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A set of new pyrimido[5,4-b]indole derivatives that are structurally related to some non-nucleside HIV-1 reverse transcriptase inhibitors were synthesized and biologically evaluated for their activity as inhibitors of wild and mutant HIV-1 RT types in an 'in vitro' recombinant HIV-1 RT screening assay, as well as anti-infectives in HLT4lacZ-1IIIB cells. Preliminary structure-activity relationships suggest that activity is promoted by simultaneous substitution in positions 2 and 4, especially when chains of alkyldiamine type are present, and by electron-releasing substituents (methoxy) in positions 7 and 8. The inactivity or the very low activity of title derivatives does not suggest interest in AIDS therapy.

Glucocorticoids and cAMP regulate, either in a synergistic or additive fashion, the transcription of multiple genes, although some antagonistic effects of dexamethasone on cAMP-activated transcription have been described. The increased glucocorticoid receptor (GR) mediated response of some cell types, as a result of augmented cAMP, has been considered to be mainly due to an increased stability of GR mRNA, although other plausible explanations should not be ruled out. We studied the possibility that GR transcription itself could be affected by cAMP levels. HeLa cells were transfected with human GR (hGR) promoter constructs and their transcriptional activity determined after inducing a cAMP increase with forskolin. We found that forskolin almost doubled the transcriptional activity of the promoter construct spanning −2995 to +38 of the hGR, whereas no significant variations were observed with shorter chimeras containing sequences downstream −979. Shift mobility showed binding of CREB in vitro to a putative cAMP responsive element located at −1000, suggesting that hGR may be upregulated by cAMP at the transcriptional level, thus adding a new mechanism ascribable to this second messenger, which in conjunction with the cAMP-induced GR mRNA increased stability, would lead to a more precise control of the amount of GR protein within the cell.

Activated and differentiated human monocytes with a CD14+CD16+ phenotype were found to contain a DNase activity associated with secretion granules. Activated cells were obtained from patients with autoimmune diseases. Activation and differentiation of monocytes were also achieved after incubation of PBMC from healthy subjects with protein A (SpA) or immunopotentiating peptides. DNase activity corresponded to a 66-kDa protein, similar to that described in granules from T lymphocytes, active preferentially on double-strand DNA. DNA fragmentation activity increased when NO donors were present; the activity was higher in the presence of Ca2+, and at low pH values. The Ca2+-dependent activity was inhibited by Zn2+. NO-dependent activity was additive with that of Ca2+-dependent and it was not inhibited by Zn2+. Dithiothreitol did not modify the effect of NO on DNase activity. Incubation of PBMC in the presence of NMLA, an inhibitor of NO synthases, decreased this DNase activity. Data reported clearly suggest a regulatory role of NO in granule-associated DNase activity.

Mobile app markets have experienced remarkable growth during the last year. The increasing number of apps available on the market and the revenue that developers and companies obtain is significant enough to seriously consider the way apps are developed. The ever-changing environment in which apps are developed makes agile methodology convenient to follow. Although agile methodologies allow the development team to quickly adjust the requisites to the new customer’s needs, there is a lack of research on how they can be explicitly adapted to develop mobile apps. There are many Websites that explain how to code a mobile app, but there is not enough information about other stages in the development process. Adapting an agile methodology for mobile apps would provide development teams with a clear guide to successfully develop an app without missing any step in the development process. This chapter proposes an agile mobile app development process, including processes and activities to be followed as well as the roles involved in these activities. Marketing issues are also considered in the proposed development process as they are necessary to publicize the mobile app. This process has been applied for over two years in the development of the institutional apps at Carlos III University of Madrid.

