Deviation of the alloimmune response toward tolerance by chimeric class I MHC molecules

Class I major histocompatibility complex (MHC) molecules induce either accelerated rejection or prolonged survival of allografts, presumably because of the presence of immunogenic or tolerogenic epitopes, respectively. To explore the molecular basis of this phenomenon, three chimeric class I molecules were constructed by substituting the rat class I RT1.A$\sp{\rm a}$ sequences with the N-terminus of HLA-A2.1 (N$\sp{\rm HLA-A2.1}$-RT1.A$\sp{\rm a}$), the $\alpha\sb1$ helix (h) with $\rm\alpha\sb{1h}\sp{u}$ sequences ( ($\rm\alpha\sb{1h}\sp{u}$) -RT1.A$\sp{\rm a}$) or the entire $\alpha\sb2$ domain (d) with $\rm\alpha\sb{2d}\sp{u}$ sequences ( ($\rm\alpha\sb{2d}\sp{u}$) -RT1.A$\sp{\rm a}$). Wild type (WT) and chimeric cDNAs were sequenced prior to transfection into Buffalo (BUF; RT1$\sp{\rm b}$) hepatoma cells. Stable transfectants were injected subcutaneously (s.c.) into different hosts 7 days prior to challenge with a heart allograft. In BUF hosts, chimeric ($\rm\alpha\sb{1h}\sp{u}$) -RT1.A$\sp{\rm a}$ accelerated the rejection of Wistar Furth (WF; RT1$\sp{\rm u}$) heart allografts, but had no effect on the survival of ACI (RT1$\sp{\rm a}$) grafts. In contrast, the ($\rm\alpha\sb{2d}\sp{u}$) -RT1.A$\sp{\rm a}$ (containing $\rm\alpha\sb{1d}\sp{a}$ sequences) immunized BUF recipients toward RT1$\sp{\rm a}$ grafts. In WF hosts, WT-RT1.A$\sp{\rm a}$ was a potent immunogen and accelerated ACI graft rejection, N$\sp{\rm HLA-A2.1}$-RT1.A$\sp{\rm a}$ was less effective and ($\rm\alpha\sb{\rm 1h}\sp{u}\rbrack$-RT1.A$\sp{\rm a}$ was not immunogenic. Thus, dominant and subdominant epitopes inducing in vivo sensitization to cardiac allografts are present in the $\alpha\sb1$ helix and the N-terminus, respectively. The failure of ($\rm\alpha\sb{2d}\sp{u}$) -RT1.A$\sp{\rm a}$ transfectants (containing recipient-type $\alpha\sb{\rm 2d}$ sequences) to sensitize WF hosts toward ACI (RT1$\sp{\rm a}$) grafts, despite the presence of donor-type immunogenic $\alpha\sb{\rm 1d}\sp{\rm a}$, suggests that "self-$\alpha\sb2$" sequences displayed on chimeric antigens interfere with immunogenicity. The ($\rm\alpha\sb{1h}\sp{u}$) -RT1.A$\sp{\rm a}$ transfectants injected s.c. prolonged the survival of WF (RT1$\sp{\rm u}$) hearts in ACI (RT1$\sp{\rm a}$) recipients. Furthermore, intra-portal injection of extracts from ($\rm\alpha\sb{1h}\sp{u}$) -RT1.A$\sp{\rm a}$, but not WT-RT1.A$\sp{\rm a}$ or RT1.A$\sp{\rm u}$, in conjunction with a brief cyclosporine course rendered ACI hosts permanently and specifically tolerant to donor-type WF cardiac allografts. Thus, immunodominant allodeterminants are present in the $\alpha\sb1$, but not the $\alpha\sb2$, domain of rat class I MHC molecules. Furthermore, the $\rm\alpha\sb{1h}\sp{u}$ immunogenic epitopes trigger tolerogenic responses when flanked by host-type N-terminal$\sp{\rm a}$ and $\rm\alpha\sb{2d}\sp{a}$ sequences. ^

Holland's secondary constructs of vocational interests and career choice readiness of secondary students

The study examined the relationship between the secondary constructs of Holland’s (1997) theory of vocational interests and career choice readiness [career maturity] attitudes with 358 Swiss secondary students. The hypothesis was tested that the secondary constructs consistency, coherence, differentiation, and congruence are measures for the degree of vocational interest development. Thus, they should belong to the content domain in career choice readiness and should show meaningful relations to career choice readiness attitudes. The hypothesis was confirmed for congruence, coherence, and differentiation. Interest profile consistency showed no relation to career choice readiness attitudes. Vocational identity emerged as a direct measure for career choice readiness attitudes. Realism of career aspirations was related to career choice readiness attitudes and coherence of career aspirations. Profile elevation was positively connected to more career planning and career exploration. Differences between gender, ethnicity, and school-types are presented. Implications for career counselling and assessment practice are discussed.

