Caracterización de sistemas vibroacústicos mediante análisis estadístico de energía

Hoy día nadie discute la importancia de predecir el comportamiento vibroacústico de estructuras (edificios, vehículos aeronaves, satélites). También se ha hecho patente, con el tiempo, que el rango espectral en el que la respuesta es importante se ha desplazado hacia alta frecuencia en prácticamente todos los campos. Esto ha hecho que los métodos de análisis en este rango alto de frecuencias cobren importancia y actualidad. Uno de los métodos más extendidos para este fin es el basado en el Análisis Estadístico de la Energía, SEA. Es un método que ha mostrado proporcionar un buen equilibrio entre potencia de calculo, precisión y fiabilidad. En un SEA el sistema (estructura, cavidades o aire circundante) se modela mediante una matriz de coeficientes que dependen directamente de los factores de pérdidas de las distintas partes del sistema. Formalmente es un método de análisis muy cómodo e intuitivo de manejar cuya mayor dificultad es precisamente la determinación de esos factores de pérdidas. El catálogo de expresiones analíticas o numéricas para su determinación no es suficientemente amplio por lo que normalmente siempre se suele acabar necesitando hacer uso de herramientas experimentales, ya sea para su obtención o la comprobación de los valores utilizados. La determinación experimental tampoco está exenta de problemas, su obtención necesita de configuraciones experimentales grandes y complejas con requisitos que pueden llegar a ser muy exigentes y en las que además, se ven involucrados problemas numéricos relacionados con los valores de los propios factores de pérdidas, el valor relativo entre ellos y las características de las matrices que conforman. Este trabajo estudia la caracterización de sistemas vibroacústicos mediante el análisis estadístico de energía. Se centra en la determinación precisa de los valores de los factores de pérdidas. Dados los problemas que puede presentar un sistema experimental de estas características, en una primera parte se estudia la influencia de todas las magnitudes que intervienen en la determinación de los factores de pérdidas mediante un estudio de incertidumbres relativas, que, por medio de los coeficientes de sensibilidad normalizados, indicará la importancia de cada una de las magnitudes de entrada (esencialmente energías y potencias) en los resultados. De esta parte se obtiene una visión general sobre a qué mensurados se debe prestar más atención, y de qué problemas pueden ser los que más influyan en la falta de estabilidad (o incoherencia) de los resultados. Además, proporciona un modelo de incertidumbres válido para los casos estudiados y ha permitido evaluar el error cometido por algún método utilizado habitualmente para la caracterización de factores de pérdidas como la aproximación a 2 subsistemas En una segunda parte se hace uso de las conclusiones obtenidas en la primera, de forma que el trabajo se orienta en dos direcciones. Una dirigida a la determi nación suficientemente fiel de la potencia de entrada que permita simplificar en lo posible la configuración experimental. Otra basada en un análisis detallado de las propiedades de la matriz que caracteriza un SEA y que conduce a la propuesta de un método para su determinación robusta, basada en un filtrado de Montecarlo y que, además, muestra cómo los problemas numéricos de la matriz SEA pueden no ser tan insalvables como se apunta en la literatura. Por último, además, se plantea una solución al caso en el que no todos los subsistemas en los que se divide el sistema puedan ser excitados. El método propuesto aquí no permite obtener el conjunto completo de coeficientes necesarios para definir un sistema, pero el solo hecho de poder obtener conjuntos parciales ya es un avance importante y, sobre todo, abre la puerta al desarrollo de métodos que permitan relajar de forma importante las exigencias que la determinación experimental de matrices SEA tiene. ABSTRACT At present there is an agreement about the importance to predict the vibroacoustic response of structures (buildings, vehicles, aircrafts, satellites, etc.). In addition, there has become clear over the time that the frequency range over which the response is important has been shift to higher frequencies in almost all the engineering fields. As a consequence, the numerical methods for high frequency analysis have increase in importance. One the most widespread methods for this type of analysis is the one based on the Statistical Energy Analysis, SEA. This method has shown to provide a good balance among calculation power, accuracy and liability. Within a SEA, a system (structure, cavities, surrounding air) is modeled by a coefficients matrix that depends directly on the loss factors of the different parts of the system. Formally, SEA is a very handy and intuitive analysis method whose greatest handicap is precisely the determination of the loss factors. The existing set of analytical or numerical equations to obtain the loss factor values is not enough, so that usually it is necessary to use experimental techniques whether it is to its determination to to check the estimated values by another mean. The experimental determination presents drawbacks, as well. To obtain them great and complex experimental setups are needed including requirements that can be very demanding including numerical problems related to the values of the loss factors themselves, their relative value and the characteristics of the matrices they define. The present work studies the characterization of vibroacousti systems by this SEA method. It focuses on the accurate determination of the loss factors values. Given all the problems an experimental setup of these characteristics can show, the work is divided in two parts. In the first part, the influence of all the quantities involved on the determination of the loss factors is studied by a relative uncertainty estimation, which, by means of the normalised sensitivity coefficients, will provide an insight about the importance of every input quantities (energies and input powers, mainly) on the final result. Besides, this part, gives an uncertainty model that has allowed assessing the error of one of the methods more widely used to characterize the loss factors: the 2-subsystem approach. In the second part, use of the former conclusions is used. An equation for the input power into the subsystems is proposed. This equation allows simplifying the experimental setup without changing the liability of the test. A detailed study of the SEA matrix properties leads to propose a robust determination method of this SEA matrix by a Monte Carlo filtering. In turn, this new method shows how the numerical problems of the SEA matrix can be overcome Finally, a solution is proposed for the case where not all the subsystems are excited. The method proposed do not allows obtaining the whole set of coefficients of the SEA matrix, but the simple fact of getting partial sets of loss factors is a significant advance and, over all, it opens the door to the development of new methods that loosen the requirements that an experimental determination of a SEA matrix have.