We have found a common structural motif in human autoantigens, heat shock proteins and viral proteins. Peptides modelled after sequences present in those molecules were synthesized and immunomodulating properties tested. They share a core of 15 amino acid residues and a common pattern ('2-6-11' motif) characterized by requirements at fixed positions with respect to a Pro (position 6); an apolar residue or a Lys at position 2; and a Glu, Asp or Lys at position 11. Any of these peptides, when added to cultures of lymphomononuclear cells, caused the activation of monocytes manifested by a release of IL-1 alpha, IL-1 beta and TNF alpha. A release of INF gamma and IL-2 took also place; this release was abolished by anti-DR antibodies. Neither IL-4 nor IL-5 could be detected. This suggests a presentation by APCs and the appearance of cells with a Th1 phenotype. Monocytes and Th1 cells freshly obtained from 12 patients of Graves' disease, 8 of Hashimoto's disease and 8 of primary biliary cirrhosis exhibited activation features similar to those found in cells from healthy subjects incubated in the presence of peptides with a "2-6-11' motif and representing fragments of autoantigens. Their immunopotentiating properties suggest their involvement in the initiation or progression of the autoimmune response mediated by activated monocytes and Th1 cells.

The presence of inducible nitric oxide synthase (iNOS) in fresh monocytes from patients with Graves' disease was demonstrated for the first time. Immunophenotypic analysis showed a profile reflecting a state of activation and differentiation of monocytes. Incubation of lymphomononuclear cells from healthy volunteers in the presence of synthetic peptides with sequences related to thyroid autoantigens (TSH receptor, thyroid peroxidase, or thyroglobulin) led to a stimulation of monocytes manifested by a change in phenotype and expression of iNOS. This expression did not take place on isolated monocytes, unless products associated with Th1 activity were present in the medium. Active peptides contained a characteristic "2-6-11" motif already described [López-Moratalla et al. (1995) Biochim. Biophys. Acta 1265, 181-188]. These results are suggestive of a new role for autoantigens in the pathogenesis of Graves' disease: that of inducing the expression of iNOS and activating the monocyte possibly underlying the autoimmune response.

La presente tesis plantea una investigación que se centra, sobre todo, en los personajes femeninos del libro Los que se van. Esta propuesta interpretativa busca renovar la metodología de la crítica tradicional merced a la atención que presta a los elementos ‘internos’ de la obra literaria (personajes, espacio, diálogos) para luego propender a una lectura asentada en el plano contextual o referencial. Se indaga en las posibles similitudes y diferencias que presentan sus formas de representar a la mujer y se realiza una interpretación de género. Su objetivo es comprobar que ciertos modelos patriarcales de representación de la mujer se mantiene en este volumen. Para ello, el trabajo se sirve de estudios como los de Benjamín Carrión, Agustín Cueva y Francisco Proaño Arandi, de concepciones sobre la mujer y el personaje literario femenino en las propuestas de Erich Neumann, Lucía Guerra‐Cunningham y Jorge O. Andrade y, de reflexiones narratológicas, principalmente de Algirdas Julius Greimas y de Mieke Bal. Por último, este trabajo se evidencia como una propuesta renovadora en la investigación y la crítica literarias.

Una de las complicaciones secundaria a los tratamientos de reproducción asistida más grave es el Síndrome de Hiperestimulación Ovárica (SHO) y consiste en una respuesta anormalmente alta de los ovarios a la estimulación hormonal. Partimos de la hipótesis de que el SHO afecta negativamente a la calidad ovocitaria y embrionaria y, por lo tanto, se obtendrán menores tasa de fecundación y de embarazo. La utilización de un análogo de la GnRH y la vitrificación de los embriones para transferirlos en un ciclo posterior, sería el tratamiento más efectivo para disminuir los riesgos del SHO y obtener unos buenos resultados. En este trabajo se evalúa la calidad ovocitaria y embrionaria en pacientes con riesgo de SHO en comparación con pacientes sanas, asimismo se analizan todos aquellos factores relacionados con el SHO, como la edad y los niveles hormonales.

Summary The synthesis and the study of the activity of new indol-2-carboxamides and pyridazino[4,5- b ]indoles as inhibitors of HIV-1 reverse transcriptase (RT) are presented. The activity of the compounds synthesized as inhibitors of different types of HIV-1 RT (wild type enzyme and mutant forms P236L, Y 181C and P236L/Y181C) was evaluated. The activity of the most active compounds was investigated in the syncytia reduction in vitro assay, in HIV-1 IIIB -infected HT4lacZ-1 cells. Their potential cytotoxicity was determined in parallel. Two lead compounds, N -[1-[2-(3-isopropylamino)pyridyl]piperazin]-5,6-methylenedioxy indol-2-carboxamide 7q and N -[1-[2-(3-ethylamino)pyridyl]piperazin]-5,6-methylenedioxyindol-2-carboxamide 7s have been identified.