An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.

In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.

DNA-Grafted Supramolecular Polymers: Helical Ribbon Structures Formed by Self-Assembly of Pyrene-DNA Chimeric Oligomers

The controlled arraying of DNA strands on adaptive polymeric platforms remains a challenge. Here, the noncovalent synthesis of DNA-grafted supramolecular polymers from short chimeric oligomers is presented. The oligomers are composed of an oligopyrenotide strand attached to the 5′-end of an oligodeoxynucleotide. The supramolecular polymerization of these oligomers in an aqueous medium leads to the formation of one-dimensional (1D) helical ribbon structures. Atomic force and transmission electron microscopy show rod-like polymers of several hundred nanometers in length. DNA-grafted polymers of the type described herein will serve as models for the development of structurally and functionally diverse supramolecular platforms with applications in materials science and diagnostics.

Biomechanical comparison between bicortical pin and monocortical screw/polymethylmethacrylate constructs in the cadaveric canine cervical vertebral column.

OBJECTIVE To compare biomechanical stiffness of cadaveric canine cervical spine constructs stabilized with bicortical stainless steel pins and polymethylmethacrylate (PMMA), monocortical stainless steel screws with PMMA, or monocortical titanium screws with PMMA. STUDY DESIGN Biomechanical cadaver study. ANIMALS Eighteen canine cervical vertebral columns (C2-C7) were collected from skeletally mature dogs (weighing 22-32 kg). METHODS Specimens were radiographed and examined by dual energy X-ray absorptiometry. Stiffness of the unaltered C4-C5 intervertebral motion unit was measured in extension, flexion and lateral bending using non-destructive 4-point bend testing. Specimens were then stabilized by (1) bicortical stainless steel pins/PMMA, (2) monocortical stainless steel screws/PMMA, or (3) monocortical titanium screws/PMMA. Mechanical testing was repeated and stiffness data from unaltered specimens and the 3 treatment groups were compared. RESULTS All 3 surgical methods significantly increased stiffness of the C4-C5 motion unit compared with the unaltered specimen (P < .001 for all treatments), but stiffness was not significantly different among the 3 fixation groups (P = .578). CONCLUSIONS In this model, monocortical screw fixation (with stainless steel or titanium screws) was biomechanically equivalent to bicortical fixation.

Comparing the factorial structure, invariance, and predictive validity of transtheoretical model constructs for alcohol use across restricted and unrestricted settings

With substance abuse treatment expanding in prisons and jails, understanding how behavior change interacts with a restricted setting becomes more essential. The Transtheoretical Model (TTM) has been used to understand intentional behavior change in unrestricted settings, however, evidence indicates restrictive settings can affect the measurement and structure of the TTM constructs. The present study examined data from problem drinkers at baseline and end-of-treatment from three studies: (1) Project CARE (n = 187) recruited inmates from a large county jail; (2) Project Check-In (n = 116) recruited inmates from a state prison; (3) Project MATCH, a large multi-site alcohol study had two recruitment arms, aftercare (n = 724 pre-treatment and 650 post-treatment) and outpatient (n = 912 pre-treatment and 844 post-treatment). The analyses were conducted using cross-sectional data to test for non-invariance of measures of the TTM constructs: readiness, confidence, temptation, and processes of change (Structural Equation Modeling, SEM) across restricted and unrestricted settings. Two restricted (jail and aftercare) and one unrestricted group (outpatient) entering treatment and one restricted (prison) and two unrestricted groups (aftercare and outpatient) at end-of-treatment were contrasted. In addition TTM end-of-treatment profiles were tested as predictors of 12 month drinking outcomes (Profile Analysis). Although SEM did not indicate structural differences in the overall TTM construct model across setting types, there were factor structure differences on the confidence and temptation constructs at pre-treatment and in the factor structure of the behavioral processes at the end-of-treatment. For pre-treatment temptation and confidence, differences were found in the social situations factor loadings and in the variance for the confidence and temptation latent factors. For the end-of-treatment behavioral processes, differences across the restricted and unrestricted settings were identified in the counter-conditioning and stimulus control factor loadings. The TTM end-of-treatment profiles were not predictive of drinking outcomes in the prison sample. Both pre and post-treatment differences in structure across setting types involved constructs operationalized with behaviors that are limited for those in restricted settings. These studies suggest the TTM is a viable model for explicating addictive behavior change in restricted settings but calls for modification of subscale items that refer to specific behaviors and caution in interpreting the mean differences across setting types for problem drinkers. ^

