Molecular analysis of $\beta$1,4-galactosyl-transferase localization to the cell surface and participation in cell adhesion

$\beta$1,4-Galactosyltransferase (GalTase) is unusual among the glycosyltransferases in that it is found in two subcellular compartments where it performs different functions. In the trans-Golgi complex, GalTase participates in oligosaccharide biosynthesis as do other glycosyltransferases. GalTase is also found on the cell surface, where it associates with the cytoskeleton and functions as a receptor for extracellular oligosaccharide ligands. Although we know much regarding GalTase function on the cell surface, little is known about the mechanisms underlying its transport to the plasma membrane. Cloning of the GalTase gene revealed that there are two GalTase proteins (i.e., long and short) with different size cytoplasmic tails. This raises the possibility that differences in the cytoplasmic domain of GalTase may influence its subcellular distribution. The object of this study was to examine this hypothesis directly through the use of molecular, immunological, and biochemical approaches.^ To examine whether the two GalTase proteins are targeted to different subcellular compartments, F9 embryonal carcinoma cells were transfected with either long or short GalTase cDNAs and intracellular and cell surface enzyme levels measured. Cell surface GalTase activity was enriched in cells overexpressing the long, but not the form of short GalTase. Furthermore, a dominant negative mutation in cell surface GalTase was created by transfecting cells with GalTase cDNAs encoding a truncated version of long GalTase devoid of the extracellular catalytic domain. Overexpressing the complete cytoplasmic and transmembrane domains of long GalTase led to a loss of GalTase-dependent cellular adhesion by specifically displacing surface GalTase from its cytoskeletal associations. In contrast, overexpressing the analogous truncated protein of short GalTase had no effect on cell adhesion. Finally, chloramphenicol acetyltransferase (CAT) reporter proteins were used to determine directly whether the cytoplasmic domains of long and short GalTase were responsible for differential subcellular distribution. The cytoplasmic and transmembrane domains of long GalTase led to CAT expression on the ceil surface and its association with the detergent-insoluble cytoskeleton; the analogous fusion protein containing short GalTase was restricted to the Golgi compartment. These results suggest that the cytoplasmic domain unique to long GalTase is responsible for targeting a portion of this protein to the cell surface and associating it with the cytoskeleton, enabling it to function as a cell adhesion molecule. ^

Expression and hormonal regulation of Muc-1 at the apical surface of mouse uterine epithelial cells and its role in modulating embryo implantation

Previous studies from our lab have established that large molecular weight mucin glycoproteins are major apically-disposed components of mouse uterine epithelial cells in vitro (Valdizan et al., (1992) J. Cell. Physiol. 151:451-465). The present studies demonstrate that Muc-1 represents one of the apically-disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and mRNA expression are regulated in the peri-implantation stage mouse uterus by ovarian steroids. Muc-1 expression is high in the proestrous and estrous stages, and decreases during diestrous. Both Muc-1 protein and mRNA levels decline to barely detectable levels by day 4 of pregnancy, i.e., prior to the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by administration of exogenous progesterone. Initiation of implantation was triggered by coinjection of progesterone maintained mice with a nidatory dose of 17$\beta$-estradiol. Muc-1 levels in the uterine epithelia of progesterone maintained mice declined to similar low levels as observed on day 4 of normal pregnancy. Coinjection of estradiol did not alter Muc-1 expression suggesting that down-regulation of Muc-1 is a progesterone dominated event. This was confirmed in ovariectomized, non-pregnant mice which displayed stimulation of Muc-1 expression following 6 hr of estradiol injection. Estradiol stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. While progesterone alone had no effect on Muc-1 expression, it antagonized estradiol action in this regard. Injection of pregnant mice with the antiprogestin, RU 486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is progesterone receptor-mediated. Muc-1 appears to function as an anti-adhesive molecule at the apical cell surface of mouse uterine epithelial cells. Treatment of polarized cultures of mouse uterine epithelial cells with O-sialoglycoprotein endopeptidase reduced mucin expression in vitro, by about 50%, and converted polarized uterine epithelia to a functionally receptive state. Similarly, ablation of Muc-1 in Muc-1 null mice resulted in polarized uterine epithelia that were functionally receptive as compared to their wild-type counterparts in vitro. Collectively, these data indicate that Muc-1 and other mucins function as anti-adhesive molecules and that reduction or removal of these molecules is a prerequisite for the generation of a receptive uterine state. ^

