RESUMEN
Currently, the incidence of tattooing is on the rise compared to the past, especially among adolescents, and it leads to the urgency of monitoring the security status of tattooing centers, as well as to inform people about the risks of tattoo practice. In our clinical experience, 20% of tattooed patients presented adverse reactions, like allergic contact dermatitis, psoriasis with Koebner's phenomena and granulomatous reactions, with the latter most prevalent and most often related to red pigment. Adverse reactions to tattoo pigments, especially the red one, are well known and described in literature. Great attention has to be focused on the pigments used, especially for the presence of new substances, often not well known. For this reason, we decided to perform a study on 12 samples of red tattoo ink, obtained by patients affected by different cutaneous reactions in the site of tattoo, to analyze their chemical composition.
Asunto(s)
Colorantes/efectos adversos , Colorantes/química , Dermatitis Alérgica por Contacto/etiología , Granuloma de Cuerpo Extraño/etiología , Tinta , Psoriasis/inducido químicamente , Tatuaje/efectos adversos , Cromatografía en Capa Delgada , Dermatitis Alérgica por Contacto/diagnóstico , Granuloma de Cuerpo Extraño/diagnóstico , Humanos , Psoriasis/diagnóstico , Factores de Riesgo , SolubilidadRESUMEN
Trichogin GA IV (GAIV) is an antimicrobial peptide of the peptaibol family, like the extensively studied alamethicin (Alm). GAIV acts by perturbing membrane permeability. Previous data have shown that pore formation is related to GAIV aggregation and insertion in the hydrophobic core of the membrane. This behavior is similar to that of Alm and in agreement with a barrel-stave mechanism, in which transmembrane oriented peptides aggregate to form a channel. However, while the 19-amino acid long Alm has a length comparable to the membrane thickness, GAIV comprises only 10 amino acids, and its helix is about half the normal bilayer thickness. Here, we report the results of neutron reflectivity measurements, showing that GAIV inserts in the hydrophobic region of the membrane, causing a significant thinning of the bilayer. Molecular dynamics simulations of GAIV/membrane systems were also performed. For these studies we developed a novel approach for constructing the initial configuration, by embedding the short peptide in the hydrophobic core of the bilayer. These calculations indicated that in the transmembrane orientation GAIV interacts strongly with the polar phospholipid headgroups, drawing them towards its N- and C-termini, inducing membrane thinning and becoming able to span the bilayer. Finally, vesicle leakage experiments demonstrated that GAIV activity is significantly higher with thinner membranes, becoming similar to that of Alm when the bilayer thickness is comparable to its size. Overall, these data indicate that a barrel-stave mechanism of pore formation might be possible for GAIV and for similarly short peptaibols despite their relatively small size.
Asunto(s)
Membrana Celular/metabolismo , Lipopéptidos/química , Péptidos/química , Aminoácidos/química , Biofisica/métodos , Membrana Celular/química , Simulación por Computador , Relación Dosis-Respuesta a Droga , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Conformación Molecular , Simulación de Dinámica Molecular , Nanopartículas , Neutrones , Estructura Terciaria de ProteínaRESUMEN
A novel method to build bicomponent peptide self-assembled monolayers (SAMs) has been developed, by exploiting helix···helix macrodipole interactions. In this work, a peptide-based self-assembled monolayer composed of two helical peptides was immobilized on a gold surface. Specifically, a pyrene-containing octapeptide, devoid of any sulfur atom (A8Pyr), and a hexapeptide, functionalized at the N-terminus with (S,R) lipoic acid, for binding to gold substrates (SSA4WA) via a Au-S linkage, have been employed. Both peptides investigated attain a helical structure, because they are almost exclusively formed by strongly folding inducer C(α)-tetrasubstituted α-amino acids. We demonstrate that the two peptides generate a stable supramolecular nanostructure (a densely packed bicomponent peptide monolayer), where A8Pyr is incorporated into the SSA4WA palisade by exploiting helix···helix macrodipole interactions. The presence of both peptides on the gold surface was investigated by spectroscopic and electrochemical techniques, while the morphology of the monolayer was analyzed by ultra high-vacuum scanning tunnelling microscopy. The composition of the bicomponent SAM on the surface was studied by a combination of electrochemical and spectroscopic techniques. In particular, the amount of Au-S linkages from the sulfur-containing peptides was quantified from reductive desorption of the peptide-based SAM, while the amount of A8Pyr was estimated by fluorescence spectroscopy. The antiparallel orientation of the A8Pyr and SSA4WA peptide chains minimizes the interaction energy between the helix dipoles, suggesting that this kind of electrostatic phenomenon is the driving force that stabilizes the bicomponent SAM.