Modelling the Impact of Built Environment, Geographical Scales and Latent Constructs On Individual Travel Behaviour

La relación entre la estructura urbana y la movilidad ha sido estudiada desde hace más de 70 años. El entorno urbano incluye múltiples dimensiones como por ejemplo: la estructura urbana, los usos de suelo, la distribución de instalaciones diversas (comercios, escuelas y zonas de restauración, parking, etc.). Al realizar una revisión de la literatura existente en este contexto, se encuentran distintos análisis, metodologías, escalas geográficas y dimensiones, tanto de la movilidad como de la estructura urbana. En este sentido, se trata de una relación muy estudiada pero muy compleja, sobre la que no existe hasta el momento un consenso sobre qué dimensión del entorno urbano influye sobre qué dimensión de la movilidad, y cuál es la manera apropiada de representar esta relación. Con el propósito de contestar estas preguntas investigación, la presente tesis tiene los siguientes objetivos generales: (1) Contribuir al mejor entendimiento de la compleja relación estructura urbana y movilidad. y (2) Entender el rol de los atributos latentes en la relación entorno urbano y movilidad. El objetivo específico de la tesis es analizar la influencia del entorno urbano sobre dos dimensiones de la movilidad: número de viajes y tipo de tour. Vista la complejidad de la relación entorno urbano y movilidad, se pretende contribuir al mejor entendimiento de la relación a través de la utilización de 3 escalas geográficas de las variables y del análisis de la influencia de efectos inobservados en la movilidad. Para el análisis se utiliza una base de datos conformada por tres tipos de datos: (1) Una encuesta de movilidad realizada durante los años 2006 y 2007. Se obtuvo un total de 943 encuestas, en 3 barrios de Madrid: Chamberí, Pozuelo y Algete. (2) Información municipal del Instituto Nacional de Estadística: dicha información se encuentra enlazada con los orígenes y destinos de los viajes recogidos en la encuesta. Y (3) Información georeferenciada en Arc-GIS de los hogares participantes en la encuesta: la base de datos contiene información respecto a la estructura de las calles, localización de escuelas, parking, centros médicos y lugares de restauración. Se analizó la correlación entre e intra-grupos y se modelizaron 4 casos de atributos bajo la estructura ordinal logit. Posteriormente se evalúa la auto-selección a través de la estimación conjunta de las elecciones de tipo de barrio y número de viajes. La elección del tipo de barrio consta de 3 alternativas: CBD, Urban y Suburban, según la zona de residencia recogida en las encuestas. Mientras que la elección del número de viajes consta de 4 categorías ordinales: 0 viajes, 1-2 viajes, 3-4 viajes y 5 o más viajes. A partir de la mejor especificación del modelo ordinal logit. Se desarrolló un modelo joint mixed-ordinal conjunto. Los resultados indican que las variables exógenas requieren un análisis exhaustivo de correlaciones con el fin de evitar resultados sesgados. ha determinado que es importante medir los atributos del BE donde se realiza el viaje, pero también la información municipal es muy explicativa de la movilidad individual. Por tanto, la percepción de las zonas de destino a nivel municipal es considerada importante. En el contexto de la Auto-selección (self-selection) es importante modelizar conjuntamente las decisiones. La Auto-selección existe, puesto que los parámetros estimados conjuntamente son significativos. Sin embargo, sólo ciertos atributos del entorno urbano son igualmente importantes sobre la elección de la zona de residencia y frecuencia de viajes. Para analizar la Propensión al Viaje, se desarrolló un modelo híbrido, formado por: una variable latente, un indicador y un modelo de elección discreta. La variable latente se denomina “Propensión al Viaje”, cuyo indicador en ecuación de medida es el número de viajes; la elección discreta es el tipo de tour. El modelo de elección consiste en 5 alternativas, según la jerarquía de actividades establecida en la tesis: HOME, no realiza viajes durante el día de estudio, HWH tour cuya actividad principal es el trabajo o estudios, y no se realizan paradas intermedias; HWHs tour si el individuo reaiza paradas intermedias; HOH tour cuya actividad principal es distinta a trabajo y estudios, y no se realizan paradas intermedias; HOHs donde se realizan paradas intermedias. Para llegar a la mejor especificación del modelo, se realizó un trabajo importante considerando diferentes estructuras de modelos y tres tipos de estimaciones. De tal manera, se obtuvieron parámetros consistentes y eficientes. Los resultados muestran que la modelización de los tours, representa una ventaja sobre la modelización de los viajes, puesto que supera las limitaciones de espacio y tiempo, enlazando los viajes realizados por la misma persona en el día de estudio. La propensión al viaje (PT) existe y es específica para cada tipo de tour. Los parámetros estimados en el modelo híbrido resultaron significativos y distintos para cada alternativa de tipo de tour. Por último, en la tesis se verifica que los modelos híbridos representan una mejora sobre los modelos tradicionales de elección discreta, dando como resultado parámetros consistentes y más robustos. En cuanto a políticas de transporte, se ha demostrado que los atributos del entorno urbano son más importantes que los LOS (Level of Service) en la generación de tours multi-etapas. la presente tesis representa el primer análisis empírico de la relación entre los tipos de tours y la propensión al viaje. El concepto Propensity to Travel ha sido desarrollado exclusivamente para la tesis. Igualmente, el desarrollo de un modelo conjunto RC-Number of trips basado en tres escalas de medida representa innovación en cuanto a la comparación de las escalas geográficas, que no había sido hecha en la modelización de la self-selection. The relationship between built environment (BE) and travel behaviour (TB) has been studied in a number of cases, using several methods - aggregate and disaggregate approaches - and different focuses – trip frequency, automobile use, and vehicle miles travelled and so