RESUMEN
Holographic three-dimensional (3D) displays provide realistic images without the need for special eyewear, making them valuable tools for applications that require situational awareness, such as medical, industrial and military imaging. Currently commercially available holographic 3D displays use photopolymers that lack image-updating capability, resulting in restricted use and high cost. Photorefractive polymers are dynamic holographic recording materials that allow updating of images and have a wide range of applications, including optical correlation, imaging through scattering media and optical communication. To be suitable for 3D displays, photorefractive polymers need to have nearly 100% diffraction efficiency, fast writing time, hours of image persistence, rapid erasure, and large area-a combination of properties that has not been shown before. Here, we report an updatable holographic 3D display based on photorefractive polymers with such properties, capable of recording and displaying new images every few minutes. This is the largest photorefractive 3D display to date (4 x 4 inches in size); it can be recorded within a few minutes, viewed for several hours without the need for refreshing, and can be completely erased and updated with new images when desired.
RESUMEN
Sleep misperception is often observed in insomnia individuals (INS). The extent of misperception varies between different types of INS. The following paper comprised sections which will be aimed at studying the sleep EEG and compares it to subjective reports of sleep in individuals suffering from either psychophysiological insomnia or paradoxical insomnia and good sleeper controls. The EEG can be studied without any intervention (thus using the raw data) via either PSG or fine quantitative EEG analyses (power spectral analysis [PSA]), identifying EEG patterns as in the case of cyclic alternating patterns (CAPs) or by decorticating the EEG while scoring the different transient or phasic events (K-Complexes or sleep spindles). One can also act on the on-going EEG by delivering stimuli so to study their impact on cortical measures as in the case of event-related potential studies (ERPs). From the paucity of studies available using these different techniques, a general conclusion can be reached: sleep misperception is not an easy phenomenon to quantify and its clinical value is not well recognized. Still, while none of the techniques or EEG measures defined in the paper is available and/or recommended to diagnose insomnia, ERPs might be the most indicated technique to study hyperarousal and sleep quality in different types of INS. More research shall also be dedicated to EEG patterns and transient phasic events as these EEG scoring techniques can offer a unique insight of sleep misperception.
Asunto(s)
Percepción/fisiología , Trastornos del Inicio y del Mantenimiento del Sueño/psicología , Sueño , Ondas Encefálicas/fisiología , Diagnóstico Diferencial , Electroencefalografía/métodos , Electroencefalografía/estadística & datos numéricos , Potenciales Evocados , Análisis de Fourier , Humanos , Registros Médicos , Polisomnografía , Trastornos Psicofisiológicos/diagnóstico , Trastornos Psicofisiológicos/fisiopatología , Trastornos Psicofisiológicos/psicología , Trastornos del Inicio y del Mantenimiento del Sueño/clasificación , Trastornos del Inicio y del Mantenimiento del Sueño/diagnóstico , Trastornos del Inicio y del Mantenimiento del Sueño/fisiopatología , Fases del Sueño/fisiología , Vigilia/fisiologíaRESUMEN
The technology of glycopeptide synthesis has recently developed into a fully mature science capable of creating diverse glycopeptides of biological interest, even in combinatorial displays. This has allowed biochemists to investigate substrate specificity in the biosynthetic processing and immunology of various protein glycoforms. The construction of all the mucin core structures and a variety of cancer-related glycopeptides has facilitated detailed analysis of the interaction between MHC-bound glycopeptides and T cell receptors. Novel dendritic neoglycopeptide ligands have been shown to demonstrate high affinity for carbohydrate receptors and these interactions are highly dendrimer specific. Large complex N-linked oligosaccharides have been introduced into glycopeptides using synthetic or chemoenzymatic procedures, both methods affording pure glycopeptides corresponding to a single glycoform in preparative quantities. The improved availability of glycosyl transferases has led to increased use of chemoenzymatic synthesis. Chemical ligation has been introduced as a method of attaching glycans to peptide templates. Combinatorial synthesis and the analysis of resin-bound glycopeptide libraries have been successfully carried out by applying the ladder synthesis principle. Direct quantitative glycosylation of peptide templates on solid phase has paved the way for the synthesis of templated glycopeptide mixtures as libraries of libraries.
