerulosa of the adrenal cortex. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 mu M and 10 mu M of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 mu M concentrations. Comparing control and REN concentration of 1 mu M, JHCO(3)(-) . nmol cm(-2) s(-1) -1,76 +/- 0.11(control) x 1,29 +/- 0,08(REN) 10 mu m: P<0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 mu M (JHCO(3)(-), nmol cm(-2) s(-1) -0.80 +/- 0.07(control) x 0.60 +/- 0.06(REN) 1 mu m; P<0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na(+)/H(+) exchanger and H(+)-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway. (c) 2009 Elsevier B.V. All rights reserved.

Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

P>Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, similar to 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, similar to 1/3 showed no change, and similar to 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.

Hemopressins and other hemoglobin-derived peptides in mouse brain: comparison between brain, blood, and heart peptidome and regulation in Cpefat/fat mice

P>Many hemoglobin-derived peptides are present in mouse brain, and several of these have bioactive properties including the hemopressins, a related series of peptides that bind to cannabinoid CB1 receptors. Although hemoglobin is a major component of red blood cells, it is also present in neurons and glia. To examine whether the hemoglobin-derived peptides in brain are similar to those present in blood and heart, we used a peptidomics approach involving mass spectrometry. Many hemoglobin-derived peptides are found only in brain and not in blood, whereas all hemoglobin-derived peptides found in heart were also seen in blood. Thus, it is likely that the majority of the hemoglobin-derived peptides detected in brain are produced from brain hemoglobin and not erythrocytes. We also examined if the hemopressins and other major hemoglobin-derived peptides were regulated in the Cpefat/fat mouse; previously these mice were reported to have elevated levels of several hemoglobin-derived peptides. Many, but not all of the hemoglobin-derived peptides were elevated in several brain regions of the Cpefat/fat mouse. Taken together, these findings suggest that the post-translational processing of alpha and beta hemoglobin into the hemopressins, as well as other peptides, is up-regulated in some but not all Cpefat/fat mouse brain regions.

Comparative effect of FGF2, synthetic peptides 1-28 N-POMC and ACTH on proliferation in rat adrenal cell primary cultures

There is evidence that pro-opiomelanocortin (POMC)-derived peptides other than adrenocorticotropic hormone (ACTH) have a role in adrenal cell proliferation. We compared the activity of synthetic rat N-terminal POMC fragment 1-28 with disulfide bridges (N-POMC(w)) and without disulfide bridges (N-POMC(w/o)), with the activity of fibroblast growth factor (FGF2), a widely studied adrenal growth factor, and ACTH, in well-characterized pure cultures of both isolated adrenal Glomerulosa (G) and Fasciculata/Reticularis (F/R) cells. Three days of FGF2-treatment had a proliferative effect similar to serum, and synthetic peptide N-POMC(w) induced proliferation more efficiently than N-POMC(w/o). Moreover, both induced proliferation via the ERK1/2 pathway. In contrast, sustained ACTH treatment decreased proliferation and viability through apoptosis induction, but not necrosis, and independently of PKA and PKC pathways. Further elucidation of 1-28 POMC signal transduction is of interest, and primary cultures of adrenal cells were found to be useful for examining the trophic activity of this peptide.

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin beta(13). It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V(H) CDR3 peptide from mAb A4 and V(L) CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

Background: Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results: An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1`. Conclusions: BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmume diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles. (C) 2009 Elsevier Inc. All rights reserved.

Cyclic Maximal Ideals of Rings of Differential Operators over Power Series Rings

In [3], Bratti and Takagi conjectured that a first order differential operator S=11 +...+ nn+ with 1,..., n, {x1,..., xn} does not generate a cyclic maximal left (or right) ideal of the ring of differential operators. This is contrary to the case of the Weyl algebra, i.e., the ring of differential operators over the polynomial ring [x1,..., xn]. In this case, we know that such cyclic maximal ideals do exist. In this article, we prove several special cases of the conjecture of Bratti and Takagi.