on. Definitely, travel is generated by the need to undertake activities and obtain services, and there is a general consensus that urban components affect TB. However researches are still needed to better understand which components of the travel behaviour are affected most and by which of the urban components. In order to fill the gap in the research, the present dissertation faced two main objectives: (1) To contribute to the better understanding of the relationship between travel demand and urban environment. And (2) To develop an econometric model for estimating travel demand with urban environment attributes. With this purpose, the present thesis faced an exhaustive research and computation of land-use variables in order to find the best representation of BE for modelling trip frequency. In particular two empirical analyses are carried out: 1. Estimation of three dimensions of travel demand using dimensions of urban environment. We compare different travel dimensions and geographical scales, and we measure self-selection contribution following the joint models. 2. Develop a hybrid model, integrated latent variable and discrete choice model. The implementation of hybrid models is new in the analysis of land-use and travel behaviour. BE and TB explicitly interact and allow richness information about a specific individual decision process For all empirical analysis is used a data-base from a survey conducted in 2006 and 2007 in Madrid. Spatial attributes describing neighbourhood environment are derived from different data sources: National Institute of Statistics-INE (Administrative: municipality and district) and GIS (circular units). INE provides raw data for such spatial units as: municipality and district. The construction of census units is trivial as the census bureau provides tables that readily define districts and municipalities. The construction of circular units requires us to determine the radius and associate the spatial information to our households. The first empirical part analyzes trip frequency by applying an ordered logit model. In this part is studied the effect of socio-economic, transport and land use characteristics on two travel dimensions: trip frequency and type of tour. In particular the land use is defined in terms of type of neighbourhoods and types of dwellers. Three neighbourhood representations are explored, and described three for constructing neighbourhood attributes. In particular administrative units are examined to represent neighbourhood and circular – unit representation. Ordered logit models are applied, while ordinal logit models are well-known, an intensive work for constructing a spatial attributes was carried out. On the other hand, the second empirical analysis consists of the development of an innovative econometric model that considers a latent variable called “propensity to travel”, and choice model is the choice of type of tour. The first two specifications of ordinal models help to estimate this latent variable. The latent variable is unobserved but the manifestation is called “indicators”, then the probability of choosing an alternative of tour is conditional to the probability of latent variable and type of tour. Since latent variable is unknown we fit the integral over its distribution. Four “sets of best variables” are specified, following the specification obtained from the correlation analysis. The results evidence that the relative importance of SE variables versus BE variables depends on how BE variables are measured. We found that each of these three spatial scales has its intangible qualities and drawbacks. Spatial scales play an important role on predicting travel demand due to the variability in measures at trip origin/destinations within the same administrative unit (municipality, district and so on). Larger units will produce less variation in data; but it does not affect certain variables, such as public transport supply, that are more significant at municipality level. By contrast, land-use measures are more efficient at district level. Self-selection in this context, is weak. Thus, the influence of BE attributes is true. The results of the hybrid model show that unobserved factors affect the choice of tour complexity. The latent variable used in this model is propensity to travel that is explained by socioeconomic aspects and neighbourhood attributes. The results show that neighbourhood attributes have indeed a significant impact on the choice of the type of tours either directly and through the propensity to travel. The propensity to travel has a different impact depending on the structure of each tour and increases the probability of choosing more complex tours, such as tours with many intermediate stops. The integration of choice and latent variable model shows that omitting important perception and attitudes leads to inconsistent estimates. The results also indicate that goodness of fit improves by adding the latent variable in both sequential and simultaneous estimation. There are significant differences in the sensitivity to the latent variable across alternatives. In general, as expected, the hybrid models show a major improvement into the goodness of fit of the model, compared to a classical discrete choice model that does not incorporate latent effects. The integrated model leads to a more detailed analysis of the behavioural process. Summarizing, the effect that built environment characteristics on trip frequency studied is deeply analyzed. In particular we tried to better understand how land use characteristics can be defined and measured and which of these measures do have really an impact on trip frequency. We also tried to test the superiority of HCM on this field. We can concluded that HCM shows a major improvement into the goodness of fit of the model, compared to classical discrete choice model that does not incorporate latent effects. And consequently, the application of HCM shows the importance of LV on the decision of tour complexity. People are more elastic to built environment attributes than level of services. Thus, policy implications must take place to develop more mixed areas, work-places in combination with commercial retails.