Asunto(s)
Glicopéptidos/química , Glicopéptidos/síntesis química , Secuencia de Aminoácidos , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicopéptidos/metabolismo , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Complejo Mayor de Histocompatibilidad , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/síntesis química , Oligosacáridos/química , Biblioteca de Péptidos , Receptores de Antígenos de Linfocitos T/metabolismo , Sialoglicoproteínas/químicaRESUMEN
Retinol circulates in the plasma bound to retinol-binding protein (RBP), but the mechanism by which retinol is transferred from RBP to target cells is not known. To study retinol delivery, human keratinocytes (HKc) were incubated with [3H]retinol added directly to the culture medium or bound to RBP and the uptake of [3H]retinol was determined at various times. During the first hour of incubation, the rate of [3H]retinol accumulation by HKc was about 40 times greater when the vitamin was added directly to the media rather than bound to RBP. Although maximal uptake of [3H]retinol added directly to the culture medium occurred at 3 h, the uptake of [3H]retinol from RBP was linear with time for at least 72 h. By 57 h, cell-associated [3H]retinol was the same whether it was added directly to the culture medium or bound to RBP. Excess unlabeled retinol or pretreatment of HKc with retinol had no effect on the uptake of [3H]retinol added directly to the culture medium or bound to RBP. Apo- but not holo-RBP was capable of competing with HKc for the uptake of [3H]retinol from RBP. No specific or saturable binding of 125I-labeled RBP to HKc cultured in the absence or the presence of retinol was found. The dose response of retinol inhibition of cholesterol sulfate synthesis and phorbol ester-induced ornithine decarboxylase activity or retinol modulation of keratin expression was the same whether the retinol was delivered to HKc bound to RBP or added directly to the medium. Our data support a mechanism for retinol delivery from RBP to HKc that does not involve cell-surface RBP receptors but instead suggest that the vitamin is first slowly released from RBP and then becomes cell-associated from the aqueous phase. This mechanism is consistent with the finding that HKc respond identically to retinol whether or not it is delivered to them bound to RBP.
Asunto(s)
Queratinocitos/metabolismo , Proteínas de Unión al Retinol/metabolismo , Vitamina A/farmacocinética , Células Cultivadas , Medios de Cultivo , Humanos , Queratinocitos/citología , Proteínas Plasmáticas de Unión al Retinol , Tritio , Vitamina A/farmacologíaRESUMEN
We have identified peptides that are relatively resistant to hydrolysis by a recombinant cysteine proteinase, CPB2.8DeltaCTE, of Leishmania mexicana, and yet exhibit inhibition constant (K(i)) values in the nanomolar range. Common to these peptides is a basic-hydrophobic-hydrophobic motif in the P3-P1 sites, which is also present in the pro-region of the enzyme. A nine-amino acid stretch, FAARYLNGA, which has good homology to the pro-region of mammalian cathepsin L was identified as the part of the pro-region most likely to interact with the active site of the parasite enzyme. This peptide is not hydrolyzed by CPB2.8DeltaCTE and inhibited it with a K(i) of 4 microM. Extension of this sequence at both the N- and C-termini and the introduction of ortho-aminobenzoic acid at the N-terminal site reduced the K(i) value to 30 nM. The best substrate for CPB2.8DeltaCTE was also well hydrolyzed by cathepsin L, however the best inhibitor of the parasite enzyme inhibit poorly cathepsin L, with K(i) value two order of magnitude higher than against the parasite enzyme. These promising data provide insights into the peculiar specificity of the parasite enzyme and will aid the design of antiparasitic drugs directed against the leishmanial enzyme.