Promising anchoring groups for single-molecule conductance measurements

The understanding of the charge transport through single molecule junctions is a prerequisite for the design and building of electronic circuits based on single molecule junctions. However, reliable and robust formation of such junctions is a challenging task to achieve. In this topical review, we present a systematic investigation of the anchoring group effect on single molecule junction conductance by employing two complementary techniques, namely scanning tunneling microscopy break junction (STM-BJ) and mechanically controllable break junction (MCBJ) techniques, based on the studies published in the literature and important results from our own work. We compared conductance studies for conventional anchoring groups described earlier with the molecular junctions formed through π-interactions with the electrode surface (Au, Pt, Ag) and we also summarized recent developments in the formation of highly conducting covalent Au–C σ-bonds using oligophenyleneethynylene (OPE) and an alkane molecular backbone. Specifically, we focus on the electron transport properties of diaryloligoyne, oligophenyleneethynylene (OPE) and/or alkane molecular junctions composed of several traditional anchoring groups, (dihydrobenzo[b]thiophene (BT), 5-benzothienyl analogue (BTh), thiol (SH), pyridyl (PY), amine (NH2), cyano (CN), methyl sulphide (SMe), nitro (NO2)) and other anchoring groups at the solid/liquid interface. The qualitative and quantitative comparison of the results obtained with different anchoring groups reveals structural and mechanistic details of the different types of single molecular junctions. The results reported in this prospective may serve as a guideline for the design and synthesis of molecular systems to be used in molecule-based electronic devices.

Effect of Nontronite Smectite Clay on the Chemical Evolution of Several Organic Molecules under Simulated Martian Surface Ultraviolet Radiation Conditions

Most of the phyllosilicates detected at the surface of Mars today are probably remnants of ancient environments that sustained long-term bodies of liquid water at the surface or subsurface and were possibly favorable for the emergence of life. Consequently, phyllosilicates have become the main mineral target in the search for organics on Mars. But are phyllosilicates efficient at preserving organic molecules under current environmental conditions at the surface of Mars? We monitored the qualitative and quantitative evolutions of glycine, urea, and adenine in interaction with the Fe3+-smectite clay nontronite, one of the most abundant phyllosilicates present at the surface of Mars, under simulated martian surface ultraviolet light (190-400 nm), mean temperature (218 +/- 2 K), and pressure (6 +/- 1 mbar) in a laboratory simulation setup. We tested organic-rich samples that were representative of the evaporation of a small, warm pond of liquid water containing a high concentration of organics. For each molecule, we observed how the nontronite influences its quantum efficiency of photodecomposition and the nature of its solid evolution products. The results reveal a pronounced photoprotective effect of nontronite on the evolution of glycine and adenine; their efficiencies of photodecomposition were reduced by a factor of 5 when mixed at a concentration of 2.6x10(-2) mol of molecules per gram of nontronite. Moreover, when the amount of nontronite in the sample of glycine was increased by a factor of 2, the gain of photoprotection was multiplied by a factor of 5. This indicates that the photoprotection provided by the nontronite is not a purely mechanical shielding effect but is also due to stabilizing interactions. No new evolution product was firmly identified, but the results obtained with urea suggest a particular reactivity in the presence of nontronite, leading to an increase of its dissociation rate. Key Words: Martian surface-Organic chemistry-Photochemistry-Astrochemistry-Nontronite-Phyllosilicates. Astrobiology 15, 221-237.

