Dual role of interleukin-12 on ultraviolet -induced immune suppression

Skin cancer is the most prevalent form of neoplasia, with over one million newcases diagnosed this year. UV radiation is a ubiquitous environmental agent that induces skin cancer. In addition to its carcinogenic effect, UV radiation also suppresses cell-mediated immune responses. This immune suppression is not only observed at the site of irradiation, but UV radiation also induces systemic immune suppression. Since UV radiation has a limited ability to penetrate the skin, the question of the mechanism of this systemic immune suppression arises. A number of studies have suggested that UV radiation induce systemic effects through the production of immunoregulatory cytokines, such as IL-4 and IL-10. These cytokines affect the immune response by altering systemic antigen presentation, specifically by suppressing the activation of Th1 cells while allowing the activation of Th2 cells. Because IL-12 is an important regulator of Th1 cell activation, we tested the hypothesis that administration of IL-12 could overcome UV-induced immune suppression. ^ The studies presented here are divided into dime specific aims. In the first specific aim, the ability of IL-12 to overcome UV-induced immune suppression was examined. IL-12 could overcome UV-induced immune suppression as well as prevent the generation of and neutralize the activity of preformed suppressor cells induced by UV radiation. In the second specific aim, the mechanism by which IL-12 overcomes UV-induced immune suppression was examined. IL-12 overcame UV-induced immune suppression by blocking the production of immunoregulatory cytokines such as IL-4, IL-10 and TNF-α. In the third specific aim, the effect of UV radiation on antigen presentation was investigated. UV radiation was found to decrease the production of biologically active IL-12. In addition, UV also increased the production of IL-12p40 homodimer, an antagonist of IL-12p70 heterodimer. This result suggests that IL-12 may have a dual role in the immune suppression induced by, UV radiation. On one hand the biologically active IL-12p70 heterodimer blocks UV-induced immune suppression. In contrast, IL-12p40 homodimer may mediate the suppressive effect of UV radiation. This paradox indicates that IL-12 may have a greater regulatory role in the immune response than was previously suspected. ^