Hoy en día, los teléfonos inteligentes forman parte de la vida de todo tipo de personas, desde los usuarios más jóvenes a los más adultos, y con objetivos diferentes, como pueden ser por cuestiones de trabajo, estudio, ocio o incluso seguridad. Debido al gran avance de las tecnologías y a los nuevos diseños de terminales ligeros e inalámbricos, cada vez son más las personas que utilizan el teléfono móvil o las tablets en lugar del ordenador, principalmente para el acceso a aplicaciones de ocio y estudio, así como para la navegación en Internet. De este modo, los mercados virtuales de estos teléfonos móviles inteligentes lanzan una gran variedad de aplicaciones destinadas a diferentes tipos de usuarios, con el propósito de conseguir un beneficio gracias a la descarga de dichas aplicaciones. Dicho beneficio puede ser directo, que consistiría en el cobro de la aplicación por cada descarga de la misma. O por otro lado, el beneficio puede ser indirecto, donde la aplicación puede ser gratuita o de coste muy bajo pero incluyendo publicidad, o simplemente que la aplicación proponga una idea interesante y bien desarrollada y aumente el prestigio de la empresa que la desarrolló. Siguiendo estos argumentos, y teniendo en cuenta el gran número de personas que utilizan los dispositivos iPhone y/o iPod, el presente trabajo muestra el desarrollo de una aplicación para dichos dispositivos. La aplicación está orientada a todas aquellas personas que se encuentran en un periodo de estudio del permiso de conducción de automóviles. Se ha desarrollado una aplicación con una interfaz sencilla y amigable, que permita a los usuarios aprender las normas de circulación con el fin de examinarse del test que demuestra que una persona es apta para continuar con las pruebas prácticas del permiso de conducción de automóviles. De este modo, el usuario de la aplicación puede evitar tener que desplazarse a la autoescuela, así como tener que estar en frente de un ordenador. Simplemente accediendo a la aplicación desde su teléfono móvil iPhone o desde su dispositivo iPod, podrá estudiar todos los temas teóricos que enseñan en las autoescuelas con la posibilidad de ver una breve descripción de cada una de ellas, así como un vídeo que orientará al usuario a entender el contenido de cada tema. Una vez aprendida la lección, se podrán realizar test relacionados con aquello que se ha estudiado de modo que el usuario de la aplicación estará preparado para realizar el examen teórico del permiso de conducción de automóviles. _______________________________________________________________________________________________________________________ Nowadays, smartphones are part of life for every kind of people, from youngest to the adult users, and with different objectives, such as work issues, study, leisure or even security. Due to great advances in technology and new terminal designs (light and wireless mobile terminals), more and more people using the mobile phone or tablets instead of computers, primarily for access to leisure and study applications and for Internet browsing. Thus, virtual markets of these smartphones launch a variety of applications for different types of users, in order to get a benefit by downloading these applications. This benefit can be a direct benefit, which would be based in charging the application by either download on the Internet. On the other hand, the benefit may be indirect benefit, where the application can be free or very low cost but including advertising or simply the application proposes an interesting and well developed idea and this causes an increase in the prestige of the company that developed it. According to these arguments, and taking into account the large number of people using the iPhone and iPod, this paper shows the development of an application for such devices. The application is aimed at people who are in a study period of car driving license. It has been developed an application with a simple and friendly interface, allowing users to learn the traffic rules in order to take the exam that demonstrate a person is able to continue with the practical tests of car driving license. In this way the user could avoid having to go to driving school as well as staying in front of a computer. Simply accessing the application from his iPhone or from his iPod, user is able to study every theoretical lesson teaching in driving schools. Application provides a brief description of each lesson, and a video that will guide the user understand the contents of each lesson. After learning the lesson, tests can be made regarding what has been studied, so the user of the application will be ready for the theoretical examination of car driving license. Ingeniería Informática

The Ca²( +) -dependent ATPase activity of spinach chloroplast coupling factor 1 (CF₁) is activated by treatment with dithiothreitol (DDT). If excess of this reagent is eliminated by gel filtration, an Eadie-Hofstee biphasic plot is obtained. These results are consistent with the existence of two active forms of the enzyme governed by the redox state. We have observed that SDS-polyacrylamide gel electrophoresis pattern is affected by the pretreatment of the samples under those two different conditions. Spontaneous activation of the samples, due to a limited proteolytic process, has also been detected. In this case the electrophoretic pattern was also affected. The protease implied in this process could be a cystein protease co-isolated with CF₁. These observations suggest that limited proteolysis, as well as redox-induced changes, are involved in the physiological regulation of the enzyme.

