PubMed Health⌕ Search

Biomedical subjects

F Reig

Publications and source records attributed to F Reig.

At least 19 recordsLinked to original sources
PubMed2005

Spectroscopic techniques applied to the study of laminin fragments inserted into model membranes.

The influence of four laminin-derived peptides on bilayer organization is studied. Spectroscopic methods applied were based on pyrene fluorescence properties (quenching, I1/I3, and monomer/excimer equilibrium), asymmetric membrane fluorescence (NBD-PE/dithionite), and polarization fluorescence (TMA-DPH). Also, the ability of these peptides to release carboxyfluorescein entrapped in vesicles was determined. Results suggest that these peptides do not noticeably modify the packing and motion of lipids (in the gel state), but coat its surface, preventing penetration of quenchers and chemical reactants. Nevertheless, their presence promotes a soft release of entrapped CF after incubation at 37 degrees C.

Fluorescence Polarization↗
PubMed2005

Synthesis and study of molecular interactions between phosphatidyl choline and two laminin derived peptides hydrophobically modified.

Two peptides based on the active SIKVAV sequence were synthesized and hydrophobically modified through attachment of a myristoyl residue to the amino terminal group. A comparative study was carried out on the physicochemical properties of both parent and hydrophobically modified structures. Properties studied were hydrophobicity, surface activity, spreadability on aqueous surfaces, penetration in lipid monolayers, aggregation and haemolytic activity. Results obtained indicate that myristoyl containing peptides form micelles at 10(-6) M concentration, whereas parent peptides start to aggregate at 10(-5) M. All of them are able to spread on aqueous surfaces forming stable monolayers. Concerning their haemolytic activity only one of the sequences promotes a partial lysis of erythrocytes after 30 min incubation at 37 degrees C and 10(-5) M concentration in the media.

Chromatography, High Pressure Liquid↗
PubMed2004

Pentapeptide YIGSR-mediated HT-1080 fibrosarcoma cells targeting of adriamycin encapsulated in sterically stabilized liposomes.

In the peptide-targeted therapy for cancer, peptides are used to reach a selective and specific target in cancer cells. Peptides are used free or coupled to chemotherapeutic drugs, phagues, proteins, polymers, liposomes, and polymer-grafted liposomes. Using this latter approach, the pentapeptide YIGSR was coupled to the distal end from carboxyl groups of liposome-grafted polyethyleneglycol (PEG) chains (YIGSR-PEG-liposome). As a control, the peptide PEAGD coupled to PEG-liposome was used. The biological activity of YIGSR-PEG-liposome was tested using HT-1080 human fibrosarcoma cells. In adhesion assays, the YIGSR-PEG-liposome coated to plastic plates promoted 30% of the specific cell attachment. In competition assays, YIGSR-PEG-liposome inhibited the specific attachment of cells to laminin-1-coated plates by 25%. Following this, we prepared peptide-PEG-liposomes encapsulating adriamycin (ADR). In vitro cytotoxicity assays against HT-1080 cells gave IC(50) values 2.1 times lower for YIGSR-PEG-liposomal ADR in comparison to PEAGD-PEG-liposomal ADR. The free peptide added in excess increased the IC(50) value of YIGSR-PEG-liposomal ADR by 72%, while the IC(50) value of control liposomal ADR was unaffected, supporting a receptor-mediated mechanism of targeting. In addition, the lower IC(50) value is correlated with a higher total of ADR accumulation in the cells.

Antineoplastic Agents↗
PubMed2003

Interfacial interactions between poly[L-lysine]-based branched polypeptides and phospholipid model membranes.

The interaction of five poly[L-lysine]-derived branched chain polypeptides of poly[Lys(X(i))] (X(i)K) or poly[Lys(X(i)-DL-Ala(m))] (XAK) with lipid bilayers (DPPC and DPPC/PG, 8:2) was studied by fluorescence polarization techniques. Two fluorescent probes, DPH and TMA-DPH, were utilized to monitor changes of motion in the internal and/or in the polar head regions, respectively. Results indicate that the interaction of polypeptides with neutral (DPPC) bilayers is mainly dependent on the polarity and electrical charge of side chains. The amphoteric E(i)K shows the highest level of interaction. Polycationic polypeptides (H(i)K, P(i)K, TAK) have a relatively small effect on the transition temperature of the lipids, while the polyanionic Succ-EAK has no effect at the alkyl chain region of the bilayer. Data with TMA-DPH indicate the lack of pronounced interaction between the polypeptides and the outer surface of the liposome. Similar tendency was documented for DPPC/PG vesicles. Polypeptides, H(i)K, and P(i)K induce significant changes in the transition temperature, thus indicating their insertion into the hydrophobic core of the bilayer without marked effect on the polar head region. Results suggest that these polypeptides (except E(i)K) have no destabilizing effect on liposomes studied. These properties are considered as beneficial for their use as safe carriers for bioactive molecules.