Surface InGaAs nanostructures for sensing applications

El desarrollo de sensores está ganando cada vez mayor importancia debido a la concienciación ciudadana sobre el medio ambiente haciendo que su desarrollo sea muy elevado en todas las disciplinas, entre las que cabe destacar, la medicina, la biología y la química. A pesar de la existencia de estos dispositivos, este área está aún por mejorar, ya que muchos de los materiales propuestos hasta el momento e incluso los ya comercializados muestran importantes carencias de funcionamiento, eficiencia e integrabilidad entre otros. Para la mejora de estos dispositivos, se han propuesto diversas aproximaciones basadas en nanosistemas. Quizá, uno de las más prometedoras son las nanoestructuras de punto cuántico, y en particular los semiconductores III-V basados en la consolidada tecnología de los arseniuros, las cuáles ofrecen excelentes propiedades para su uso como sensores. Además, estudios recientes demuestran su gran carácter sensitivo al medio ambiente, la posibilidad de funcionalizar la superficie para la fabricación de sensores interdisciplinares y posibilididad de mejorar notablemente su eficiencia. A lo largo de esta tesis, nos centramos en la investigación de SQD de In0.5Ga0.5As sobre substratos de GaAs(001) para el desarrollo de sensores de humedad. La tesis abarca desde el diseño, crecimiento y caracterización de las muestras hasta la el posterior procesado y caracterización de los dispositivos finales. La optimización de los parámetros de crecimiento es fundamental para conseguir una nanoestructura con las propiedades operacionales idóneas para un fin determinado. Como es bien sabido en la literatura, los parámetros de crecimiento (temperatura de crecimiento, relación de flujos del elemento del grupo V y del grupo I II (V/III), velocidad de crecimiento y tratamiento térmico después de la formación de la capa activa) afectan directamente a las propiedades estructurales, y por tanto, operacionales de los puntos cuánticos (QD). En esta tesis, se realiza un estudio de las condiciones de crecimiento para el uso de In0.5Ga0.5As SQDs como sensores. Para los parámetros relacionados con la temperatura de crecimiento de los QDs y la relación de flujos V / I I I se utilizan los estudios previamente realizados por el grupo. Mientras que este estudio se centrará en la importancia de la velocidad de crecimiento y en el tratamiento térmico justo después de la nucleación de los QDs. Para ello, se establece la temperatura de creciemiento de los QDs en 430°C y la relación de flujos V/III en 20. Como resultado, los valores más adecuados que se obtienen para la velocidad de crecimiento y el tratamiento térmico posterior a la formación de los puntos son, respectivamente, 0.07ML/s y la realización de una bajada y subida brusca de la temperatura del substrato de 100°C con respecto a la temperatura de crecimiento de los QDs. El crecimiento a una velocidad lo suficientemente alta que permita la migración de los átomos por la superficie, pero a su vez lo suficientemente baja para que se lleve a cabo la nucleación de los QDs; en combinación con el tratamiento brusco de temperatura que hace que se conserve la forma y composición de los QDs, da lugar a unos SQDs con un alto grado de homogeneidad y alta densidad superficial. Además, la caracterización posterior indica que estas nanoestructuras de gran calidad cristalina presentan unas propiedades ópticas excelentes incluso a temperatura ambiente. Una de las características por la cual los SQD de Ino.5Gao.5As se consideran candidatos prometedores para el desarrollo de sensores es el papel decisivo que juega la superficie por el mero hecho de estar en contacto directo con las partículas del ambiente y, por tanto, por ser capaces de interactuar con sus moléculas. Así pues, con el fin de demostrar la idoneidad de este sistema para dicha finalidad, se evalúa el impacto ambiental en las propiedades ópticas y eléctricas de las muestras. En un primer lugar, se analiza el efecto que tiene el medio en las propiedades ópticas. Para dicha evaluación se compara la variación de las propiedades de emisión de una capa de puntos enterrada y una superficial en distintas condiciones externas. El resultado que se obtiene es muy claro, los puntos enterrados no experimentan un cambio óptico apreciable cuando se varían las condiciones del entorno; mientras que, la emisión de los SQDs se modifica significativamente con las condiciones del medio. Por una parte, la intensidad de emisión de los puntos superficiales desaparece en condiciones de vacío y decrece notablemente en atmósferas secas de gases puros (N2, O2). Por otra parte, la fotoluminiscencia se conserva en ambientes húmedos. Adicionalmente, se observa que la anchura a media altura y la longitud de onda de emisión no se ven afectadas por los cambios en el medio, lo que indica, que las propiedades estructurales de los puntos se conservan al variar la atmósfera. Estos resultados apuntan directamente a los procesos que tienen lugar en la superficie entre estados confinados y superficiales como responsables principales de este comportamiento. Así