Peptides from 10 to 22 amino acids containing sequences encompassed by Staphylococcus aureus protein A were synthesized. Some of these peptides, when present in cultures of lymphomononuclear cells from healthy donors or from cancer patients (melanoma, breast carcinoma, non-Hodgkin lymphoma and renal cell carcinoma) promoted: (i) changes in the phenotype of the lymphomononuclear population, (ii) stimulation of monocytes (release of IL-1 and TNF-alpha), and (iii) an increase in cytotoxicity against K562, Daudi and HT-29 cells. Isolated monocytes responded also to those peptides with a release of IL-1 and TNF alpha and an increase of cytotoxicity against HT-29 cells. It was found that the active peptides had the following structural pattern: a length of at least 15 amino-acid residues with a proline at position 6, valine, leucine, isoleucine, glycine, alanine or lysine at position 2, and glutamic or aspartic acid at position 11. Replacement of Pro at position 6 with any other residue turned the peptide inactive. Replacement of residues at positions 2 and 11 with amino-acid residues other than those required for activity resulted in compounds with a marked decrease in the immunomodulating properties described, or lacking these properties altogether.

The ability to detect chromosomal or genetic defects in embryos in vitro, associated with assisted human reproduction techniques before his possible transfer to the uterus to complete its development was presented as an alternative to eugenic abortion. And an option for older women to procreate, to avoid pregnancy of embryos with chromosomal defects. Genetic diagnosis before implantation (PGD) and screening of embryos in vitro (by the acronym, PSC), offers the image of the disabled person as an individual excluded from society. It assumes a direct human experimentation without therapeutic purposes or to manipulate the embryo that is chosen or discarded according to diagnosis or for advancement in perinatal medicine. Because these techniques can have multiple embryos, eugenics has also generated a ″positive eugenics″ that seeks to select embryos according to a third party, having certain characteristics, sex, or lack of predisposition to disease. Several issues demand unavoidable ethical duty to report on this form of eugenics, in addition to be directly and intentionally directed to destroy human life in its early stages, and does not meet the minimum requirements of rigorous scientific research or biotechnology. There have been no previous animal tests to validate the techniques so that there are serious errors in diagnosis with false positives and false negatives. Recently it has been shown that some discarded embryos can eliminate their detected defects two days after the biopsy. Moreover, the study about what may or may not be diagnosed is retrospective and unrecoverable damage. And, of particular importance is the fact that it is not known with certainty the effects that an embryo biopsy may cause to those diagnosed.

In the period between puberty and maturity takes place the process of brain maturation. Hormone levels induce changes in neurons and direct the architecture and structural functionality thus affecting patterns of development of different brain areas. The onset of puberty brings with it the invasion of the female brain by high levels of hormones, cyclic surges of estrogen and progesterone in addition to steroids produced in situ. Control centers of emotions (amygdala), memory and learning (hippocampus) and sexual activity (hypothalamus) are modified according to the cyclical concentrations of both hormones. Sex hormones stimulate multimodal actions, both short and longer terms, because neurons in various brain areas have different types of receptors, membrane, cytoplasmic and nuclear. The composition of emergency contraceptive pill (postcoital pill) with high hormonal content raises the urgency of a thorough knowledge about the possible effect that the lack of control of the menstrual cycle in a time of consolidation of brain maturation, can bring in structuring and development of brain circuitry. Changes in the availability of sex steroids during puberty and adolescence underlie psychiatric disorders whose prevalence is typically feminine, such as depression, anxiety disorders. It is a fundamental ethical duty to present scientific data about the influence of estrogen in young female brain maturation, both for full information to potential users, and also to induce the appropriate public health measures.