Impact assessment of an integrated approach for thermal power generation plants asset management in the current energy market

El sector energético, en España en particular, y de forma similar en los principales países de Europa, cuenta con una significativa sobrecapacidad de generación, debido al rápido y significativo crecimiento de las energías renovables en los últimos diez años y la reducción de la demanda energética, como consecuencia de la crisis económica. Esta situación ha hecho que las centrales térmicas de generación de electricidad, y en concreto los ciclos combinados de gas, operen con un factor de utilización extremadamente bajo, del orden del 10%. Además de la reducción de ingresos, esto supone para las plantas trabajar continuamente fuera del punto de diseño, provocando una significativa pérdida de rendimiento y mayores costes de explotación. En este escenario, cualquier contribución que ayude a mejorar la eficiencia y la condición de los equipos, es positivamente considerada. La gestión de activos está ganando relevancia como un proceso multidisciplinar e integrado, tal y como refleja la reciente publicación de las normas ISO 55000:2014. Como proceso global e integrado, la gestión de activos requiere el manejo de diversos procesos y grandes volúmenes de información, incluso en tiempo real. Para ello es necesario utilizar tecnologías de la información y aplicaciones de software. Esta tesis desarrolla un concepto integrado de gestión de activos (Integrated Plant Management – IPM) aplicado a centrales de ciclo combinado y una metodología para estimar el beneficio aportado por el mismo. Debido a las incertidumbres asociadas a la estimación del beneficio, se ha optado por un análisis probabilístico coste-beneficio. Así mismo, el análisis cuantitativo se ha completado con una validación cualitativa del beneficio aportado por las tecnologías incorporadas al concepto de gestión integrada de activos, mediante una entrevista realizada a expertos del sector de generación de energía. Los resultados del análisis coste-beneficio son positivos, incluso en el desfavorable escenario con un factor de utilización de sólo el 10% y muy prometedores para factores de utilización por encima del 30%. ABSTRACT The energy sector particularly in Spain, and in a similar way in Europe, has a significant overcapacity due to the big growth of the renewable energies in the last ten years, and it is seriously affected by the demand decrease due to the economic crisis. That situation has forced the thermal plants and in particular, the combined cycles to operate with extremely low annual average capacity factors, very close to 10%. Apart from the incomes reduction, working in out-of-design conditions, means getting a worse performance and higher costs than expected. In this scenario, anything that can be done to improve the efficiency and the equipment condition is positively received. Asset Management, as a multidisciplinary and integrated process, is gaining prominence, reflected in the recent publication of the ISO 55000 series in 2014. Dealing Asset Management as a global, integrated process needs to manage several processes and significant volumes of information, also in real time, that requires information technologies and software applications to support it. This thesis proposes an integrated asset management concept (Integrated Plant Management-IPM) applied to combined cycle power plants and develops a methodology to assess the benefit that it can provide. Due to the difficulties in getting deterministic benefit estimation, a statistical approach has been adopted for the cot-benefit analysis. As well, the quantitative analysis has been completed with a qualitative validation of the technologies included in the IPM and their contribution to key power plant challenges by power generation sector experts. The cost- benefit analysis provides positive results even in the negative scenario of annual average capacity factor close to 10% and is promising for capacity factors over 30%.