mismo, se ha llevado a cabo un análisis más detallado de la influencia de la calidad y composición de la atmósfera en las propiedades ópticas de los puntos cuánticos superficiales. Para ello, se utilizan distintas sustancias con diferente polaridad, composición atómica y masa molecular. Como resultado se observa que las moléculas de menor polaridad y más pesadas causan una mayor variación en la intensidad de emisión. Además, se demuestra que el oxígeno juega un papel decisivo en las propiedades ópticas. En presencia de moléculas que contienen oxígeno, la intensidad de fotoluminiscencia disminuye menos que en atmósferas constituidas por especies que no contienen oxígeno. Las emisión que se observa respecto a la señal en aire es del 90% y del 77%, respectivamente, en atmósferas con presencia o ausencia de moléculas de oxígeno. El deterioro de la señal de emisión se atribuye a la presencia de defectos, enlaces insaturados y, en general, estados localizados en la superficie. Estos estados actúan como centros de recombinación no radiativa y, consecuentemente, se produce un empeoramiento de las propiedades ópticas de los SQDs. Por tanto, la eliminación o reducción de la densidad de estos estados superficiales haría posible una mejora de la intensidad de emisión. De estos experimentos de fotoluminiscencia, se deduce que las interacciones entre las moléculas presentes en la atmósfera y la superficie de la muestra modifican la superficie. Esta alteración superficial se traduce en un cambio significativo en las propiedades de emisión. Este comportamiento se atribuye a la posible adsorción de moléculas sobre la superficie pasivando los centros no radiativos, y como consecuencia, mejorando las propiedades ópticas. Además, los resultados demuestran que las moléculas que contienen oxígeno con mayor polaridad y más ligeras son adsorbidas con mayor facilidad, lo que hace que la intensidad óptica sufra variaciones despreciables con respecto a la emisión en aire. Con el fin de desarrollar sensores, las muestras se procesan y los dispositivos se caracterizan eléctricamente. El procesado consiste en dos contactos cuadrados de una aleación de Ti/Au. Durante el procesado, lo más importante a tener en cuenta es no realizar ningún ataque o limpieza que pueda dañar la superficie y deteriorar las propiedades de las nanostructuras. En este apartado, se realiza un análisis completo de una serie de tres muestras: GaAs (bulk), un pozo cuántico superficial (SQW) de Ino.5Gao.5As y SQDs de Ino.5Gao.5As. Para ello, a cada una de las muestras se le realizan medidas de I-V en distintas condiciones ambientales. En primer lugar, siguiendo los resultados obtenidos ópticamente, se lleva a cabo una comparación de la respuesta eléctrica en vacío y aire. A pesar de que todas las muestras presentan un carácter más resistivo en vacío que en aire, se observa una mayor influencia sobre la muestra de SQD. En vacío, la resistencia de los SQDs decrece un 99% respecto de su valor en aire, mientras que la variación de la muestras de GaAs e Ino.5Gao.5As SQW muestran una reducción, respectivamente, del 31% y del 20%. En segundo lugar, se realiza una evaluación aproximada del posible efecto de la humedad en la resistencia superficial de las muestras mediante la exhalación humana. Como resultado se obtiene, que tras la exhalación, la resistencia disminuye bruscamente y recupera su valor inicial cuando dicho proceso concluye. Este resultado preliminar indica que la humedad es un factor crítico en las propiedades eléctricas de los puntos cuánticos superficiales. Para la determinación del papel de la humedad en la respuesta eléctrica, se somete a las muestras de SQD y SQW a ambientes con humedad relativa (RH, de la siglas del inglés) controlada y se analiza el efecto sobre la conductividad superficial. Tras la variación de la RH desde 0% hasta el 70%, se observa que la muestra SQW no cambia su comportamiento eléctrico al variar la humedad del ambiente. Sin embargo, la respuesta de la muestra SQD define dos regiones bien diferenciadas, una de alta sensibilidad para valores por debajo del 50% de RH, en la que la resistencia disminuye hasta en un orden de magnitud y otra, de baja sensibilidad (>50%), donde el cambio de la resistencia es menor. Este resultado resalta la especial relevancia no sólo de la composición sino también de la morfología de la nanostructura superficial en el carácter sensitivo de la muestra. Por último, se analiza la influencia de la iluminación en la sensibilidad de la muestra. Nuevamente, se somete a las muestras SQD y SQW a una irradiación de luz de distinta energía y potencia a la vez que se varía controladamente la humedad ambiental. Una vez más, se observa que la muestra SQW no presenta ninguna variación apreciable con las alteraciones del entorno. Su resistencia superficial permanece prácticamente inalterable tanto al modificar la potencia de la luz incidente como al variar la energía de la irradiación. Por el contrario, en la muestra de SQD se obtiene una reducción la resistencia superficial de un orden de magnitud al pasar de condiciones de oscuridad a iluminación. Con