Fluorescence Polarization↗
PubMed2003

Synthesis and physicochemical study of the laminin active sequence: SIKVAV.

The synthesis, physicochemical characterization, and interaction with membrane model systems of a peptide derived from the PA22-2 region of laminin are described. Surface activity studies indicate that this peptide is able to spread at the air-water interface being the maximal spreading pressure 20 mN/m at subphase concentrations around 10 micro M. Besides, these peptide molecules are also able to form stable monolayers. Physicochemical studies concerning the interaction of this peptide with lipids, organized in mono and bilayers, were carried out using Langmuir balance experiments and polarization fluorescence techniques. The peptide penetrates better in monolayers of DPPC than in those of PC and forms condensed mixed monolayers with DPPC. Energies of mixing are small thus indicating that deviations from ideality were almost negligible. Interactions with bilayers were studied through microviscosity changes (DPH and TMA-DPH probes), membrane permeability alterations (CF, NBD-PE/dithionite), and fusion promotion (NBD-PE/Rh-PE, resonance energy transfer). Results indicate that this sequence interacts very softly with bilayers without promoting changes in their organization. These data as well as the lack of interaction with erythrocytes suggest that coating liposomes with this peptide through chemical amide bonds can render stable inmunoliposomes for further biological applications.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2003

Physicochemical characterization of branched chain polymeric polypeptide carriers based on a poly-lysine backbone.

A systematic study is reported on the physicochemical characteristics of two branched chain polymers (based on a poly-L-lysine backbone) with a general formula poly[Lys-(DL-Alam-Xi)], where X = Orn (OAK) or N-acetyl-Glu (Ac-EAK) and m approximately equal to 3, using surface pressure and fluorescence polarization methods. These data are compared with those of the linear poly(L-Lys) from which OAK and Ac-EAK are derived. These two polymers show a moderate surface activity, able to form stable monomolecular layers at the air-water interface. Poly(L-Lys), the most hydrophilic, has the lowest surface activity. The interaction of these polymers with phospholipid bilayers either neutral or negatively charged was studied with vesicles labeled with two fluorescent probes: ANS and DPH. Results indicate that these polymers are able to accommodate in their internal structure, mainly through electrostatic interactions, a certain amount of ANS marker molecules, but fluorescence increases of the ANS-polypeptide complexes were so low that its influence in further polarization measurements could be discarded. After interaction with liposomes, these polymers induce an increase in the polarization of the probes, thus indicating a rigidification of the bilayers. Electrostatic forces seem to be very important in this interaction; cationic polymers are clearly more active, with PG-containing liposomes, than Ac-EAK. Moreover, in these assays poly(L-Lys) behaves as the more active compound. This fact is probably due to its major ability to form alpha-helical structures that could insert easily in the bilayers. These results indicate that the polymeric structures studied can be used as carriers for biologically active molecules, because their interactions with bilayers remain soft and have a positive effect on the stability of the membranes.

Fluorescence Polarization↗
PubMed2003

Synthesis of peptide sequences related to thrombospondin: factors affecting aspartimide by-product formation.

Aspartimide formation is one of the most common secondary reactions on solid phase peptide synthesis. In the present work, we describe the optimization of the synthesis of two thrombospondin fragments containing an Asp-Gly sequence that show a strong tendency to form cyclic aspartimide derivatives in an unusual high percentage. Several different strategies were applied changing type of resin, Fmoc-deprotection reagents, coupling additives, resin cleavage cocktails and the use of Hmb-Gly derivative to minimize the extension of this byproduct. Best results were obtained with cross-linked ethoxylate acrylate (CLEAR-cross-linked ethoxylate Acrylate Resin)-type resin and pip/dimethylformamide deprotection. Besides, as in biological assays the aspartimide containing sequence resulted to be more active than the linear one, the optimization of its synthesis was also carried out.

Amino Acid Sequence↗
PubMed2002

[Giant cholesteatoma of the external ear canal after myringoplasty].

Cholesteatoma are specially localized in middle ear and mastoid process, although sometimes is the external ear canal which shows the disorder. Usually its a appearance is spontaneous. We report a rare case of gigantic cholesteatoma which origin was the previous otologic surgery done because of the inclusion of a bit of epithelial tissue inside the surgical incision. Similar outcomes justify periodical checking after ear surgery with outlining a tympanomeatal flap.