An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

soc-2 encodes a leucine-rich repeat protein implicated in fibroblast growth factor receptor signaling

Activation of fibroblast growth factor (FGF) receptors elicits diverse cellular responses including growth, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-15 FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-15 FGF receptor, consistent with the soc genes functioning downstream of EGL-15. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein–protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5/GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans.

Overexpression of transforming growth factor β1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia

Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor β1 (TGF-β1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-β1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-β1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-β1 withdrawal is associated with regression of vascular lesions. These data suggest an unexpected plasticity of the adult vascular smooth muscle cell phenotype and provide an etiology for cartilaginous metaplasia of the arterial wall. Our observations may help to reconcile divergent views of the role of TGF-β1 in vascular disease.

A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA

The topology of signal transduction is particularly important for neurons. Neurotrophic factors such as nerve growth factor (NGF) interact with receptors at distal axons and a signal is transduced by retrograde transport to the cell body to ensure survival of the neuron. We have discovered an organelle that may account for the retrograde transport of the neurotrophin signal. This organelle is derived from endocytosis of the receptor tyrosine kinase for NGF, TrkA. In vitro reactions containing semi-intact PC12 cells and ATP were used to enhance recovery of a novel organelle: small vesicles containing internalized NGF bound to activated TrkA. These vesicles were distinct from clathrin coated vesicles, uncoated primary endocytic vesicles, and synaptic vesicles, and resembled transport vesicles in their sedimentation velocity. They contained 10% of the total bound NGF and almost one-third of the total tyrosine phosphorylated TrkA. These small vesicles are compelling candidates for the organelles through which the neurotrophin signal is conveyed down the axon.

Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.

ARF-GEP100, a guanine nucleotide-exchange protein for ADP-ribosylation factor 6

A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP100, which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a ≈8-kb mRNA that hybridized with an ARF-GEP100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [35S]GTPγS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP100 Sec7 domain contains Asp543 and Met555, corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe535 and Ala536, associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP100 activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP100, a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP100 in those areas. No similar coincidence of ARF-GEP100 with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

Studying Early Nodulin Gene ENOD40 Expression and Induction by Nodulation Factor and Cytokinin in Transgenic Alfalfa1

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated MsENOD40–1 and MsENOD40–2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40–1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40–2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-(naphthyl)phthalamic acid on roots of plants containing the MsENOD40–1 promoter construct, whereas pseudonodules from plants containing the MsENOD40–2 promoter construct stained blue. A 616-bp region at the distal 5′ end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.

Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.

Sleep electroencephalogram delta-frequency amplitude, night plasma levels of tumor necrosis factor alpha, and human immunodeficiency virus infection.