DNA alters the bilayer structure of cationic lipid diC14-amidine: A spin label study

Cationic lipids-DNA complexes (lipoplexes) have been used for delivery of nucleic acids into cells in vitro and in vivo. Despite the fact that, over the last decade, significant progress in the understanding of the cellular pathways and mechanisms involved in lipoplexes-mediated gene transfection have been achieved, a convincing relationship between the structure of lipoplexes and their in vivo and in vitro transfection activity is still missing. How does DNA affect the lipid packing and what are the consequences for transfection efficiency is the point we want to address here. We investigated the bilayer organization in cationic liposomes by electron spin resonance (ESR). Phospholipids spin labeled at the 5th and 16th carbon atoms were incorporated into the DNA/diC14-amidine complex. Our data demonstrate that electrostatic interactions involved in the formation of DNA-cationic lipid complex modify the packing of the cationic lipid membrane. DNA rigidifies the amidine fluid bilayer and fluidizes the amidine rigid bilayer just below the gel-fluid transition temperature. These effects were not observed with single nucleotides and are clearly related to the repetitive charged motif present in the DNA chain and not to a charge-charge interaction. These modifications of the initial lipid packing of the cationic lipid may reorient its cellular pathway towards different routes. A better knowledge of the cationic lipid packing before and after interaction with DNA may therefore contribute to the design of lipoplexes capable to reach specific cellular targets. (c) 2009 Elsevier B.V. All rights reserved.

Leishmanicidal Activity and Immobilization of dermaseptin 01 antimicrobial peptides in ultrathin films for nanomedicine applications

Antimicrobial peptides (AMPs) are essential for the innate immune system of eukaryotes, imparting protection against pathogens and their proliferation in host organisms. The recent interest in AMPs as active materials in bionanostructures is due to the properties shown by these biological molecules, such as the presence of an alpha-helix structure and distribution of positive charges along the chain. In this study the antimicrobial peptide dermaseptin 01 (DS 01), from the skin secretion of Phyllomedusa hypochondrialis frogs was immobilized in nanostructured layered films in conjunction with nickel tetrasulfonated phthalocyanines. The leishmanicidal activity of DS 01 was confirmed using kinetic essays, in which DS 01 promoted death of all metacyclic promastigote cells in 45 minutes. Surprisingly, the immobilized DS 01 molecules displayed electroactivity, as revealed by electrochemical experiments, in which an oxidation peak at about 0.61 V was observed for a DS 01 monolayer deposited on top of a conductive electrode. Such electroactivity was used to investigate the sensing abilities of the nanostructured films toward Leishmania. We observed an increase in the oxidation current as a function of number of Leishmania cells in the electrolytic solution at concentrations down to 10(3) cells/mL. The latter is indicative that the use of AMPs immobilized in electroactive nanostructured films may be of interest for applications in the pharmaceutical industry and diagnosis.

CoMFA and CoMSIA 3D QSAR Models for a Series of Cyclic Imides with Analgesic Activity

Three-dimensional quantitative structure-activity relationships (3D-QSAR) were performed for a series of analgesic cyclic imides using the CoMFA and CoMSIA methods. Significant correlation coefficients ( CoMFA, r(2) = 0.95 and q(2) = 0.72; CoMSIA, r(2) = 0.96 and q(2) = 0.76) were obtained, and the generated models were externally validated using test sets. The final QSAR models as well as the information gathered from 3D contour maps should be useful for the design of novel cyclic imides having improved analgesic activity.

Novel manganese (III) porphyrin containing peripheral [RuCl(dppb)(X-bipy)](+) cations [dppb=1,4-bis(diphenylphosphino) butane and X = -CH3, -OMe, -Cl]. X-ray structure of the cis-[RuCl(dppb)(bipy)(4-Mepy)]PF6 complex

New tetraruthenated manganese (III) porphyrins were synthesized and characterized (P-31 NMR, cyclic voltammetry, UV-Vis). This new system presents four units of cationic ``[RuCl(dppb)(X-bipy)](+)``. The electrochemical and catalytic properties of the central manganese (III) show dependence on the characteristics of the peripheral ruthenium complexes as evidenced by the Mn-(III)/Mn-(II) reduction potential. The catalytic oxidation reactions of olefins, cyclohexene and cyclohexane, were carried out in the presence of tetrapyridyl manganese (III) porphyrins containing cationic ruthenium complex and using iodosylbenzene as oxygen donor. The performance of these new tetraruthenated porphyrins systems were evaluated and compared with the manganese porphyrin. (C) 2007 Elsevier Ltd. All rights reserved.