respecto a la potencia y energía de la luz incidente, se observa que a pesar de que la muestra no experimenta variaciones notables con la potencia de la irradiación, esta sufre cambios significativos con la energía de la luz incidente. Cuando se ilumina con energías por encima de la energía de la banda prohibida (gap) del GaAs (Eg ~1.42 eV ) se produce una reducción de la resistencia de un orden de magnitud en atmósferas húmedas, mientras que en atmósferas secas la conductividad superficial permanece prácticamente constante. Sin embargo, al inicidir con luz de energía menor que Eg, el efecto que se produce en la respuesta eléctrica es despreciable. Esto se atribuye principalmente a la densidad de portadores fotoactivados durante la irradiación. El volumen de portadores excita dos depende de la energía de la luz incidente. De este modo, cuando la luz que incide tiene energía menor que el gap, el volumen de portadores generados es pequeño y no contribuye a la conductividad superficial. Por el contrario, cuando la energía de la luz incidente es alta (Eg), el volumen de portadores activados es elevado y éstos contribuyen significantemente a la conductividad superficial. La combinación de ambos agentes, luz y humedad, favorece el proceso de adsorción de moléculas y, por tanto, contribuye a la reducción de la densidad de estados superficiales, dando lugar a una modificación de la estructura electrónica y consecuentemente favoreciendo o dificultando el transporte de portadores. ABSTRACT Uncapped three-dimensional (3D) nanostructures have been generally grown to assess their structural quality. However, the tremendous growing importance of the impact of the environment on life has become such nanosystems in very promising candidates for the development of sensing devices. Their direct exposure to changes in the local surrounding may influence their physical properties being a perfect sign of the atmosphere quality. The goal of this thesis is the research of Ino.5Gao.5As surface quantum dots (SQDs) on GaAs(001), covering from their growth to device fabrication, for sensing applications. The achievement of this goal relies on the design, growth and sample characterization, along with device fabrication and characterization. The first issue of the thesis is devoted to analyze the main growth parameters affecting the physical properties of the Ino.5Gao.5As SQDs. It is well known that the growing conditions (growth temperature , deposition rate, V/III flux ratio and treatment after active layer growth) directly affect the physical properties of the epilayer. In this part, taking advantage of the previous results in the group regarding Ino.5Gao.5As QD growth temperature and V/III ratio, the effect of the growth rate and the temperature treatment after QDs growth nucleation is evaluated. Setting the QDs growth temperature at 430°C and the V/III flux ratio to ~20, it is found that the most appropriate conditions rely on growing the QDs at 0.07ML/s and just after QD nucleation, rapidly dropping and again raising 100°C the substrate temperature with respect to the temperature of QD growth. The combination of growing at a fast enough growth rate to promote molecule migration but sufficiently slow to allow QD nucleation, together with the sharp variation of the temperature preserving their shape and composition yield to high density, homogeneous Ino.5Gao.5As SQDs. Besides, it is also demonstrated that this high quality SQDs show excellent optical properties even at room temperature (RT). One of the characteristics by which In0.5Ga0.5As/GaAs SQDs are considered promising candidates for sensing applications is the crucial role that surface plays when interacting with the gases constituting the atmosphere. Therefore, in an attempt to develop sensing devices, the influence of the environment on the physical properties of the samples is evaluated. By comparing the resulting photoluminescence (PL) of SQDs with buried QDs (BQDs), it is found that BQDs do not exhibit any significant variation when changing the environmental conditions whereas, the external conditions greatly act on the SQDs optical properties. On one hand, it is evidenced that PL intensity of SQDs sharply quenches under vacuum and clearly decreases under dry-pure gases atmospheres (N2, O2). On the other hand, it is shown that, in water containing atmospheres, the SQDs PL intensity is maintained with respect to that in air. Moreover, it is found that neither the full width at half maximun nor the emission wavelength manifest any noticeable change indicating that the QDs are not structurally altered by the external atmosphere. These results decisively point to the processes taking place at the surface such as coupling between confined and surface states, to be responsible of this extraordinary behavior. A further analysis of the impact of the atmosphere composition on the optical characteristics is conducted. A sample containing one uncapped In0.5Ga0.5As QDs layer is exposed to different environments. Several solvents presenting different polarity, atomic composition and molecular mass, are used to change the