Adult↗
PubMed2001

Differences in secondary structure of HAV-synthetic peptides induced by the sequential order of T- and B-cell epitopes.

The present study was undertaken to examine the structural features of two peptide constructs designed on the basis of linear combination of B and T-cell epitopes in different orientations (BT and TB) that may be important to explain the differences in the elicited antihepatitis A virus immune response and in the interaction with biological model membranes. A CD study was carried out and the corresponding quantitative analysis of the experimental data was done using deconvolution computer programs. Moreover, fluorescence experiments were performed to analyze differences in the fluorescence emission spectra of both molecules. The main conformational difference by CD studies was obtained working in aqueous medium. Although the TB sequence adopted a preferably random coil structure, the BT peptide was best fitted with beta-type structures. These results are further supported by fluorescence studies. These findings have relevance for the design of synthetic immunopeptides.

Amino Acid Sequence↗
PubMed2000

Hepatitis A Synthetic Peptide VP3(110-121) Miscibility with Dipalmitoylphosphatidylcholine, Dipalmitoylphosphatidylglycerol, and Stearylamine Monolayers.

To prepare liposomes containing a synthetic hepatitis A virus antigen (HAV) [VP3(110-121)] as a vaccine, the miscibility of this peptide (with negative net charge) with a neutral lipid [dipalmitoylphosphatidylcholine (DPPC)], a negatively charged lipid [dipalmitoylphosphatidylglycerol (DPPG)], and a positively charged lipid [Stearylamine (SA)] was studied through compression isotherms of monolayers. Mixtures with DPPC and SA showed a low degree of interaction with the peptide, the composition of the monolayer being stable through compression. For DPPG-containing monolayers larger positive deviations from ideality were found, and the peptide was squeezed out from the monolayer at a DPPG/VP3(110-121) mole fraction of 0.8/0.2. All this suggests that besides hydrophobic interactions between the peptide and the lipid, electrostatic forces also play a role; thus it seems that neutral and positively charged lipids would be more suitable for preparing stable liposomes with VP3(110-121). Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Fluorescence study on the interaction of a multiple antigenic peptide from hepatitis A virus with lipid vesicles.

The interaction of the multiple antigenic peptide MAP4VP3 with lipid membranes has been studied by spectroscopic techniques. MAP4VP3 is a multimeric peptide that corresponds to four units of the sequence 110-121 of the capsid protein VP3 of hepatitis A virus. In order to evaluate the electrostatic and hydrophobic components on the lipid-peptide interaction, small unilamelar vesicles of different compositions, including zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic dipalmitoylphosphatidylcholine/phatidylinositol (DPPC:PI 9:1), and cationic dipalmitoylphosphatidylcholine/stearylamine (DPPC:SA 9.5:0.5), were used as membrane models. Intrinsic tryptophan fluorescence changes and energy transfer experiments show that MAP4VP3 binds to all three types of vesicles with the same stoichiometry, indicating that the electrostatic component of the interaction is not important for binding of this anionic peptide. Steady-state polarization experiments with vesicles labeled with 1,6-diphenyl-1,3,5-hexatriene or with 1-anilino-8-naphtalene sulphonic acid indicate that MAP4VP3 induces a change in the packing of the bilayers, with a decrease in the fluidity of the lipids and an increase in the temperature of phase transition in all the vesicles. The percentage of lipid exposed to the bulk aqueous phase is around 60% in intact vesicles, and it does not change upon binding of MAP4VP3 to DPPC vesicles, indicating that the peptide does not alter the permeability of the membrane. An increase in the amount of lipid exposed to the aqueous phase in cationic vesicles indicates either lipid flip-flop or disruption of the vesicles. Binding to DPPC vesicles occurs without leakage of entrapped carboxyfluorescein, even at high mol fractions of peptide. However, a time-dependent leakage is seen with cationic DPPC/SA and anionic DPPC/PI vesicles, indicating that the peptide induces membrane destabilization and not lipid flip-flop. Resonance energy transfer experiments show that MAP4VP3 leakage from cationic vesicles is due to membrane fusion, whereas leakage from anionic vesicles is not accompanied by lipid mixing. Results show that MAP4VP3 interacts strongly with the lipid components of the membrane, and although binding is not of electrostatic nature, the bound form of the peptide has different activity depending on the membrane net charge; thus, it is membrane disruptive in cationic and anionic vesicles, whereas no destabilizing effect is seen in DPPC vesicles.

