[Eukaryotic antibiotic peptides: a evolutionary relic or therapeutic remedy?].
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Anti-Bacterial Agents↗
Biomedical subjects
D Andreu
Publications and source records attributed to D Andreu.
[Eukaryotic antibiotic peptides: A evolutionary relic or therapeutic remedy?]
Explore the source record for details and available documents.
Journal Article↗
[Vitreus incarceration after using iris retractors].
PURPOSE/METHODS: Biomicroscopic and ophthalmoscopic evaluation of an aphakic patient treated for a retmal detachment with scleral surgery and iris retractors. To describe a complication of the use of iris retractors in retinal surgery in aphakic patients. RESULTS/CONCLUSIONS: On physical examination, a vitreus incarceration in the paracentesis performed for the iris retractors was observed. Nevertheless retinal application and good visual acuity was achieved. We conclude that the use of flexible iris retractors during scleral surgery in aphakic patients is not exempt from complications.
Aged↗
Identification of an anti-mycobacterial domain in NK-lysin and granulysin.
NK-lysin and granulysin are homologous cationic anti-bacterial peptides produced by pig and human cytolytic lymphocytes, respectively. The solution structure of NK-lysin comprises five amphipathic alpha-helices. To investigate the properties of a helix-loop-helix region postulated to be a membrane-docking part of NK-lysin, we synthesized 22- and 29-residue peptides reproducing this region for both NK-lysin and granulysin. CD spectroscopy of the synthetic peptides in a liposomal solution showed spectra typical of alpha-helical peptides. The peptides were active against Gram-positive and Gram-negative bacteria, with the two NK-lysin peptides showing higher anti-bacterial activities than the two from granulysin. One NK-lysin peptide was active against Pseudomonas aeruginosa and Staphylococcus aureus, two organisms against which NK-lysin is inactive. Granulysin peptides were inactive against these bacteria, in contrast with granulysin, which is known to be active against them. Both NK-lysin and all synthetic analogues killed Mycobacterium tuberculosis and K562 tumour cells, but did not display haemolytic activity. These results identify a potent anti-mycobacterial domain in NK-lysin and granulysin consisting of a 22-residue (helix 3) sequence plus a disulphide-constrained loop.
Amino Acid Sequence↗
Interaction of mitochondrial presequences with DnaK and mitochondrial hsp70.
Mitochondrial heat shock protein 70 (mt-hsp70) functions as a molecular chaperone in mitochondrial biogenesis. The chaperone in co-operation with its co-proteins acts as a translocation motor pulling the mitochondrial precursor into the matrix. Mt-hsp70s are highly conserved when compared to the bacterial hsp70 homologue, DnaK. Here we have used DnaK as a model to study the interaction of mitochondrial presequences with mt-hsp70 applying a DnaK-binding algorithm, computer modeling and biochemical investigations. DnaK-binding motifs have been analysed on all available, statistically relevant mitochondrial presequences found in the OWL database by running the algorithm. A total of 87 % of mammalian, 97 % of plant, 71 % of yeast and 100 % of Neurospora crassa presequences had at least one DnaK binding site. Based on the prediction, five 13-mer presequence peptides have been synthesized and their inhibitory effect on the molecular chaperone (DnaK/DnaJ/GrpE) assisted refolding of luciferase has been analysed. The peptide with the highest predicted binding likelihood showed the strongest inhibitory effect, whereas the peptide with no predicted binding capacity showed no inhibitory effect. A 3D structure of the pea mt-hsp70 has been constructed using homology modeling. The binding affinities of the 13-mer presequence peptides and additional control peptides to DnaK and pea mt-hsp70 have been theoretically estimated by calculating the buried hydrophobic surface area of the peptides docked to DnaK and to the mt-hsp70 structural model. These results suggest that mitochondrial presequences interact with the mt-hsp70 during or after mitochondrial protein import.
Amino Acid Sequence↗
Synthesis of cyclic Herpes simplex virus peptides containing 281-284 epitope of glycoprotein D-1 in endo- or exo-position.
We have prepared two types of cyclopeptides containing the 281DPVG284 sequence from the 276-284 region of glycoprotein gD-1 of the Herpes simplex virus (HSV). The syntheses were performed by solid phase methodology using MBHA or BHA resin and orthogonal protection schemes. Head-to-side-chain cyclization included the N-terminal part of the epitope, while side-chain-to-side-chain lactam bridge formation resulted in a peptide containing a C-terminal cycle. Peptides elongated by Cys at the N-terminal of the sequence were also prepared. Boc chemistry using Fmoc and OFm orthogonal protection was applied for on-resin cyclization. Based on the orthogonality of Bzl and cHex esters under a 1 M TMSOTf-thioanisole/TFA cleavage condition, a new approach for the cyclization on BHA-resin has also been developed. Preliminary studies on solution conformation of the cyclic peptides by CD spectroscopy indicated the importance of the location and the size of the cycle within the epitope sequence.