Asunto(s)
Nanoestructuras/química , Péptidos/química , Oro/química , Sustancias Macromoleculares/síntesis química , Sustancias Macromoleculares/química , Membranas Artificiales , Modelos Moleculares , Conformación Molecular , Péptidos/síntesis químicaRESUMEN
Peptide-induced vesicle leakage is a common experimental test for the membrane-perturbing activity of antimicrobial peptides. The leakage kinetics is usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibits a biphasic behavior. We report here that, in the case of the peptaibol trichogin GA IV, all processes involved in peptide-membrane interaction, such as peptide-membrane association, peptide aggregation, and peptide translocation, take place on a timescale much shorter than the leakage kinetics. On the basis of these findings, we propose a stochastic model in which the leakage kinetics is determined by the discrete nature of a vesicle suspension: peptides are continuously exchanging among vesicles, producing significant fluctuations over time in the number of peptide molecules bound to each vesicle, and in the formation of pores. According to this model, the fast initial leakage is caused by vesicles that contain at least one pore after the peptides are randomly distributed among the liposomes, whereas the slower release is associated with the time needed to occasionally reach in an intact vesicle the critical number of bound peptides necessary for pore formation. Fluctuations due to peptide exchange among vesicles therefore represent the rate-limiting step of such a slow mechanism.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Liposomas Unilamelares/metabolismo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Cinética , Modelos Biológicos , Transporte de Proteínas , Procesos Estocásticos , TermodinámicaRESUMEN
The 310-helix, first predicted as a reasonably stable polypeptide secondary structure fifty years ago, has only recently attracted the attention of structural biochemists and protein crystallographers. It represents the third principal structure occurring in globular proteins and has been described at atomic resolution in model peptides and in peptaibol antibiotics.
Asunto(s)
Péptidos/química , Conformación Proteica , Humanos , Modelos QuímicosRESUMEN
Nowadays, practice of tattooing is very common worldwide and, along with this increasing trend, there is also an increased risk of adverse reactions to tattoo pigments that are well known and described in literature. Previous studies have reported that cutaneous and allergic reactions to a particular pigment can manifest in several ways (allergic contact dermatitis and photo-allergic dermatitis). In this paper, a new high-throughput method is presented, in order to achieve a new system for the quality control on tattoo inks based on chromatographic-spectroscopic approach. The samples, twenty-one tattoo inks and three permanent makeup, comprised the following colors: black inks, yellow, blue, green, white, pink and various shades of red (pigment that gives many allergic responses) were analyzed through the combination of chromatographic and spectroscopic techniques, the HPTLC-Raman. In particular, Raman technique has been chosen because of its high sensitivity towards the inorganic and organic pigments, main constituents of tattoo inks. Moreover, the advantage of this hyphenated technique is to overcome the problem of analysing the complex mixture of tattoo inks, allowing to obtain a Raman spectrum of each single component, isolated by chromatographic separation. This approach aims at developing a powerful instrument to establish the nature of tattoo inks and substances that could be cause adverse reactions in tattooed patients.
Asunto(s)
Cromatografía en Capa Delgada/métodos , Colorantes/análisis , Tinta , Espectrometría Raman/métodos , Tatuaje , Colorantes/química , Colorantes/normas , Ensayos Analíticos de Alto Rendimiento , Control de CalidadRESUMEN
The secondary structures of thirteen fish protamines have been predicted by the statistical method of Chou and Fasman as well as by two modifications of it. The occurrence of phosphorylatable residues in predicted beta-turns is discussed. The results are compared with available spectroscopic observations.
Asunto(s)
Protaminas , Secuencia de Aminoácidos , Animales , Peces , Fragmentos de Péptidos/análisis , Conformación ProteicaRESUMEN
The retro-all-D analog and retro isomer of the formyl-methionyl-carboxamide-tripeptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2, namely CHO-D-Phe-D-Leu-D-Met-NH2 and CHO-L-Phe-L-Leu-L-Met-NH2, respectively, have been synthesized in solution by classical methods and fully characterized. The tetrapeptide CHO-L-Phe-Gly-L-Leu-L-Met-NH2, representing the C-terminal portion of the tachykinin, Substance P, and resembling the sequence of the retro isomer, has also been synthesized and characterized. The three N alpha-formylated tripeptide amides, prepared in order to obtain a deeper insight into the model of binding at the formyl peptide chemotactic receptor on rabbit neutrophils, have been tested for their ability to induce granule enzyme secretion from rabbit peritoneal neutrophils. The retro isomer, CHO-L-Phe-L-Leu-L-Met-NH2 is approximately 100-fold less active, the retro-all-D analog, CHO-D-Phe-D-Leu-D-Met-NH2 approximately 10,000-fold less active and the Substance P analog CHO-L-Phe-Gly-L-Leu-L-Met-NH2 1000-fold less active than the parent formyl peptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2. We interpret these results to indicate that a precise alignment of amino acid side chains as well as backbone amide bonds is an important factor involved in the receptor recognition of the formyl tripeptide chemoattractant.