We tested the hypothesis that increases in tumor necrosis factor alpha (TNF-alpha) induced by human immunodeficiency virus (HIV) are associated with the increases in slow-wave sleep seen in early HIV infection and the decrease with sleep fragmentation seen in advanced HIV infection. Nocturnal sleep disturbances and associated fatigue contribute to the disability of HIV infection. TNF-alpha causes fatigue in clinical use and promotes slow-wave sleep in animal models. With slow progress toward a vaccine and weak effects from current therapies, efforts are directed toward extending productive life of HIV-infected individuals and shortening the duration of disability in terminal illness. We describe previously unrecognized nocturnal cyclic variations in plasma levels of TNF-alpha in all subjects. In 6 of 10 subjects (1 control subject, 3 HIV-seropositive patients with CD4+ cell number > 400 cells per microliters, and 2 HIV-positive patients with CD4+ cell number < 400 cells per microliters), these fluctuations in TNF-alpha were coupled to the known rhythm of electroencephalogram delta amplitude (square root of power) during sleep. This coupling was not present in 3 HIV-positive subjects with CD4+ cell number < 400 cells per microliters and 1 control subject. In 5 HIV subjects with abnormally low CD4+ cell counts ( < 400 cells per microliters), the number of days since seroconversion correlated significantly with low correlation between TNF-alpha and delta amplitude. We conclude that a previously unrecognized normal, physiological coupling exists between TNF-alpha and delta amplitude during sleep and that the lessened likelihood of this coupling in progressive HIV infection may be important in understanding fatigue-related symptoms and disabilities.

Activation of a serine/threonine kinase signaling pathway by transforming growth factor type beta.

Transforming growth factor type beta (TGF-beta) is a multifunctional factor that regulates proliferation and differentiation of many cell types. TGF-beta mediates its effects by binding to and activating cell surface receptors that possess serine/threonine kinase activity. However, the intracellular signaling pathways through which TGF-beta receptors act remain largely unknown. Here we show that TGF-beta activates a 78-kDa protein (p78) serine/threonine kinase as evidenced by an in-gel kinase assay. Ligand-induced activation of the kinase was near-maximal 5 min after TGF-beta addition to the cells and occurred exclusively on serine and threonine residues. This kinase is distinct from TGF-beta receptor type II, as well as several cytoplasmic serine/threonine kinases of similar size, including protein kinase C, Raf, mitogen-activated protein kinase kinase kinase, and ribosomal S6 kinase. Indeed, these kinases can be separated almost completely from p78 kinase by immunoprecipitation with specific antibodies. Furthermore, using different cell lines, we demonstrate that p78 kinase is activated only in cells for which TGF-beta can act as a growth inhibitory factor. These data raise the interesting possibility that protein serine/threonine kinases contribute to the intracellular relay of biological signals originating from receptor serine/threonine kinases such as the TGF-beta receptors.

The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library.

Type I and II receptors for the transforming growth factor beta (TGF-beta) are transmembrane serine/threonine kinases that are essential for TGF-beta signaling. However, little is known about their in vivo substrates or signal transduction pathways. To determine the substrate specificity of these kinases, we developed combinatorial peptide libraries synthesized on a hydrophilic matrix that is easily accessible to proteins in aqueous solutions. When we subjected these libraries to phosphorylation by the cAMP-dependent protein kinase, we obtained the optimal peptide sequence RRXS (I/L/V), in perfect agreement with the substrate sequence deduced from mutagenesis and crystal structure analyses. By using the same libraries, we showed that the optimal substrate peptide for both the type I and II TGF-beta receptors was KKKKKK(S/T)XXX. Since the two kinases are thought to play different roles in intracellular signal transduction, it was a surprise to find that they have almost identical substrate specificity. Our method is direct, sensitive, and simple and provides information about the kinase specificity for all the amino acid residues at each position.

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.