Amino Acid Sequence↗
Surface plasmon resonance screening of synthetic peptides mimicking the immunodominant region of C-S8c1 foot-and-mouth disease virus.
The main antigenic site (site A) of foot-and-mouth disease virus (FMDV, strain C-S8c1) may be adequately reproduced by a 15-peptide with the amino acid sequence H-YTASARGDLAHLTTT-NH(2) (A15), corresponding to the residues 136-150 of the viral protein VP1. The effect of amino acid substitutions within A15 on its antigenicity towards monoclonal antibodies (MAb) raised against antigenic site A, has been studied by means of BIAcore technology, based on surface plasmon resonance (SPR). Although these antigenicities have previously been determined from enzyme-linked immunosorbent assays (ELISA), the SPR-based technique is superior in that it allows a fast and straightforward screening of antigens while simultaneously providing kinetic data of the antigen-antibody interaction. With a view to screening fairly large libraries of individual peptides, we have inverted the typical SPR experiment by immobilizing the MAb on the sensor surface and using peptides as soluble analytes. We report the validation of this approach through the screening of 44 site A peptides, with results generally in good agreement with the relative antigenicities previously determined by competition ELISA.
Amino Acid Sequence↗
Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides.
We have investigated the solution conformation of the functionally relevant C-terminal extremes of alpha- and beta-tubulin, employing the model recombinant peptides RL52alpha3 and RL33beta6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotypes of alpha- and beta-tubulin, respectively, and synthetic peptides with the alpha-tubulin(430-443) and beta-tubulin(412-431) internal sequences. Alpha(404-451) and beta(394-445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of beta(394-445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining the proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced markedly helical CD spectra in alpha(404-451) and beta(394-445). A substantial part of the helicity of beta(394-445) was found to be in the CD spectrum of the shorter peptide beta(412-431) with TFE. Two-dimensional 1H-NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational C alphaH shifts) in 30% TFE permitted to conclude that about 25% of alpha(404-451) and 40% of beta(394-451) form well-defined helices encompassing residues 418-432 and 408-431, respectively, flanked by disordered N- and C-segments. The side chains of beta(394-451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both alpha and beta chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of alpha(404-451) is the same as that described in the electron crystallographic model structure of alphabeta-tubulin, while in beta(394-451) it extends for nine residues more, supporting the possibility of a functional coil --> helix transition at the C-terminus of beta-tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.
Amino Acid Sequence↗
A comparative study of cyclization strategies applied to the synthesis of head-to-tail cyclic analogs of a viral epitope.
A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to approximately 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.
Antibodies, Monoclonal↗
Tolerability and safety of 0.1% diclofenac plus 0.3% tobramycin fixed-dose ophthalmic solution: a randomized, comparative, controlled study in healthy volunteers.
The purpose of this double-blind, observer-masked, randomized, crossover trial was to compare the tolerability and safety of a fixed-dose ophthalmic solution of 0.3% tobramycin plus 0.1% diclofenac versus Tobrex (tobramycin sulfate ophth) and Voltaren (diclofenac sodium). Control treatments included a saline solution and a control solution of 0.3% tobramycin prepared by Alcon Cusí. Ten healthy volunteers received three consecutive instillations of 1 drop of a given ophthalmic solution at 08:00, 11:00 and 14:00 h to the same eye; after a washout period of 18 h, the next ophthalmic solution was tested according to a randomized sequence. Occurrence, intensity, and duration of ocular irritation and conjunctival hyperemia at baseline and after the three instillations were recorded. Slit lamp biomicroscopy examination, measurement of intraocular pressure (IOP) changes, visual acuity, and examination of the fundus of the eye were performed after each third instillation by an ophthalmologist. Side effect incidence and patient and investigator opinions were also recorded. Results showed that Voltaren instillation induced statistically significant ocular irritation (p = 0.0077); the remaining ophthalmic solutions tested caused no ocular irritation (Physiological Saline Braun, p = 0.9808; Tobrex, p = 0.8826; control 0.3% tobramycin solution, p = 0.8327; and 0.1% diclofenac plus 0.3% tobramycin, p = 0.5399). None of the ophthalmic solutions tested caused severe conjunctival hyperemia. Analysis of the sum of conjunctival parameters of both eyes for all ophthalmic solutions studied showed no statistically significant differences (p = 0.4688). Moderate superficial punctate keratitis was observed after instillation of Voltaren and of 0.1% diclofenac plus 0.3% tobramycin (1 subject each) that spontaneously resolved within 2 days. Slit lamp biomicroscopy, visual acuity and IOP values showed no statistically significant changes. No systemic side effects related to the study treatments were recorded. In conclusion, the ophthalmic solution containing 0.1% diclofenac plus 0.3% tobramycin was well tolerated under the study conditions. Its tolerability was equivalent to that of Braun physiological saline, Tobrex and a control 0.3% tobramycin solution and was better than that of Voltaren.