Asunto(s)
Quimiotaxis de Leucocito , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/síntesis química , Neutrófilos/fisiología , Receptores Inmunológicos/metabolismo , Animales , Cinética , Muramidasa/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Conejos , Receptores de Formil Péptido , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The various diastereomers of the N alpha-formylated(CHO) and tert-butyloxycarbonylated (t-Boc) Phe-(Leu-Phe)n and (Leu-Phe)n methyl esters, where n = 1-2 and 1-3, respectively, have been newly synthesized and their physical properties described. The CHO-blocked peptides are all able to release beta-glucosaminidase from rabbit peritoneal neutrophils in a concentration-dependent manner. There is a strong effect of primary structure and of chirality on their biology activity; lengthening the peptide chain distinctly increases activity in each series and within a series the activity decreases in the order: all-L greater than D-L much greater than all-D. Of the t-Boc protected synthetic precursors, the all-L isomers have definite but weak agonist activity; the agonist activity of the other isomers is equivocal or not detectable. All the t-Boc peptides, however, are capable of acting as weak, specific antagonists. There is a dependence of antagonist activity on primary structure, but this is variable and contingent on the nature of the peptide. Similarly, an effect of chirality on antagonist activity, although present, also depends on the structure of the peptide. In the one instance directly tested, t-Boc-L-Phe-(D-Leu-L-Phe)2-OMe (OMe, methoxy) was found to be distinctly less active than the corresponding free acid.
Asunto(s)
N-Formilmetionina Leucil-Fenilalanina/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Péptidos/farmacología , Receptores Inmunológicos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , Conejos , Receptores de Formil Péptido , Relación Estructura-ActividadRESUMEN
Monodispersed N- and C-protected linear homo-oligomethionines (n = 2- -7) are studied by measurements of circular dichroism in the vacuum ultraviolet region. In the solid state higher members of the series take up a beta-conformation in which both parallel and antiparallel chain arrangements are present. The strong beta-forming tendency of the methionine residue is demonstrated.
Asunto(s)
Metionina , Oligopéptidos , Fenómenos Químicos , Química , Dicroismo Circular , Membranas Artificiales , Conformación ProteicaRESUMEN
The three main components YI, YII, and Z of clupeine, a protamine from herring, have been purified and characterized. The conformational preferences of clupeines have been examined as a funciton of pH, temperature, added salts, and presence of structure-disrupting agents and helix-supporting solvents using circular dichroism. It was found that these small basic proteins assume predominantly an unordered conformation in aqueous solution. Addition of counter ions, in particular perchlorate, and 2-chloroethanol induces in various amounts the onset of the right-handed alpha-helical conformation. Urea favors the statistical coil state. It was also demonstrated that in the 0.1--4.0 . 10(-1) M range, in contrast to clupeines YI and Z, the circular dichroic properties of the YII component do not seem to be sensitive to the addition of mono- and diphosphate.
Asunto(s)
Clupeína , Protaminas , Aminoácidos/análisis , Dicroismo Circular , Conformación Proteica , Espectrofotometría UltravioletaRESUMEN
The interaction of the three clupeine fractions, YI, YII, Z, and salmine fraction AI with mononucleotides has been examined by means of 1H nuclear magnetic resonance. The results obtained are interpreted in terms of electrostatic interactions between positive arginine guanidinyl groups and negative nucleotide phosphates. In addition, clupeine fraction YI and salmine fraction AI exhibit with guanine and adenine nucleotides a more specific interaction that leads to the formation of large aggregates in solution. The experimental data presented in this work demonstrate that the strength of interaction between clupeine YI and salmine AI with mononucleotides follows the order: 5'-dTMP approximately equal to 5'-dCMP much less than 5'-dAMP less than 5'-dGMP approximately equal to 5'-GMP.