Antigens, Viral↗
PubMed1999

Liposomes as vehicles for the presentation of a synthetic peptide containing an epitope of hepatitis A virus.

Previous work from our group showed that the entrapment of HAV-related synthetic peptides into multilamellar liposomes yielded satisfactory immunoresponses when administered to mice. In the present work we report investigative results for several liposome formulations of a 20-mer peptide related to VP3 capsid protein of HAV. In this sense, the recently introduced surface plasmon resonance technique has been applied to compare the different strategies of association between the synthetic peptide and phospholipid vesicles and to demonstrate that no significant alteration in antigenicity is produced when the peptide sequence is covalently coupled to the surface of small unilamellar vesicles. In addition, conformational data obtained by the circular dichroism technique have shown a decrease in the helical contribution of peptide-once linked to phospholipid; probably this change is due to the restriction introduced at the N-terminus of the sequence when coupled to the derivatized phospholipids at the surface of vesicle bilayers.

Animals↗
PubMed1998

Physicochemical Study of Hydrophobic Derivatives of Collagen Fragments.

Collagen fragments of molecular weights ranging from 700 to 3500 were prepared and allowed to react with hexyl and lauryl aldehydes in order to obtain derivatives of different hydrophobicities. Lauryl collagen forms, in aqueous solutions, aggregates that have low tendency to interact with phospholipid mono and bilayers, with reduced surface activity but the ability to entrap ANS molecules. The hexyl derivative shows a greater hydrophobicity in terms of surface activity and interaction with lipids but a lower ability to incorporate ANS. Underivatized collagen shows no lipid interaction in any of the experiments. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Conformational behavior of the HAV-VP3(110-121) peptidic sequence and synthetic analogs in membrane environments studied by CD and computational methods.

The present study was undertaken to examine the structural features that may be important to explain the immunogenicity of the (110-121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus. A conformational analysis of the preferred conformations by CD and molecular mechanics was carried out. Present results suggest that the interaction with liposomes as biomembrane model induces and stabilizes the amphipathic beta-structure of the peptide. To study the contribution of amino acid replacements at the RGD tripeptide as well as the influence of the peptide chain length on peptide conformation, solid-phase peptide synthesis of several peptide analogs was carried out and the peptide conformation was studied using CD spectroscopy. The results show that the RGD sequence is necessary to induce the beta-structure in the presence of liposomes.

Capsid↗
PubMed1998

Physico-chemical characterization of liposomes with covalently attached hepatitis A VP3(101-121) synthetic peptide.

The covalent conjugation of a 20-mer peptide belonging to the VP3 capsid protein of hepatitis A virus to the surface of preformed liposomes was investigated. Three different bonds (disulfide, thioether and amide) were established between the peptide sequence and liposomes bearing at their surface appropriate reactive groups. The effect of the relative concentration of the N-[4-(p-maleimidophenyl)butyryl]dipalmitoylphosphatidylethanolamine anchor in liposomes on stability during coupling of the peptide sequence was studied. The interaction of the three liposomal preparations with phospholipids in a biomembrane model system, monolayers at the air-water interface, is also reported. The results showed that although the peptides associate with liposomes in similar yields for the three strategies studied, differences can be observed when their interaction with phospholipid monolayers composed of dipalmitoylphosphatidylcholine is analysed.

Adjuvants, Immunologic↗
PubMed1996

Coating liposomes with collagen (Mr 50,000) increases uptake into liver.

Collagen-coated small unilamellar liposomes were prepared by incubation of two hydrophobic derivatives of collagen (average Mr 50 000) with preformed vesicles. The introduction of hexyl and lauryl residues to the collagen molecule improved by 10-fold the ability of collagen to coat liposomes. In vitro stability of the different coated vesicles prepared, was studied by their ability to retain entrapped carboxyfluorescein as a function of the time. Coated vesicles were clearly more stable in vitro than control liposomes, except for those containing the lauryl derivative in a protein/phospholipid weight ratio higher than 10(-3). Vesicle clearance from circulation as well as tissue distribution were also determined. Pharmacokinetics (determined by both fluorescence and radioactive techniques) were highly dependent on the injected dose, phospholipids used and the content of collagen. Half-lives were maximum for liposomes composed of saturated phospholipids injected at a dose of 2 micromol phospholipid. Besides, blood elimination of collagen-containing vesicles was about 2-fold faster and liver uptake 1.5 to 2-fold higher than control liposomes.

Animals↗