Adolescent↗
Human CD5 signaling and constitutive phosphorylation of C-terminal serine residues by casein kinase II.
CD5 is a lymphocyte surface glycoprotein with a long cytoplasmic domain suitable for phosphorylation and signal transduction, which is involved in the modulation of Ag-specific receptor-mediated activation and differentiation signals. In this study, we use Jurkat T cell transfectants of CD5 cytoplasmic tail mutants to reveal phosphorylation sites relevant to signal transduction. Our results show that casein kinase II (CKII) is responsible for the constitutive phosphorylation of CD5 molecules at a cluster of three serine residues located at the extreme C terminus (S458, S459, and S461). Furthermore, the yeast two-hybrid system demonstrates the specific association between the C-terminal regions of the CD5 cytoplasmic tail and the regulatory beta subunit of CKII. We demonstrate that CKII associates with and phosphorylates the C-terminal region of CD5, a conserved domain known to be relevant for the generation of second lipid messengers, and thereby enables at least one component of its signaling function.
Amino Acid Sequence↗
The plasma membrane of Leishmania donovani promastigotes is the main target for CA(1-8)M(1-18), a synthetic cecropin A-melittin hybrid peptide.
Reports on the lethal activity of animal antibiotic peptides have largely focused on bacterial rather than eukaryotic targets. In these, involvement of internal organelles as well as mechanisms different from those of prokaryotic cells have been described. CA(1-8)M(1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal activity. Using Leishmania donovani promastigotes as a model system we have studied the mechanism of action of CA(1-8)M(1-18), its two parental peptides and two analogues. At micromolar concentration CA(1-8)M(1-18) induces a fast permeability to H+/OH-, collapse of membrane potential and morphological damage to the plasma membrane. Effects on other organelles are related to the loss of internal homeostasis of the parasite rather than to a direct effect of the peptide. Despite the fast kinetics of the process, the parasite is able to deactivate in part the effect of the peptide, as shown by the higher activity of the d-enantiomer of CA(1-8)M(1-18). Electrostatic interaction between the peptide and the promastigote membrane, the first event in the lethal sequence, is inhibited by polyanionic polysaccharides, including its own lipophosphoglycan. Thus, in common with bacteria, the action of CA(1-8)M(1-18) on Leishmania promastigotes has the same plasma membrane as target, but is unique in that different peptides show patterns of activity that resemble those observed on eukaryotic cells.
Adenosine Triphosphate↗
Animal antimicrobial peptides: an overview.
Antibiotic peptides are a key component of the innate immune systems of most multicellular organisms. Despite broad divergences in sequence and taxonomy, most antibiotic peptides share a common mechanism of action, i.e., membrane permeabilization of the pathogen. This review provides a general introduction to the subject, with emphasis on aspects such as structural types, post-translational modifications, mode of action or mechanisms of resistance. Some of these questions are treated in depth in other reviews in this issue. The review also discusses the role of antimicrobial peptides in nature, including several pathological conditions, as well as recent accounts of their application at the preclinical level.
Amino Acid Sequence↗
Long-lasting remission of cytomegalovirus retinitis without maintenance therapy in human immunodeficiency virus-infected patients.