Asunto(s)
Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/farmacología , Leishmania mexicana/enzimología , Oligopéptidos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Catepsina L , Catepsinas/química , Cisteína Endopeptidasas/genética , Inhibidores de Cisteína Proteinasa/química , Humanos , Cinética , Mamíferos , Datos de Secuencia Molecular , Oligopéptidos/química , Fragmentos de Péptidos/química , Proteínas Protozoarias/química , Proteínas Recombinantes/antagonistas & inhibidores , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The primary S(1) subsite specificity of a recombinant cysteine proteinase, CPB2.8 Delta CTE, of Leishmania mexicana was investigated in a systematic way using a series of peptides derived from Abz-KLRFSKQ-EDDnp in which Arg was substituted by all natural amino acids (where Abz is ortho-amino-benzoyl and EDDnp is N-[2,4-dinitrophenyl]-ethylenediamine). The peptides from this series with charged side chain amino acids, Cys, Cys(SBzl), and Thr(OBzl) were well hydrolysed. All other substitutions resulted in peptides that were resistant or hydrolysed very slowly and inhibited the enzyme with K(i) values in the range of 9--400 nM. Looking for natural substrates for CPB2.8, we observed that the recombinant enzyme failed to release kinin from human kininogen, an activity earlier observed with cruzipain from Trypanosoma cruzi (Del Nery et al., J. Biol. Chem. 272 (1997) 25713.). This lack of activity seems to be a result of the resistance to hydrolysis of the sequence at the N-terminal site of bradykinin in the human kininogen. The preferences for the S(3), S(2) and S(1)'-S(3)' for some amino acids were also examined using substrates derived from Abz-KLRFSKQ-EDDnp with variations at Lys, Leu, Phe, Ser and Lys, using the amino acids Ala, Phe, Leu, His or Pro. Peptides with Phe at P(1)' presented the highest affinity to the leishmanial enzyme. For comparison, some of the obtained peptides were also assayed with recombinant human cathepsin L and cruzain. The best substrates for CPB2.8 Delta CTE were also well hydrolysed by cathepsin L, however, the best inhibitors of the parasite enzyme have low affinity to cathepsin L. These promising data provide leads for the design of anti-parasitic drugs directed against the leishmanial enzyme.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Leishmania mexicana/enzimología , Secuencia de Aminoácidos , Animales , Catepsina L , Catepsinas/metabolismo , Cisteína Endopeptidasas/genética , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Quininógenos/metabolismo , Leishmania mexicana/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
In response to body demands, retinol (ROL) is secreted from the liver into the circulation bound to serum retinol-binding protein (RBP). The mechanism by which ROL is transferred from RBP to target cells remains controversial. To study ROL delivery, we have used a model system of cultured human foreskin keratinocytes (HKc) to compare the uptake, metabolism and biologic effects of ROL added either directly to the medium or bound to RBP. [3H]ROL added directly to the medium was rapidly taken up by HKc, and maximal accumulation of [3H]ROL occurred by 3 h. In contrast, [3H]ROL delivered bound to RBP was taken up very slowly but at a linear rate for at least 72 h. Several experimental approaches indicated that ROL uptake from RBP was not mediated by a cell surface receptor for RBP. The rate and extent of [3H]ROL metabolism to retinyl esters was the same whether the ROL was added directly to the medium or bound to RBP. In addition, several biologic responses by HKc to ROL showed the same dose response curves whether the ROL was added directly to the medium or bound to RBP. Overall, the results support a model of ROL uptake from RBP in which the vitamin is first slowly released from RBP into the aqueous phase and then becomes cell associated. In this manner, the cells are provided with a slow but constant supply of ROL and are protected from the rapid and potentially toxic accumulation of ROL that occurs in the absence of RBP.
Asunto(s)
Queratinocitos/metabolismo , Proteínas de Unión al Retinol/metabolismo , Vitamina A/farmacocinética , Células Cultivadas , Ésteres del Colesterol/análisis , Medio de Cultivo Libre de Suero , Humanos , Recién Nacido , Inhibidores de la Ornitina Descarboxilasa , Tritio , Vitamina A/farmacologíaRESUMEN
Ultrasonic velocity measurement in bone can be used as a clinical tool that will accurately and objectively monitor the progress of fracture healing. This technique may provide a rational basis for evaluating the effectiveness of various modalities in clinical treatment. The eventual goal of this technique is to determine the status of healing fractures, prescribe appropriate treatment, give the rationale for the timing and form of a rehabilitative program that would accelerate the patient's return to productivity. An instrument has been developed to measure ultrasonic velocity in the long bones of the extremities. Serial measurements of tibial fractures and the measurement of a large group of normals shows no statistically significant differences in ultrasonic measurements have been found on the basis of race, sex, or pregnancy among normals. But a numerically small but statistically significant variation with age was found. While the number of fracture cases observed was small, the evidence is clear that ultrasonic measurement in fractured tibia may give a more reliable indication of the general pattern of healing response than the clinical and roentgenographic examination.