Los paisajes del desecho: reactivación de los lugares del deterioro

Actualmente en nuestro planeta producimos 1.300 millones de toneladas de residuos urbanos al año. Si los extendemos sobre la superficie de un cuadrado de lado 100 m (una hectárea) alcanzarían una altura de 146 km. ¿Cuál es el origen de nuestros residuos? ¿A dónde va esta basura? ¿Cómo nos afecta? ¿Tiene alguna utilidad? Se trata de un problema antiguo que, en los últimos tiempos, ha adquirido una nueva dimensión por el tipo y la cantidad de residuos generados. Las primeras preocupaciones de la ciudad por ordenar estos problemas dieron lugar al establecimiento de espacios o lugares específicos para la acumulación de los residuos urbanos: los vertederos. Los desechos hoy se generan más rápidamente que los medios disponibles para reciclarlos o tratarlos. Los vertederos de residuos urbanos son y seguirán siendo, a corto y medio plazo, soluciones válidas por ser un método de gestión relativamente barato, sobre todo en los países en vías de desarrollo. Como consecuencia y necesidad de lo anterior, se plantea demostrar que la recuperación y la transformación de estos vertederos de residuos urbanos (lugares del deterioro), una vez abandonados, es posible y que además pueden dar lugar a nuevos espacios públicos estratégicos de la ciudad contemporánea. Son espacios de oportunidad, vacíos monumentales producto de una reactivación arquitectónica y paisajística realizada a partir de complejos procesos de ingeniería medioambiental. Pero las soluciones aplicadas a los vertederos de residuos urbanos desde mediados del siglo XX se han realizado exclusivamente desde la ingeniería para tratar de resolver cuestiones técnicas, un modelo agotado que ya no puede gestionar la magnitud que este problema ha alcanzado, haciéndose necesaria e inevitable la participación de la arquitectura para abrir nuevas líneas de investigación y de acción. En estos primeros compases del siglo XXI existe una “nueva” preocupación, un “nuevo” interés en los paradigmas de lo ecológico y de la sostenibilidad, también un interés filosófico (que igualmente otorga un nuevo valor al residuo como recurso), que dirigen su mirada hacia un concepto de paisaje abierto y diferente a modelos anteriores más estáticos, recuperando como punto de partida el ideal pintoresco. El landscape urbanism se consolida como una disciplina capaz de dar respuesta a lo natural y artificial simultáneamente, que sustituye a las herramientas tradicionales de la arquitectura para solucionar los problemas de la ciudad contemporánea, incorporando las infraestructuras de gran escala, como un vertedero de residuos urbanos, y los paisajes públicos que generan como el verdadero mecanismo de organización del urbanismo de hoy. No se trata solo de un modelo formal sino, lo que es más importante: de un modelo de procesos. Esta nueva preocupación permite abordar la cuestión del paisaje de manera amplia, sin restricciones, con un alto grado de flexibilidad en las nuevas propuestas que surgen como consecuencia de estos conceptos, si bien los esfuerzos, hasta la fecha, parecen haberse dirigido más hacia el fenómeno de lo estético, quedando todavía por explorar las consecuencias políticas, sociales, económicas y energéticas derivadas de los residuos. También las arquitectónicas. El proyecto del landscape urbanism se ocupa de la superficie horizontal, del plano del suelo. Desde siempre, la preparación de este plano para desarrollar cualquier actividad humana ha sido un gesto fundacional, un gesto propio necesario de toda arquitectura, que además ahora debe considerarlo como un medio o soporte biológicamente activo. En términos contemporáneos, el interés disciplinar radica en la continuidad y en la accesibilidad del suelo, diluyendo los límites; en que funcione a largo plazo, que se anticipe al cambio, a través de la flexibilidad y de la capacidad de negociación, y que sea público. La recuperación de un vertedero de residuos urbanos ofrece todas estas condiciones. Un breve recorrido por la historia revela los primeros ejemplos aislados de recuperación de estos lugares del deterioro, que han pasado por distintas fases en función de la cantidad y el tipo de los desechos producidos, evolucionando gracias a la tecnología y a una nueva mirada sobre el paisaje, hasta desarrollar una verdadera conciencia de lo ecológico (nacimiento de una ideología). El Monte Testaccio en Roma (siglos I-III d.C.) constituye un caso paradigmático y ejemplar de vertedero planificado a priori no solo como lugar en el que depositar los residuos, sino como lugar que será recuperado posteriormente y devuelto a la ciudad en forma de espacio público. Una topografía de desechos generada por acumulación, organizada y planificada durante tres siglos, que nos hace reflexionar sobre los temas de producción, consumo y proyecto arquitectónico. El Monte Testaccio revela una fuente de inspiración, un arquetipo de gestión sostenible de los recursos y del territorio. A través de la experiencia en la recuperación y transformación en espacios públicos de casos contemporáneos, como el antiguo vertedero de Valdemingómez en Madrid o el de El Garraf en Barcelona, se han analizado las técnicas y las soluciones empleadas para establecer nuevas herramientas de proyecto planteadas en clave de futuro, que revelan la importancia de los procesos frente a la forma, en los cuales intervienen muchos factores (tanto naturales como artificiales), entre ellos la vida y el tiempo de la materia viva acumulada. Son lugares para nuevas oportunidades y ejemplos de una nueva relación con la naturaleza. La reactivación de los vertederos de residuos, a través del proyecto, nos propone una nueva topografía construida en el tiempo, el suelo como soporte, como punto de encuentro de la naturaleza y los sistemas tecnológicos de la ciudad que posibilitan nuevos modos de vida y nuevas actividades. Los vertederos de residuos son inmensas topografías naturales surgidas de procesos artificiales, atalayas desde las que divisar un nuevo horizonte, un nuevo mundo, un nuevo futuro donde sea posible lograr la reversibilidad de nuestros actos del deterioro. Pero la voluntad de estas recuperaciones y transformaciones no consiste