Asunto(s)
Nucleótidos , Protaminas , Adamantano/análogos & derivados , Secuencia de Aminoácidos , Arginina , Clupeína , Desoxicitidina Monofosfato , Nucleótidos de Desoxiguanina , Espectroscopía de Resonancia Magnética , Salmina , Timidina MonofosfatoRESUMEN
A synthetic, terminally blocked homodecapeptide from the C alpha, alpha-dimethylated glycyl residue alpha-aminoisobutyric acid has been analyzed by single-crystal X-ray diffraction and the structure refined to R = 0.073. The compound crystallizes as a perfect 3(10) helix, stabilized by eight consecutive intramolecular N-H . . . O = C hydrogen bonds. This is the first observation at atomic resolution of a regular polypeptide 3(10) helix as long as three complete turns.
Asunto(s)
Ácidos Aminoisobutíricos , Oligopéptidos , Fenómenos Químicos , Química Física , Enlace de Hidrógeno , Estructura Molecular , Conformación Proteica , Difracción de Rayos XRESUMEN
The highly hydrophobic C60 (buckminsterfullerene) was water solubilized by covalently linking the synthon 1,2-dihydro-1,2-methanofullerene [60]-61-carboxylic acid to the alpha-amino group of the hydrophilic 4-8 sequence of peptide T, known to display potent human monocyte chemotaxis. The resulting compound, characterized by a variety of analytical techniques, including a UV spectrum in aqueous solution, exhibits remarkable chemotactic potency, comparable to that of the parent pentapeptide. Furthermore, this fullerene-peptide conjugate inhibits, albeit weakly, HIV-1 protease.
Asunto(s)
Péptido T/análogos & derivados , Secuencia de Aminoácidos , Quimiotaxis de Leucocito/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/inmunología , Péptido T/farmacologíaRESUMEN
A series of analogues of desArg(9)-Lys-bradykinin (BK), Lys-Arg-X-Ac(n)c-X-Ser-Pro-Phe, in which the spacer X-Ac(n)c-X replaces the central tetrapeptide Pro-Pro-Gly-Phe of BK, have been synthesized and functionally characterized at the B1 receptor. The 1-aminocycloalkane-1-carboxylic acids (Ac(6)c, Ac(7)c, Ac(8)c, Ac(9)c, Ac(12)c) were incorporated to impart conformational constraint and probe the importance of the hydrophobicity of the residue in the central position. The linker is varied in length (X = Gly, betaAla, gammaAbu) to examine the optimal distance between the biologically important residues at the N- and C-termini. The biological assays indicate that the optimal length is obtained with X = Gly, with reduced activities for the longer linkers. Although the size of the central cyclic amino acid does not significantly alter the biological activity, the hydrophobic residue Ac(n)c which may tether the peptide in the membrane environment is required (Lys-Arg-Gly-Gly-Gly-Ser-Pro-Phe is inactive). Two of the analogues, Lys-Arg-Gly-Ac(7)c-Gly-Ser-Pro-Phe and Lys-Arg-gammaAbu-Ac(7)c-gammaAbu-Ser-Pro-Phe, have been structurally characterized in the presence of a zwitterionic lipid environment by high-resolution NMR. Both compounds have similar structural features, differing greatest in the distance between the termini (9 and 15 A for the Gly- and gammaAbu-containing analogues, respectively). The correlation of the smaller distance with activity at the B1 receptor is in complete accord with the results from our previous examination of Lys-Arg-NH-(CH(2))(11)-CO-Ser-Pro-Phe. With the results from this series of compounds we are beginning to define some of the molecular descriptors important for activity at the B1 BK receptor.
Asunto(s)
Bradiquinina/análogos & derivados , Bradiquinina/síntesis química , Oligopéptidos/química , Fragmentos de Péptidos/química , Receptores de Bradiquinina/agonistas , Animales , Bradiquinina/química , Bradiquinina/farmacología , Íleon/efectos de los fármacos , Íleon/fisiología , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Ratas , Receptor de Bradiquinina B1 , Relación Estructura-ActividadRESUMEN
Four previously reported kinin receptor peptide antagonists, including the B1 receptor-selective peptides desArg10-HOE 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-OH) and B-9858 (H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-OH), have been modified by replacement of the central tetrapeptide Pro-Hyp-Gly-Xaa with linear alkyl spacers of variable length. The analogue of desArg10-HOE 140 containing the 11-aminoundecanoic acid as spacer, MEN 11575 [H-D-Arg-Arg-NH-(CH2)10-CO-Ser-D-Tic-Oic-OH], was found to be slightly more potent than the unmodified peptide (pA2 = 7.1) as a kinin B1 receptor antagonist in the rat ileum longitudinal smooth muscle assay. Moreover, MEN 11575 is devoid of residual agonist activity at the kinin B1 receptor (rat ileum) and antagonist activity at the kinin B2 receptor (guinea pig ileum longitudinal smooth muscle). Both these activities are displayed by the parent peptide desArg10-HOE 140. Therefore, despite its greatly simplified chemical structure, MEN 11575 shows an improved pharmacological profile in terms of both potency and selectivity, and it represents a good template for the development of new peptidomimetic kinin B1 receptor antagonists. We also report an attempt to investigate the conformational role of the flexible, linear spacer of MEN 11575 and to design more constrained analogues, possibly locked in the bioactive conformation, using semirigid spacers based on Calpha-tetrasubstituted alpha-amino acids of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc).