A set of new pyrimido[5,4-b]indole derivatives that are structurally related to some non-nucleside HIV-1 reverse transcriptase inhibitors were synthesized and biologically evaluated for their activity as inhibitors of wild and mutant HIV-1 RT types in an 'in vitro' recombinant HIV-1 RT screening assay, as well as anti-infectives in HLT4lacZ-1IIIB cells. Preliminary structure-activity relationships suggest that activity is promoted by simultaneous substitution in positions 2 and 4, especially when chains of alkyldiamine type are present, and by electron-releasing substituents (methoxy) in positions 7 and 8. The inactivity or the very low activity of title derivatives does not suggest interest in AIDS therapy.

Acercamiento a la figura del literato veneciano Lodovico Dolce (ca. 1510-1568) a través de las obras conservadas en la Biblioteca Histórica Marqués de Valdecilla, prestando especial atención a su Dialogo della memoria (1ª ed. 1562) y a las relaciones entre imagen y texto allí presentes, que se rastrean también en otras de sus publicaciones.[ABSTRAC]Approach to the figure of the Venetian man of letters Lodovico Dolce (ca. 1510-1568) through the works kept at the Biblioteca Histórica Marqués de Valdecilla, paying special attention to his Dialogo della memoria (1st ed. 1562) and to the connections between image and text present in it, which are also traced in some of his other publications.

Approach to the prehistory of the Dialogo della pittura by Lodovico Dolce (1557), paying special attention to its connection with the letters by Michelangelo, Raphael and Titian included in the Lettere di diversi (1554) compiled by the writter. In Appendix also the translation into Spanish and comment of the letters of artistic argument that appeared in the second edition of the Lettere (1555) which Dolce addressed to Gasparo Ballini and Alessandro Contarini. Acercamiento a la prehistoria del Dialogo della pittura de Lodovico Dolce (1557), prestando especial atención a su relación con las cartas de Miguel Ángel, Rafael y Tiziano incluidas en las Lettere di diversi (1554) recopiladas por el literato. Se traducen y comentan en Apéndice las cartas de sujeto artístico aparecidas en la segunda edición de las Lettere (1555) que Dolce dirige a Gasparo Ballini y Alessandro Contarini.

A medida que las nuevas tecnologías evolucionan en una era basada en la informática y las telecomunicaciones, la lógica de negocio de las compañías corre el riesgo de quedarse obsoleta debido a las preferencias del mercado. Los productos o servicios ofertados deben orientarse y especializarse cada vez más en función del tipo de cliente que requiere de ellos. Los clientes ya no se conforman con lo que encuentran, ya que actualmente tienen múltiples opciones para satisfacer sus necesidades. Hoy en día, la competencia, además de ser muy numerosa, es de clases muy distintas, por lo que muchas empresas necesitan realizar un cambio de mentalidad y dar un giro al modo de llevar su negocio. La necesidad de las empresas de adaptarse a los cambios continuos en la forma de trabajo, así como de hacer frente a la presión ejercida por la competencia, otorga una importancia significativa al fenómeno de reingeniería. En el presente proyecto se pretende mostrar como la reingeniería ayuda al desarrollo y evolución de los productos software de una empresa, sobre todo si se trata de un sistema amplio con gran complejidad. Para ello, se mostrará cómo aplicar un proceso de reingeniería del software en un sistema de información, basado en una migración del software. De este modo se obtendrá un sistema adaptado a las nuevas tecnologías que pueda enfrentarse a la competencia.------------------------------------------------------------------------- As new technologies evolve in an era based on informatics and telecommunications, the business logic of the companies run the risk of becoming obsolete due to market preferences. The products or services offered should be increasingly designed and specialized depending on the type of customer that requires them. Customers are no longer satisfied with what they find, as they currently have multiple options to meet their needs. Nowadays, rival companies, as well as being many, are very different kinds, so many companies need to change their mind and vary the way they run their business. The need for companies to adapt themselves to continuous changes in the way of work and to cope with the pressure of competition, gives a significant importance to the phenomenon of reengineering. The present project aims to show how reengineering helps to develop any company’s software products, especially if it is a comprehensive system with great complexity. To do this, will show how putting into practice a software reengineering process in the system based on a technology migration plan. This will provide us with a system adapted to new technologies that may face competition. Ingeniería en Informática