exclusivamente en su reintegración al paisaje, sino que han servido como muestra de las nuevas actitudes que la sociedad ha de emprender en relación a los temas medio ambientales. ABSTRACT Here on our planet we currently produce 1.3 billion tonnes of urban waste per year. If we were to spread this over a surface of 100m2 (one hectare), it would reach a height of 146km. What is the origin of this waste? Where does our refuse go? How does it affect us? Does it have any uses? We are dealing with an old problem which, in recent times, has taken on a new dimension due to the type of waste and the amount generated. Cities’ first concerns in resolving these problems gave rise to the establishment of areas or specific places for the accumulation of urban waste: landfills. These days, waste is generated more quickly than the available resources can recycle or process it. Urban waste landfills are and will continue to be, in the short and mid-term, valid solutions, given that they constitute a relatively cheap method for waste management, especially in developing countries. Consequently and necessarily, we plan to demonstrate that it is possible to recover and transform these urban waste landfills (areas of deterioration) once they have been abandoned and that they can give rise to new strategic public areas in contemporary cities. They are areas of opportunity, monumental vacancies produced by an architectural reactivation of the landscape, which is achieved using complex processes of environmental engineering. But the solutions applied to urban waste landfills throughout the 20th century have used engineering exclusively in the attempt to resolve the technical aspects. This is a worn-out model which can no longer handle the magnitude which the problem has attained and therefore, there is an inevitable need for the participation of architecture, which can open new lines of research and action. In these first steps into the 21st century, there is a “new” concern, a “new” interest in the paradigms of environmentalism and sustainability. There is also a philosophical interest (which assigns the new value of ‘resource’ to waste) and all is aimed towards the concept of an open landscape, unlike the previous, more static models, and the intention is to recover picturesque ideals as the starting point. Landscape urbanism has been established as a discipline capable of simultaneously responding to the natural and the artificial, replacing the traditional tools of architecture in order to resolve contemporary cities’ problems. It incorporates large scale infrastructures, such as urban waste landfills, and public landscapes which are generated as the true organisational mechanism of modern day urbanism. It is not merely a formal model, it is more important than that: it is a model of processes. This new concern allows us to address the matter of landscape in a broad way, without restrictions, and with a great degree of flexibility in the new proposals which come about as a consequence of these concepts. However, efforts to date seem to have been more directed at aesthetic aspects and we have yet to explore the political, social, economic and energetic consequences derived from waste – nor have we delved into the architectural consequences. The landscape urbanism project is involved with the horizontal surface, the ground plane. Traditionally, the preparation of this plane for the development of any human activity has been a foundational act, a necessary act of all architecture, but now this plane must be considered as a biologically active medium or support. In contemporary terms, the discipline’s interest lies in the continuity and accessibility of the land, diffusing the limits; in long term functionality; in the anticipation of change, via flexibility and the ability to negotiate; and in it being a public space. The recovery of an urban waste landfill offers all of these conditions. A brief look through history reveals the first isolated examples of recovery of these spaces of deterioration. They have gone through various phases based on the quantity and type of waste produced, they have evolved thanks to technology and a new outlook on the landscape, and a real environmental awareness has been developed (the birth of an ideology). Monte Testaccio in Rome (1st to 3rd Century AD) constitutes a paradigmatic and exemplary case of a landfill that was planned a priori not only as a place to deposit waste but also as a place that would be subsequently recovered and given back to the city in the form of a public space. This spoil mound, generated by organised and planned accumulation over three centuries, makes us reflect on the themes of production, consumption and architectural planning. Monte Testaccio reveals a source of inspiration, an archetype of the sustainable management of resources and land. Using our experience of contemporary cases of land recovery and its transformation into public spaces, such as the former Valdemingómez landfill in Madrid or the Garraf in Barcelona, we analysed the techniques and solutions used in order to establish new project tools. These are proposed with an eye on the future, seeing as they reveal the importance of the processes over the form and involve many factors (both natural and artificial), including the life and age of the accumulated living matter. They are places for new opportunities and examples of our new relationship with nature. The reactivation of landfills, via this project, is a proposal for a new topography built within time, using the ground as the support, as the meeting point between nature and the technological systems of the city which make it possible for new ways of life and new activities to come about. Landfills are immense natural topographical areas produced by artificial processes, watchtowers from which to discern a new horizon, a new world, a new future in which it will be possible to reverse our acts of deterioration. But the intention behind these recoveries and transformations does not only hope for landscape reintegration but it also hopes that they will also serve as a sign of the new attitudes that must be adopted by society with regard to environmental matters.

Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans

Difficulties in determining composition and sequence of glycosaminoglycans, such as those related to heparin, have limited the investigation of these biologically important molecules. Here, we report methodology, based on matrix-assisted laser desorption ionization MS and capillary electrophoresis, to follow the time course of the enzymatic degradation of heparin-like glycosaminoglycans through the intermediate stages to the end products. MS allows the determination of the molecular weights of the sulfated carbohydrate intermediates and their approximate relative abundances at different time points of the experiment. Capillary electrophoresis subsequently is used to follow more accurately the abundance of the components and also to measure sulfated disaccharides for which MS is not well applicable. For those substrates that produce identical or isomeric intermediates, the reducing end of the carbohydrate chain was converted to the semicarbazone. This conversion increases the molecular weight of all products retaining the reducing terminus by the “mass tag” (in this case 56 Da) and thus distinguishes them from other products. A few picomoles of heparin-derived, sulfated hexa- to decasaccharides of known structure were subjected to heparinase I digestion and analyzed. The results indicate that the enzyme acts primarily exolytically and in a processive mode. The methodology described should be equally useful for other enzymes, including those modified by site-directed mutagenesis, and may lead to the development of an approach to the sequencing of complex glycosaminoglycans.

Immunotargeting of liposomes to activated vascular endothelial cells: A strategy for site-selective delivery in the cardiovascular system

Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1β (IL-1β). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1β-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2–4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.

Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood–brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-β-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-β-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

Proteinase inhibitor-inducing activity of the prohormone prosystemin resides exclusively in the C-terminal systemin domain

Prosystemin is the 200-amino acid precursor of the 18-amino acid polypeptide defense hormone, systemin. Herein, we report that prosystemin was found to be as biologically active as systemin when assayed for proteinase inhibitor induction in young tomato plants and nearly as active in the alkalinization response in Lycopersicon esculentum suspension-cultured cells. Similar to many animal prohormones that harbor multiple signals, the systemin precursor contains five imperfect repetitive domains N-terminal to a single systemin domain. Whether the five repetitive domains contain defense signals has not been established. N-terminal deletions of prosystemin had little effect on its activity in tomato plants or suspension-cultured cells. Deletion of the C-terminal region of prosystemin containing the 18-amino acid systemin domain completely abolished its proteinase inhibitor induction and alkalinization activities. The apoplastic fluid from tomato leaves and the medium of cultured cells were analyzed for proteolytic activity that could process prosystemin to systemin. These experiments showed that proteolytic enzymes present in the apoplasm and medium could cleave prosystemin into large fragments, but the enzymes did not produce detectable levels of systemin. Additionally, inhibitors of these proteolytic enzymes did not affect the biological activity of prosystemin. The cumulative data indicated that prosystemin and/or large fragments of prosystemin can be active inducers of defense responses in both tomato leaves and suspension-cultured cells and that the only region of prosystemin that is responsible for activating the defense response resides in the systemin domain.

Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological (≈1 μM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.

Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm

Under physiological conditions, the Escherichia coli cytoplasm is maintained in a reduced state that strongly disfavors the formation of stable disulfide bonds in proteins. However, mutants in which the reduction of both thioredoxins and glutathione is impaired (trxB gor mutants) accumulate oxidized, enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate extragenic suppressors at a high frequency. One such suppressor strain, FA113, grows almost as rapidly as the wild type in the absence of reductant, exhibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substantially higher yields of properly oxidized proteins compared with wild-type or trxB mutant strains. For polypeptides with very complex patterns of disulfide bonds, such as vtPA and the full-length tPA, the amount of active protein was further enhanced up to 15-fold by co-expression of TrxA (thioredoxin 1) mutants with different redox potentials, or 20-fold by the protein disulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of E. coli FA113 compared with what could be achieved via secretion into the periplasm of a wild-type strain, even under optimized conditions. These results demonstrate that the cytoplasm can be rendered sufficiently oxidizing to allow efficient formation of native disulfide bonds without compromising cell viability.