Asunto(s)
Oligopéptidos/farmacología , Péptidos/farmacología , Receptores de Neuroquinina-3/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Bradiquinina/análogos & derivados , Bradiquinina/química , Bradiquinina/farmacología , Cobayas , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Músculo Liso/efectos de los fármacos , Oligopéptidos/química , Péptidos/química , RatasRESUMEN
The two diastereomeric sulphoxides and the sulphone derived from the formyl-methionyl tripeptide chemoattractant CHO-L-Met-L-Phe-OMe have been synthesized and fully characterized. The diastereomeric sulphoxide tripeptides have been separated at the stage of their N-tert-butyloxycarbonyl synthetic precursors. All of the oxidized sulphur derivatives induce secretion of granule enzymes with ED50s from 1-2 x 10(-9) M with no significant differences in activity among them. They are also active to the same relative extent in inducing chemotaxis. In parallel, a solution conformational analysis has been performed in solvents of widely different polarities and capabilities of H-bond formation using circular dichroism, infrared absorption and 1H nuclear magnetic resonance. No significant propensity for formation of intramolecularly (C = O...H-N) H-bonded folded forms has been detected in any of the four tripeptides. Intermolecular S = O...H-N interactions are postulated to tentatively explain the 1H nuclear magnetic resonance behavior of the Met and, particularly, Leu NH resonances of the two sulphoxide tripeptides in CDCl3 solution. The biological and conformational data agree with the recently proposed model of the chemotactic peptide receptor of rabbit neurotrophils, which involves the extended backbone of the integrity of the Met side-chain sulphide sulphur atom as a corollary point of ligand interaction.
Asunto(s)
N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/síntesis química , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Conformación Proteica , Estereoisomerismo , Relación Estructura-Actividad , Sulfonas , SulfóxidosRESUMEN
The infrared absorption and 1H nuclear magnetic resonance analyses of chloroform solutions of the terminally-blocked segment corresponding to the 2-9 sequence of emerimicins III and IV, -(Aib)3-L-Val-Gly-L-Leu-(Aib)2-, are consistent with the presence of a 3(10)-helical structure of high thermal stability. The crystal structure of the octapeptide, obtained by X-ray diffraction indicates the formation of a right-handed 3(10)-helix, stabilized by six consecutive intramolecular N-H....O:C H-bonds, slightly distorted at the level of the L-Leu residue.
Asunto(s)
Antibacterianos , Péptidos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Estructura Molecular , Oligopéptidos , Peptaiboles , Conformación ProteicaRESUMEN
The crystal-state preferred conformation of the terminally blocked hepta- and octapeptides with the general formula -(Aib)n L-Leu-(Aib)2- (n = 4 and 5, respectively), determined by X-ray diffraction, was found to be a right-handed 3(10)-helix stabilized by five and six consecutive intramolecular NH...O = C H-bonds of the C(10)-III type, respectively. The octapeptide structure represents the first observation at atomic resolution of a regular, chiral 3(10)-helix larger than two complete turns. In both cases the right handed screw sense of the helix is dictated by the presence of the single, internal L-residue. This study confirms the propensity of short peptides rich in Aib, the prototype of the amino acid residues dialkylated at the alpha carbon, to adopt a 3(10)-helical structure and is expected to help our understanding of the conformational preferences of the membrane-active, channel-forming, ion-transporting peptaibol antibiotics.
Asunto(s)
Péptidos , Butiratos/síntesis química , Enlace de Hidrógeno , Leucina/análogos & derivados , Leucina/síntesis química , Modelos Moleculares , Péptidos/síntesis química , Conformación Proteica , Difracción de Rayos XRESUMEN
To assess the minimal peptide length required for the stabilization of the alpha-helix relative to the 3(10)-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula-(Aib-L-Ala)n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3(10)-helical with four 1----4 intramolecular N-H . . . O = C H-bonds. On the other hand, the octapeptide molecules are essentially alpha-helical with four 1----5 H-bonds; however, the helix is elongated at the N-terminus, with two 1----4 H-bonds, giving these molecules a mixed alpha/3(10)-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3(10)----alpha-helix conversion in the crystal state induced by peptide backbone lengthening only.