Asunto(s)
Fracturas Óseas/terapia , Ultrasonografía , Cicatrización de Heridas , Adulto , Factores de Edad , Anciano , Población Negra , Femenino , Humanos , Masculino , Persona de Mediana Edad , Embarazo , Factores Sexuales , Factores de Tiempo , Ultrasonido/instrumentación , Ultrasonido/métodos , Población BlancaRESUMEN
A study of the binding of the Shiga-like toxin 1 (SLT-1) to the P(k) trisaccharide [methyl 4-O-(4-O-alpha-D-galactopyranosyl)-4-O-beta-D- glucopyranoside] and its constituent dissacharides was carried out. The trisaccharide represents the carbohydrate recognition domain of the neutral glycolipid receptor of the SLT-1, globotriosylceramide (GbOse3). The binding constant for soluble trisaccharide to the soluble pentameric B-subunit is weak, with a K(a) of (0.5-1) x 10(3) M-1 for B-subunit monomer. Scatchard analysis of the binding data indicates five identical non-interacting carbohydrate binding sites per B-subunit pentamer and no cooperativity in binding. Despite weak binding (delta G = -3.6 kcal mol-1), the enthalpy of binding (delta H = -12 kcal mol-1) and the change in molar heat capacity accompanying binding (delta C(p) = -40 eu) are comparable to other protein-carbohydrate interactions. Dynamic light scattering studies indicate that carbohydrate binding induces protein aggregation. At carbohydrate concentrations where > 90% of B-subunit monomers are bound, the far-UV CD spectra were unchanged, whereas a change in the near-UV CD, maximal near 270 nm, titrated to give an apparent binding constant in good agreement with that obtained by isothermal microcalorimetry. Steady-state fluorescence and fluorescence lifetime measurements indicated that the environments of the central tryptophans are perturbed during saccharide binding, and the changes correlate with the extent of protein aggregation. On the basis of the thermodynamics of binding, optical spectroscopy, and binding-induced aggregation, we propose a model of SLT-1-membrane interaction that relies on protein-carbohydrate interaction for specificity and protein-lipid interaction for tight binding.
Asunto(s)
Toxinas Bacterianas/metabolismo , Receptores de Superficie Celular/metabolismo , Trihexosilceramidas/metabolismo , Toxinas Bacterianas/química , Calorimetría , Secuencia de Carbohidratos , Dicroismo Circular , Disacáridos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Agregación de Receptores , Receptores de Superficie Celular/química , Proteínas Recombinantes , Toxina Shiga I , Espectrometría de Fluorescencia , Termodinámica , Trisacáridos/metabolismo , Triptófano/químicaRESUMEN
The products of a wide variety of organic reactions were rapidly identified by their masses through a combination of thin layer chromatography (TLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Crude mixtures of peptides and glycopeptides, complex carbohydrate reactions, and a classical organic reaction were analysed using the following standard protocol. The components of the reaction mixtures were first separated on the TLC plate, scraped off, extracted and analysed by MALDI-TOFMS. The technique used is easy and applicable to most organic reactions, becoming a very powerful technique in reaction optimization once composition and identity of TLC spots have been established by MS. Moreover, the TLC/MALDI-TOFMS method was used to identify low molecular weight compounds with masses within the matrix region (100-500 u), a goal which is normally difficult to achieve. We successfully detected low molecular weight compounds by suppression of the matrix peaks using a relatively low matrix:analyte ratio (15:1 or lower). Doping both matrix and analyte solutions with [Cs]+ ions resulted in suppression of both [Na]+ and [K]+ peaks, thus enhancing the spectral signals and making identification of low molecular weight compounds more facile.