Constructs and evaluation strategies for intelligent speculative parallelism - armageddon revisited

This report addresses speculative parallelism (the assignment of spare processing resources to tasks which are not known to be strictly required for the successful completion of a computation) at the user and application level. At this level, the execution of a program is seen as a (dynamic) tree —a graph, in general. A solution for a problem is a traversal of this graph from the initial state to a node known to be the answer. Speculative parallelism then represents the assignment of resources to múltiple branches of this graph even if they are not positively known to be on the path to a solution. In highly non-deterministic programs the branching factor can be very high and a naive assignment will very soon use up all the resources. This report presents work assignment strategies other than the usual depth-first and breadth-first. Instead, best-first strategies are used. Since their definition is application-dependent, the application language contains primitives that allow the user (or application programmer) to a) indícate when intelligent OR-parallelism should be used; b) provide the functions that define "best," and c) indícate when to use them. An abstract architecture enables those primitives to perform the search in a "speculative" way, using several processors, synchronizing them, killing the siblings of the path leading to the answer, etc. The user is freed from worrying about these interactions. Several search strategies are proposed and their implementation issues are addressed. "Armageddon," a global pruning method, is introduced, together with both a software and a hardware implementation for it. The concepts exposed are applicable to áreas of Artificial Intelligence such as extensive expert systems, planning, game playing, and in general to large search problems. The proposed strategies, although showing promise, have not been evaluated by simulation or experimentation.

Molecular coevolution within a Drosophila clock gene

The period (per) gene in Drosophila melanogaster provides an integral component of biological rhythmicity and encodes a protein that includes a repetitive threonine-glycine (Thr-Gly) tract. Similar repeats are found in the frq and wc2 clock genes of Neurospora crassa and in the mammalian per homologues, but their circadian functions are unknown. In Drosophilids, the length of the Thr-Gly repeat varies widely between species, and sequence comparisons have suggested that the repeat length coevolves with the immediately flanking amino acids. A functional test of the coevolution hypothesis was performed by generating several hybrid per transgenes between Drosophila pseudoobscura and D. melanogaster, whose repetitive regions differ in length by about 150 amino acids. The positions of the chimeric junctions were slightly altered in each transgene. Transformants carrying per constructs in which the repeat of one species was juxtaposed next to the flanking region of the other were almost arrhythmic or showed a striking temperature sensitivity of the circadian period. In contrast, transgenes in which the repeat and flanking regions were conspecific gave wild-type levels of circadian rescue. These results support the coevolutionary interpretation of the interspecific sequence changes in this region of the PER molecule and reveal a functional dimension to this process related to the clock’s temperature compensation.