atmosphere composition. It is revealed that low polarity and heavy molecules cause a greater variation on the PL intensity. Besides, oxygen is demonstrated to play a decisive role on the PL response. Results indicate that in presence of oxygen-containing molecules, the PL intensity experiments a less reduction than that suffered in presence of nonoxygen-containing molecules, 90% compared to 77% signal respect to the emission in air. In agreement with these results, it is demonstrated that high polarity and lighter molecules containing oxygen are more easily adsorbed, and consequently, PL intensity is less affected. The presence of defects, unsaturated bonds and in general localized states in the surface are proposed to act as nonradiative recombination centers deteriorating the PL emission of the sample. Therefore, suppression or reduction of the density of such states may lead to an increase or, at least, conservation of the PL signal. This research denotes that the interaction between sample surface and molecules in the atmosphere modifies the surface characteristics altering thus the optical properties. This is attributed to the likely adsoption of some molecules onto the surface passivating the nonradiative recombination centers, and consequently, not deteriorating the PL emission. Aiming for sensors development, samples are processed and electrically characterized under different external conditions. Samples are processed with two square (Ti/Au) contacts. During the processing, especial attention must be paid to the surface treatment. Any process that may damage the surface such as plasma etching or annealing must be avoided to preserve the features of the surface nanostructures. A set of three samples: a GaAs (bulk), In0.5Ga0.5As SQDs and In0.5Ga0.5As surface quantum well (SQW) are subjected to a throughout evaluation. I-V characteristics are measured following the results from the optical characterization. Firstly, the three samples are exposed to vacuum and air. Despite the three samples exhibit a more resistive character in vacuum than in air, it is revealed a much more clear influence of the pressure atmosphere in the SQDs sample. The sheet resistance (Rsh) of SQDs decreases a 99% from its response value under vacuum to its value in air, whereas Rsh of GaAs and In0.5Ga0.5As SQW reduces its value a 31% and a 20%, respectively. Secondly, a rough analysis of the effect of the human breath on the electrical response evidences the enormous influence of moisture (human breath is composed by several components but the one that overwhelms all the rest is the high concentration of water vapor) on the I-V characteristics. Following this result, In0.5Ga0.5As SQDs and In0.5Ga0.5As SQW are subjected to different controlled relative humidity (RH) environments (from 0% to 70%) and electrically characterized. It is found that SQW shows a nearly negligible Rsh variation when increasing the RH in the surroundings. However, the response of SQDs to changes in the RH defines two regions. Below 50%, high sensitive zone, Rsh of SQD decreases by more than one order of magnitude, while above 50% the dependence of Rsh on the RH becomes weaker. These results remark the role of the surface and denote the existence of a finite number of surface states. Nevertheless, most significantly, they highlight the importance not only of the material but also of the morphology. Finally, the impact of the illumination is determined by means of irradiating the In0.5Ga0.5As SQDs and In0.5Ga0.5As SQW samples with different energy and power sources. Once again, SQW does not exhibit any correlation between the surface conductivity and the external conditions. Rsh remains nearly unalterable independently of the energy and power of the incident light. Conversely, Rsh of SQD experiences a decay of one order of magnitude from dark-to-photo conditions. This is attributed to the less density of surface states of SQW compared to that of SQDs. Additionally, a different response of Rsh of SQD with the energy of the impinging light is found. Illuminating with high energy light results in a Rsh reduction of one order of mag nitude under humid atmospheres, whereas it remains nearly unchanged under dry environments. On the contrary, light with energy below the bulk energy bandgap (Eg), shows a negligible effect on the electrical properties regardless the local moisture. This is related to the density of photocarriers generated while lighting up. Illuminating with excitation energy below Eg affects a small absorption volume and thus, a low density of photocarriers may be activated leading to an insignificant contribution to the conductivity. Nonetheless, irradiating with energy above the Eg can excite a high density of photocarriers and greatly improve the surface conductivity. These results demonstrate that both illumination and humidity are therefore needed for sensing. The combination of these two agents improves the surface passivation by means of molecule adsorption reducing the density of surface states, thus modifying the electronic structures, and consequently, promoting the carrier motion.