Asunto(s)
Química Orgánica/instrumentación , Carbohidratos/química , Cromatografía en Capa Delgada , Metales/química , Conformación Molecular , Peso Molecular , Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S3 subsite and hydrophobic residues in S2; the most prevalent residue not being Phe but Val. The S1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S1'-S4' subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the kcat/KM's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO2)PMPPLCTSMK(Abz) (kcat/KM = 2109 (mM s)-1), Y(NO2)PYAVQSPQK(Abz) (kcat/KM = 1524 (mM s)-1), and Y(NO2)PVLRQQRSK(Abz) (kcat/KM = 1450 (mM s)-1). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Oligopéptidos/síntesis química , Papaína/química , Papaína/metabolismo , Biblioteca de Péptidos , Péptidos/síntesis química , Especificidad por Sustrato , Secuencia de Aminoácidos , Sitios de Unión , Técnicas Químicas Combinatorias/métodos , Cinética , Modelos Moleculares , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
A combinatorial split-and-mix library of peptide isosters based on a Diels-Alder reaction was synthesized as a "one-bead-two-compounds" library and encoded by ladder synthesis for facile analysis by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In the "one-bead-two-compounds" library approach, each bead contains a library member as a putative protease inhibitor along with a fluorescence-quenched substrate for the protease. When the library was screened with CPB2.8 DeltaCTE, a recombinant cysteine protease from L. mexicana, several beads containing compounds with inhibitory activity could be selected from the library and analyzed by MALDI-TOF MS for structure elucidation. Two types of inhibitors were revealed. One novel class of inhibitors had the bicyclic Diels-Alder product isosteric element incorporated internally in a peptide, while the other type was an N-terminal alpha,beta-unsaturated ketone Michael acceptor used as starting material for the Diels-Alder reaction. Selected hit sequences and constructed consensus sequences based on the observed frequencies of amino acids in different subsites were resynthesized and assayed in solution for inhibitor activity and were shown to have IC(50) values in the high nanomolar to low micromolar range.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/síntesis química , Leishmania mexicana/enzimología , Péptidos/síntesis química , Animales , Técnicas Químicas Combinatorias , Cisteína Endopeptidasas/química , Isomerismo , Cinética , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The substrate specificity of CPB2.8DeltaCTE, a recombinant cysteine protease from Leishmania mexicana, was mapped by screening a fluorescence-quenched combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S(3) subsite and for hydrophobic residues, both aliphatic and aromatic, in S(2). The S(1) subsite exhibited a specificity for the basic residues Arg and Lys. Generally, the specificity of the primed subsites was less strict compared with the non-primed side which showed preference for Arg, Lys and Ala in S'(1), Arg, Pro and Gly in S'(2) and Lys, Arg and Ser in S'(4). By contrast, a strict preference for the basic residues Arg and Lys was found for S'(3). Overall, there was a trend for basic residues in alternating subsites and smaller residues in the primed sites compared with the non-primed sites. In addition, there were strict requirements for the amino acids in subsites S(3)--S(1). Fluorescence-quenched peptides from the library with the highest on-resin cleavage were resynthesised and their kinetics of hydrolysis by CPB2.8DeltaCTE assessed in solution phase assays. Several good substrates containing the quintessential dipeptide particular to cathepsin-L-like enzymes, -F-R/K-, in P(2) and P(1) were identified (e.g. Y(NO(2))-EKFR down arrow RGK-K(Abz)G, Abz=2-aminobenzoyl; k(cat)K(m)(-1)=4298 mM(-1)s(-1)). However, novel substrates containing the dipeptide -L/I-Q- in P(2) and P(1) were also well hydrolysed (e.g. Y(NO(2))-YLQ down arrow GIQK-K(Abz)G; k(cat)K(m)(-1)=2583 mM(-1)s(-1)). The effect of utilising different fluorescent donor--quencher pairs on the value of k(cat)K(m)(-1) was examined. Generally, the use of the Abz/Q-EDDnp donor--quencher pair (EDDnp=N-(2,4-dinitrophenyl)ethylenediamine) instead of K(Abz)/Y(NO(2)) resulted in higher k(cat)K(m)(-1) values for analogous substrates.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Colorantes Fluorescentes/metabolismo , Leishmania mexicana/enzimología , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismo , Aminoácidos/química , Animales , Sitios de Unión , Técnicas Químicas Combinatorias/métodos , Cinética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Especificidad por SustratoRESUMEN
The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb3 and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10(5) compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galalpha1-4Galbeta1-4Glc trisaccharide portion of Gb3. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.