Chimeric anti-angiogenin antibody cAb 26–2F inhibits the formation of human breast cancer xenografts in athymic mice

Angiogenin (Ang), an inducer of neovascularization, is secreted by several types of human tumor cells and appears critical for their growth. The murine anti-Ang monoclonal antibody (mAb) 26–2F neutralizes the activities of Ang and dramatically prevents the establishment and metastatic dissemination of human tumor cell xenografts in athymic mice. However, for use clinically, the well-documented problem of the human anti-globulin antibody response known to occur with murine antibodies requires resolution. As a result, chimeric as well as totally humanized antibodies are currently being evaluated as therapeutic agents for the treatment of several pathological conditions, including malignancy. Therefore, we have constructed a chimeric mouse/human antibody based on the structure of mAb 26–2F. Complementary DNAs from the light and heavy chain variable regions of mAb 26–2F were cloned, sequenced, and genetically engineered by PCR for subcloning into expression vectors that contain human constant region sequences. Transfection of these vectors into nonproducing mouse myeloma cells resulted in the secretion of fully assembled tetrameric molecules. The chimeric antibody (cAb 26–2F) binds to Ang and inhibits its ribonucleolytic and angiogenic activities as potently as mAb 26–2F. Furthermore, the capacities of cAb 26–2F and its murine counterpart to suppress the formation of human breast cancer tumors in athymic mice are indistinguishable. Thus cAb 26–2F, with its retained neutralization capability and likely decreased immunogenicity, may be of use clinically for the treatment of human cancer and related disorders where pathological angiogenesis is a component.

A general strategy to enhance the potency of chimeric transcriptional activators

Efforts to increase the potency of transcriptional activators are generally unsuccessful because poor expression of activators in mammalian cells limits their delivery to target promoters. Here we report that the effectiveness of chimeric activators can be dramatically improved by expressing them as noncovalent tetrameric bundles. Bundled activation domains are much more effective at activating a reporter gene than simple monomeric activators, presumably because, at similar expression levels, up to 4 times as many the activation domains are delivered to the target promoter. These bundled activation domains are also more effective than proteins in which activation domains are tandemly reiterated in the same polypeptide chain, because such proteins are very poorly expressed and therefore not delivered effectively. These observations suggest that there is a threshold number of activation domains that must be bound to a promoter for activation, above which promoter activity is simply a function of the number of activators bound. We show that bundling can be exploited practically to enhance the sensitivity of mammalian two-hybrid assays, enabling detection of weak interactions or those between poorly expressed proteins. Bundling also dramatically improves the performance of a small-molecule-regulated gene expression system when the expression level of regulatory protein is limiting, a situation that may be encountered in gene therapy applications.

AP-2-null cells disrupt morphogenesis of the eye, face, and limbs in chimeric mice

The homozygous disruption of the mouse AP-2 gene yields a complex and lethal phenotype that results from defective development of the neural tube, head, and body wall. The severe and pleiotropic developmental abnormalities observed in the knockout mouse suggested that AP-2 may regulate several morphogenic pathways. To uncouple the individual developmental mechanisms that are dependent on AP-2, we have now analyzed chimeric mice composed of both wild-type and AP-2-null cells. The phenotypes obtained from these chimeras indicate that there is an independent requirement for AP-2 in the formation of the neural tube, body wall, and craniofacial skeleton. In addition, these studies reveal that AP-2 exerts a major influence on eye formation, which is a critical new role for AP-2 that was masked previously in the knockout mice. Furthermore, we also have uncovered an unexpected influence of AP-2 on limb pattern formation; this influence is typified by major limb duplications. The range of phenotypes observed in the chimeras displays a significant overlap with those caused by teratogenic levels of retinoic acid, strongly suggesting that AP-2 is an important component of the mechanism of action of this morphogen.

Regulated expression of the diphtheria toxin A chain by a tumor-specific chimeric transcription factor results in selective toxicity for alveolar rhabdomyosarcoma cells

Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was ≈250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3–6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3–6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)- or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion protein-regulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.

Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.