Seven AIDS patients who were receiving suppressive therapy for previously diagnosed cytomegalovirus (CMV) retinitis were offered treatment with protease inhibitors (PIs). Secondary prophylaxis for CMV was discontinued after 3 months of therapy with PIs if patients had >150 CD4 cells/mm3 and a human immunodeficiency virus (HIV) load of <200 copies/mL and if they were negative for CMV as determined by qualitative CMV polymerase chain reaction (PCR). Ophthalmologic exams were done periodically. After a median follow-up of 9 months (range, 9-12), no new episodes of CMV retinitis were observed. CD4 cell counts were >150 cells/mm3 in all cases, HIV loads were <200 copies/mL, and results for qualitative CMV PCRs remained negative. These observations suggest that for selected patients with healed CMV retinitis who have immunologic and virologic evidence of a clinical response to potent combination antiretroviral therapy, temporary discontinuation of a chronic anti-CMV suppressive therapy may not result in further retinal necrosis. However, the long-term immunologic benefit of PIs and hence the safety of prolonged withdrawal of anti-CMV therapy is unknown.
AIDS-Related Opportunistic Infections↗
Cecropin A-derived peptides are potent inhibitors of fungal plant pathogens.
Cecropins are naturally occurring peptides that play an important role in the immune response of insects. Cecropin A-derived and cecropin A-melittin hybrid peptides, all smaller than the natural compound cecropin A, were synthesized and tested for their ability to inhibit growth of several agronomically important fungal pathogens. We found that an 11-amino-acid sequence, corresponding to the N-terminal amphipathic alpha-helix domain of cecropin A, exhibited antifungal activity. Differences in susceptibility of the various pathogens were observed, Phytophthora infestans being particularly sensitive to the shortened cecropin A peptides (IC50 = 2 x 10(-6) M). Biotoxicity of the shortest cecropin A-derived peptide was variously affected by the presence of proteins extracted from leaves of tobacco and tomato plants, either total extracts or intercellular fluids (ICFs). Overall, there was a greater tolerance to tomato protein extracts than to tobacco extracts. These findings suggest that tobacco should not be used as a model for testing the possible protective effects of transgenically expressed, cecropin-based genes. The feasibility of tailoring cecropin A genes to enhance crop protection in particular plant/fungus combinations is discussed.
Amino Acid Sequence↗
A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation.
The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C1, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.
Amino Acid Sequence↗
Macrophage triggering with cecropin A and melittin-derived peptides induces type II nitric oxide synthase expression.
Triggering of RAW 264.7 cells with a cecropin A-melittin hybrid peptide (CA(1-8)M(1-18)) promoted a rapid rise in the intracellular calcium concentration that was followed, after a lag period of 6 h, by nitric oxide synthesis through the expression of the cytokine-inducible form of nitric oxide synthase (type II NOS or iNOS). The maximal effect was obtained at peptide concentrations in the 2 to 5-microM range. Simultaneous incubation with the peptide and LPS abrogated the nitric oxide synthesis elicited after LPS treatment of the cells. CA(1-8)M(1-18) induced a rapid activation of nuclear factor kappaB as evidenced by the presence of p50/p65 heterodimers of the nuclear factor kappaB/c-Rel family in the nuclei of activated cells. This peptide also activated the reporter activity of cells transfected with a plasmid harboring a 1-kb fragment corresponding to the 5'-flanking region of the murine iNOS gene. CA(1-8)M(1-18) promoted apoptotic cell death at concentrations below 1 to 2 microM, whereas higher concentrations altered the plasma membrane integrity. These results suggest the involvement of multiple intracellular signaling pathways in the mechanism by which this peptide elicits macrophage triggering.
Animals↗
A cyclic disulfide peptide reproduces in solution the main structural features of a native antigenic site of foot-and-mouth disease virus.
A cyclic disulfide peptide corresponding to the G-H loop sequence 134-155 [replacement Tyr136 and Arg153 with Cys] of the capsid protein VP1 of foot-and-mouth disease virus (FMDV) isolate C-S8c1 was examined by proton 2D-NMR spectroscopy in water and in 25% HFIP/water. In water, NMR data supported the presence of a non-canonical turn in the central, conserved cell adhesion RGD motif and suggested the presence of a nascent helix in the C-terminal part, stabilized and slightly extended upon addition of 25% HFIP, a secondary structure stabilizing cosolvent. The formation of the C-terminal helix was evidenced by combined analysis of NOE connectivities, H alpha chemical shifts, 3JNH-H alpha coupling constants and amide temperature coefficients. Surprisingly, these global structural features of the cyclic peptide in solution show similarities to previous X-ray structure analysis of (a) a shortened linear peptide complexed with a antivirus antibody and (b) the G-H loop represented on the chemical reduced viral surface of a different serotype. Thus, even in entirely different biological environments the cyclic peptide reflect similar structural features, reinforcing the concept that this viral loop behaves as an independent structural and functional unit.
1-Propanol↗