It has been reported previously that a short synthetic immunomodulating peptide (Pa) and the neuropeptide beta-endorphin modulate the immune system. We have found now that NF-kappaB participates in the stimulation of monocytes by both peptides and we investigated the molecular mechanism by which these stimuli activate NF-kappaB. Pa and beta-endorphin induce accumulation of cyclic 3('),5(')-adenosine monophosphate (cAMP) in a calcium/calmodulin-dependent fashion since it was completely inhibited by the calmodulin antagonist W-7. The effect of these complexes seems to be mediated, at least in part, by nitric oxide (NO) synthesized by constitutive NO synthase since the NO synthase inhibitor N-methyl-L-arginine (NMLA) reduced the elevation of cAMP. Furthermore, the NO donor SIN-1 provoked nitration of G(S)alpha, leading to the cAMP elevation that was suppressed by the G(S)alpha-selective antagonist NF-449. Interestingly, the rapid degradation of NF-kappaB inhibitor IkappaBalpha induced by Pa- and beta-endorphin was reversed by a pretreatment with H-89 and cyclosporin A, inhibitors of protein kinase A (PKA) and protein phosphatase 2B (PP2B), respectively. These observations are consistent with the inhibition caused by W-7, NMLA, H-89, and cyclosporin A on NF-kappaB induction by these agonists, indicating the involvement of PKA and PP2B in the regulation of NF-kappaB in human monocytes.

A short immunomodulating peptide (Pa) containing a defined structural motif present in a number of extracellular matrix proteins and autoantigens was found to stimulate human monocytes. Pa-induced apoptosis of isolated monocytes, as indicated by internucleosomal DNA cleavage, increased annexin V binding capacity and cleavage of caspase substrates, such as poly(ADP)ribosylpolymerase. In addition, Bcl-2 protein levels were downregulated during Pa-induced cell death. Nuclear extracts of monocytes incubated with Pa showed higher neutral, Ca(2+)-dependent DNase activity than those obtained from nontreated monocytes. Caspase inhibitors prevented Pa-induced apoptosis, Bcl-2 depletion, and DNase activation. Treatment of monocytes with Pa activated c-Jun N-terminal kinases and p38 kinase, in an acidic sphingomyelinase- and caspase-dependent fashion. Pa-induced apoptosis was blocked by selective inhibitors of p38 kinase (SB203580) and acidic sphingomyelinase (SR33557). These results indicate that JNK and p38 kinase stimulation as well as monocyte apoptosis induced by Pa could depend, at least in part, on early activation of acidic sphingomyelinase.

Human monocyte subsets, isolated from cultures of mononuclear cells, or freshly obtained from patients with multiple sclerosis, Graves' disease or pemphigus vulgaris, differed in phenotype, apoptotic features, mRNA levels of arginase II (A-II) and the inducible form of nitric oxide synthase (iNOS). Liver-type arginase I mRNA was present in all subsets. Apoptosis was followed by the expression of T cell intracellular antigen (TIA) and the simultaneous detection of DNA stainability by propidium iodine and annexin V binding. Apoptosis was practically absent both in activated CD14(++)CD33(++)DR(++)CD25(++)CD69(++)CD71(++/+) CD16(-) cells, expressing A-II mRNA and having arginase activity, but not iNOS mRNA, and in not fully mature large CD14(++)CD16(+)CD23(+)DR(++) monocytes, expressing simultaneously both mRNAs and having both enzyme activities. However, differentiated small CD14(+/++)CD16(+)CD69(+)CD25(+/-)CD71(++)CD23(+) DR(++) monocytes, expressing high levels of iNOS mRNA, exhibited apoptotic signs. Amounts of NO synthesised by monocytes co-expressing iNOS and arginase changed with the addition of arginine or an iNOS inhibitor; in that case a correlation of NO production and apoptotic features was observed. Data suggest a regulatory role for endogenous NO in apoptosis of stimulated and differentiated monocytes, and also that iNOS and A-II, when simultaneously present, could control the production of NO as a consequence of their competition for arginine.