A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1

The 3.0-Å structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A′ strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5

HIV-1 entry into CD4+ cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

Streptavidin facilitates internalization and pulmonary targeting of an anti-endothelial cell antibody (platelet-endothelial cell adhesion molecule 1): A strategy for vascular immunotargeting of drugs

Conjugation of drugs with antibodies to surface endothelial antigens is a potential strategy for drug delivery to endothelium. We studied antibodies to platelet-endothelial adhesion molecule 1 (PECAM-1, a stably expressed endothelial antigen) as carriers for vascular immunotargeting. Although 125I-labeled anti-PECAM bound to endothelial cells in culture, the antibody was poorly internalized by the cells and accumulated poorly after intravenous administration in mice and rats. However, conjugation of biotinylated anti-PECAM (b-anti-PECAM) with streptavidin (SA) markedly stimulated uptake and internalization of anti-PECAM by endothelial cells and by cells expressing PECAM. In addition, conjugation with streptavidin markedly stimulated uptake of 125I-labeled b-anti-PECAM in perfused rat lungs and in the lungs of intact animals after either intravenous or intraarterial injection. The antioxidant enzyme catalase conjugated with b-anti-PECAM/SA bound to endothelial cells in culture, entered the cells, escaped intracellular degradation, and protected the cells against H2O2-induced injury. Anti-PECAM/SA/125I-catalase accumulated in the lungs after intravenous injection or in the perfused rat lungs and protected these lungs against H2O2-induced injury. Thus, modification of a poor carrier antibody with biotin and SA provides an approach for facilitation of antibody-mediated drug targeting. Anti-PECAM/SA is a promising candidate for vascular immunotargeting of bioactive drugs.

E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.

An in vitro study of the dynamic features of the major histocompatibility complex class I complex relevant to its role as a versatile peptide-receptive molecule