The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments

Our recent studies have shown that deregulated expression of R2, the rate-limiting component of ribonucleotide reductase, enhances transformation and malignant potential by cooperating with activated oncogenes. We now demonstrate that the R1 component of ribonucleotide reductase has tumor-suppressing activity. Stable expression of a biologically active ectopic R1 in ras-transformed mouse fibroblast 10T½ cell lines, with or without R2 overexpression, led to significantly reduced colony-forming efficiency in soft agar. The decreased anchorage independence was accompanied by markedly suppressed malignant potential in vivo. In three ras-transformed cell lines, R1 overexpression resulted in abrogation or marked suppression of tumorigenicity. In addition, the ability to form lung metastases by cells overexpressing R1 was reduced by >85%. Metastasis suppressing activity also was observed in the highly malignant mouse 10T½ derived RMP-6 cell line, which was transformed by a combination of oncogenic ras, myc, and mutant p53. Furthermore, in support of the above observations with the R1 overexpressing cells, NIH 3T3 cells cotransfected with an R1 antisense sequence and oncogenic ras showed significantly increased anchorage independence as compared with control ras-transfected cells. Finally, characteristics of reduced malignant potential also were demonstrated with R1 overexpressing human colon carcinoma cells. Taken together, these results indicate that the two components of ribonucleotide reductase both are unique malignancy determinants playing opposing roles in its regulation, that there is a novel control point important in mechanisms of malignancy, which involves a balance in the levels of R1 and R2 expression, and that alterations in this balance can significantly modify transformation, tumorigenicity, and metastatic potential.

Rat heart: A site of oxytocin production and action

We report here that the rat heart is a site of oxytocin (OT) synthesis and release. Oxytocin was detected in all four chambers of the heart. The highest OT concentration was in the right atrium (2128 ± 114 pg/mg protein), which was 19-fold higher than in rat uterus but 3.3-fold lower than in the hypothalamus. OT concentrations were significantly greater in the right and left atria than in the corresponding ventricles. Furthermore, OT was released into the effluent of isolated, perfused rat heart (34.5 ± 4.7 pg/min) and into the medium of cultured atrial myocytes. Reverse-phase HPLC purification of the heart extracts and heart perfusates revealed a main peak identical with the retention time of synthetic OT. Southern blots of reverse transcription–PCR products from rat heart revealed gene expression of specific OT mRNA. OT immunostaining likewise was found in atrial myocytes and fibroblasts, and the intensity of positive stains from OT receptors paralleled the atrial natriuretic peptide stores. Our findings suggest that heart OT is structurally identical, and therefore derived from, the same gene as the OT that is primarily found in the hypothalamus. Thus, the heart synthesizes and processes a biologically active form of OT. The presence of OT and OT receptor in all of the heart’s chambers suggests an autocrine and/or paracrine role for the peptide. Our finding of abundant OT receptor in atrial myocytes supports our hypothesis that OT, directly and/or via atrial natriuretic peptide release, can regulate the force of cardiac contraction.

Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derived senescence retardant

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

Isolation and characterization of a tobacco mosaic virus-inducible myb oncogene homolog from tobacco

Salicylic acid (SA) plays an important role in signaling the activation of plant defense responses against pathogen attack including induction of pathogenesis-related (PR) proteins. To gain further insight into the SA-mediated signal transduction pathway, we have isolated and characterized a tobacco mosaic virus (TMV)-inducible myb oncogene homolog (myb1) from tobacco. The myb1 gene was induced upon TMV infection during both the hypersensitive response and development of systemic acquired resistance in the resistant tobacco cultivar following the rise of endogenous SA, but was not activated in the susceptible cultivar that fails to accumulate SA. The myb1 gene was also induced by incompatible bacterial pathogen Pseudomonas syringae pv. syringae during the hypersensitive response. Exogenous SA treatment rapidly (within 15 min) activated the expression of myb1 in both resistant and susceptible tobacco cultivars with the subsequent induction of PR genes occurring several hours later. Biologically active analogs of SA and 2,6-dichloroisonicotinic acid (a synthetic functional analog of SA), which induce PR genes and enhanced resistance, also activated the myb1 gene. In contrast, biologically inactive analogs were poor inducers of myb1 gene expression. Furthermore, the recombinant Myb1 protein was shown to specifically bind to a Myb-binding consensus sequence found in the promoter of the PR-1a gene. Taken together, these results suggest that the tobacco myb1 gene encodes a signaling component downstream of SA that may participate in transcriptional activation of PR genes and plant disease resistance.

A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

Death-associated protein kinase (DAP-kinase) is a Ca+2/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-γ to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-α-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the “linker” region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.

Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces

A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein–protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds.

Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor

Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor α and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor α release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.