Involvement of phosphodiesterase isoenzymes (PDEs) in guanosine-3',5'-cyclic monophosphate (cGMP) hydrolysis was analyzed in aortic smooth muscle cells. Four families of PDEs were separated from pig aorta: PDE1 (calcium-calmodulin-activated), PDE3 (cGMP-inhibited), PDE4 (adenosine 3',5'-cyclic monophosphate [cAMP]-specific), and PDE5 (cGMP-specific). Within this PDE complement, PDE1 and PDE5 mostly contributed to the hydrolysis of cGMP both in the presence and absence of calcium-calmodulin. The role of these isoenzymes in cGMP degradation was analyzed in primary cultures of porcine aortic smooth muscle cells after stimulation with sodium nitroprusside (SNP) or atrial natriuretic factor (ANF). Pretreatment with 10 microM zaprinast, a concentration that selectively inhibits PDE5, did not potentiate the SNP- or ANF-induced rise of cGMP, questioning the widespread opinion that only PDE5 accounts for cGMP hydrolysis in this tissue. Further evidence came from experiments assessing the effect of zaprinast or 3-isobutyl-1-methylxanthine at concentrations inhibiting both type 1 and type 5 isoenzymes, in which this potentiation was clearly seen. Contribution of cGMP egression to the control of intracellular cGMP levels after SNP or ANF stimulation was also investigated. Shortly after guanylate cyclase activation, extracellular cGMP levels surpassed intracellular levels. However, comparison of the amounts of cGMP extruded to the extracellular medium with those degraded by PDEs leads to the conclusion that efflux is of relatively minor importance in regulating intracellular cGMP levels. In cells made tolerant to SNP, selective PDE5 inhibition synergistically increased intra- and extracellular cGMP amounts after SNP stimulation. These results indicate a previously undescribed greater relevance of PDE5 after tolerance development in aortic smooth muscle cells.

Lipophilic and hydrophilic extracts of four Argentine plants (Gamochaeta simplicaulis Cabr. 1, Achyrocline flaccida Wein. D. C. 2, Eupatorium buniifolium H. et A. 3, and Phyllanthus sellowianus Muell. Arg. 4) were examined in vitro for their ability to inhibit the polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (wild and Y181C mutant types). The active extracts were also examined as inhibitors of viral replication in HLT4LacZ-1 IIIB cell cultures, evaluating their cytotoxicity in parallel. Infusions 21 and 41, among the crude extracts, showed the highest activity. These extracts were refractioned into four fractions; 2I 4 and 4I 4 were active as inhibitors of DNA-polymerase (wild and Y181C types) and RNase H activities. These fractions were potent as inhibitors of viral replication and were not cytotoxic. Refractionation of 214 yielded five new fractions, two of which, 2I 4 -4 and 2I 4 -5, showed notable activity. Refractionation of 4I 4 yielded four new fractions; of these, 414-3 and 414-4 were active. The marked biological activity found in the infusion of A. flaccida and P. sellowianus makes them sufficiently attractive to be considered in the combined chemotherapy of the disease. Copyright © 1999 John Wiley & Sons, Ltd.

Evidence suggesting a relationship between neuroendocrine and immune systems is steadily growing. We demonstrate now that inducible nitric oxide synthase (iNOS) is expressed in human peripheral blood monocytes after incubation of lymphomononuclear cells in the presence of beta-endorphin, a neuropeptide released by the pituitary in response to mental or physical stress or by activated lymphocytes. beta-endorphin raised cAMP level in monocytes. The possible relationship between cAMP and iNOS expression on monocytes was investigated. Immunostaining for iNOS decreased, when besides beta-endorphin an inhibitor of protein kinase A (H-89) was added to the medium at the beginning of the incubation. The cAMP level raised by beta-endorphin was lowered by naloxone, which also reduced slightly iNOS expression. These results clearly point to the monocyte as a link between neuroendocrine and immune systems, an observation of potential relevance in our understanding of how stress and autoimmunity could be interconnected.