The major histocompatibility complex class I complex consists of a heavy chain and a light chain (β2-microglobulin, β2m), which assemble with a short endogenously derived peptide in the endoplasmic reticulum. The class I peptide can be directly exchanged, either at the cell surface or, as recently described, in vesicles of the endocytic compartments, thus allowing exogenous peptides to enter the class I presentation pathway. To probe the interactions between the components of the class I molecule, we analyzed the exchange of peptide and β2m by using purified, recombinant H2-Kb/peptide complexes in a cell-free in vitro system. The exchange of competitor peptide was primarily dependent on the off-rate of the original peptide in the class I binding groove. Peptide exchange was not enhanced by the presence of exogenous β2m, as exchange occurred to the same extent in its absence. Thus, the exchange of peptide and β2m are independent events. The exchange rate of β2m also was not affected by the dissociation rates of the original peptides. Furthermore, peptides could substantially exchange into class I molecules over a pH range of 5.5 to 7.5, conditions prevalent in certain endocytic compartments. We conclude that the dynamic properties of the components of class I molecules explain its function as a highly peptide-receptive molecule. The major histocompatibility complex class I can readily receive peptides independent of the presence of exogenous β2m, even at a low pH. Such properties are relevant to class I peptide acquisition, which can occur at the cell surface, as well as in specialized endosomes.

A short segment within the cytoplasmic domain of the neural cell adhesion molecule (N-CAM) is essential for N-CAM-induced NF-κB activity in astrocytes

The neural cell adhesion molecule (N-CAM) is expressed on the surface of astrocytes, where its homophilic binding leads to the activation of the transcription factor NF-κB. Transfection of astrocytes with a construct encompassing the transmembrane region and the cytoplasmic domain of N-CAM (designated Tm-Cyto, amino acids 685–839 in the full-length molecule) inhibited this activation up to 40%, and inhibited N-CAM-induced translocation of NF-κB to the nucleus. N-CAM also activated NF-κB in astrocytes from N-CAM knockout mice, presumably through binding to a heterophile. This activation, however, was not blocked by Tm-Cyto expression, indicating that the inhibitory effect of the Tm-Cyto construct is specific for cell surface N-CAM. Deletions and point mutations of the cytoplasmic portion of the Tm-Cyto construct indicated that the region between amino acids 780 and 800 were essential for inhibitory activity. This region contains four threonines (788, 793, 794, and 797). Mutation to alanine of T788, T794, or T797, but not T793, abolished inhibitory activity, as did mutation of T788 or T797 to aspartic acid. A Tm-Cyto construct with T794 mutated to aspartic acid retained inhibitory activity but did not itself induce a constitutive NF-κB response. This result suggests that phosphorylation of T794 may be necessary but is not the triggering event. Overall, these findings define a short segment of the N-CAM cytoplasmic domain that is critical for N-CAM-induced activation of NF-κB and may be important in other N-CAM-mediated signaling.

An isolated, surface-expressed I domain of the integrin αLβ2 is sufficient for strong adhesive function when locked in the open conformation with a disulfide bond

We introduced disulfide bonds to lock the integrin αLβ2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing αLβ2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated αLβ2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal α-helix of the I domain, inhibited binding to ICAM-1 by αLβ2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the αL I domain for adhesive function and show that this domain is sufficient for full adhesive activity.

Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the α3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-γ

The equilibrium dissociation of recombinant human IFN-γ was monitored as a function of pressure and sucrose concentration. The partial molar volume change for dissociation was −209 ± 13 ml/mol of dimer. The specific molar surface area change for dissociation was 12.7 ± 1.6 nm2/molecule of dimer. The first-order aggregation rate of recombinant human IFN-γ in 0.45 M guanidine hydrochloride was studied as a function of sucrose concentration and pressure. Aggregation proceeded through a transition-state species, N*. Sucrose reduced aggregation rate by shifting the equilibrium between native state (N) and N* toward the more compact N. Pressure increased aggregation rate through increased solvation of the protein, which exposes more surface area, thus shifting the equilibrium away from N toward N*. The changes in partial molar volume and specific molar surface area between the N* and N were −41 ± 9 ml/mol of dimer and 3.5 ± 0.2 nm2/molecule, respectively. Thus, the structural change required for the formation of the transition state for aggregation is small relative to the difference between N and the dissociated state. Changes in waters of hydration were estimated from both specific molar surface area and partial molar volume data. From partial molar volume data, estimates were 25 and 128 mol H2O/mol dimer for formation of the aggregation transition state and for dissociation, respectively. From surface area data, estimates were 27 and 98 mol H2O/mol dimer. Osmotic stress theory yielded values ≈4-fold larger for both transitions.