The authors have already described that a series of short peptides, modelled after sequences related to human extracelluar matrix (ECM) proteins and sharing some common structural features, activate Th1 clones through a process involving peptide presentation in HLA-DR proteins. Those peptides induce also LAK- and NK-dependent cytotoxicity as well as activation of monocytes/macrophages present in human peripheral blood mononuclear cell (PBMC) populations. The release of interleukin 2 (IL-2) and interferon γ (IFN-γ) by Th cells present in PBMC depleted of macrophages, or B cells is reported, after incubation in the presence of those peptides, fibronectin or Staphylococcus aureus protein A. The authors found that all the molecules tested needed at least the presence of a type of antigen-presenting cell (APC) to exert their stimulatory effect. Some peptides seem to be preferentially presented to Th cells by B cells, while others seem to depend on monocyte/macrophages for this presentation. The dependence on one or another APC seems to be due to differences in the sequences of these peptides. The immunomodulatory agents studied also gave rise to a clear increase in a DNase activity associated with secretion granules of PBMC. That there is a correlation between the release of IL-2 and this DNase activity when using a complete PBMC population, B cell-depleted PBMC or macrophage-depleted PBMC stimulated with the peptides tested has been found.

Different methods for studying the concurrent effects of two linear inhibitors on a single enzyme have been published, including the fractional product of Webb, the Yonetani-Theorell plot or the method of Chou and Talalay. Recently the use of combination plots has also been advocated for this purpose. We have evaluated the applicability of these methods and found that most of them depend on assumptions about the mechanism of action of the inhibitors. If the mechanism of action is not completely understood, or if some assumptions about the mechanism are unfounded, the parameters obtained may be meaningless. Even if these assumptions are correct, the interaction can be advantageously measured using an alternative representation that does not require a knowledge of the inhibition constants and allows experimental data to be retrieved from the plot. In other cases it is the interpretation of the results rather than the validity of the method that is misleading. A common mistake is to take the exclusivity of the effects of two inhibitors as exclusivity of their binding. We show that this assumption is seldom justified. In any case, it is possible to decide whether the combination of two or more inhibitors is more effective than their individual use by means of isobolographic analysis, even when no information about their mechanism of action is available.

We analyzed the phenotypic characteristics of PBMC from 34 patients with Graves' disease (GD) at different stages of the disease to explore the sequence of immunological events associated with it. In all cases their monocytes were in a state of activation and differentiation more advanced than those of a group of 23 healthy individuals. Strikingly, some patients had CD14++DR- immature monocytes, which were absent in healthy individuals. CD14+CD16+DRhigh monocytes were more abundant in patients. We found a positive correlation between the CD14++DR- monocyte and CD4+CD45RA- helper cells and a negative correlation between the same monocyte subset and CD4+CD45RA+ naive cells. CD14+/++DRlow monocytes directly correlated with this latter T4 subset and CD14+ CD16+DRhigh with CD4+CD45RO+ memory lymphocytes. There was also a positive correlation between memory T4 cells and the subset of activated B lymphocytes (CD19+CD5+) and suppressor T8 cells (CD8+CD11b+). T8 cytotoxic cells (CD8+CD11b-) positively correlated with T4 naive cells. The circulating levels of T3 and TSI (thyroid-stimulating immunoglobulin) directly correlated with a decrease in naive cells and an increase in T8 suppressors. The results suggest that the imbalance suppression/cytotoxicity in GD may be due to a reiterated presentation of autoantigens, or mimetic antigens, to T helpers by mature monocytes and activated B cells.

Immunophenotypic analysis of peripheral blood leukocytes from patients with multiple sclerosis (MS) showed a profile reflecting a state of activation and differentiation of monocytes. A subset of CD16+ monocytes with high HLA-DR expression was more prominent in patients with MS than in healthy subjects. The presence of the inducible form of nitric oxide synthase (iNOS) in these differentiated and activated monocytes freshly obtained from patients with MS was demonstrated by immunocytochemistry and flow cytometry analysis with two different antibodies. Incubation of lymphomononuclear cells from healthy volunteers in the presence of an immunomodulating peptide (NVLGAPKKLNESQAV) led to stimulation and maturation of monocytes manifested by changes in phenotype and an increase in both iNOS mRNA and protein, as well as HLA-DR expression. In this case also iNOS was expressed mainly on subsets of CD16+ monocytes with high HLA-DR expression. NO produced by human monocytes seems to have a function in the upregulation of membrane HLA-DR. These results are suggestive of a role for monocytic iNOS in the autoimmune response underlying the pathogenesis of multiple sclerosis.