Constitutive activity and inverse agonism at the alpha1adrenoceptors.

Mutations of G protein-coupled receptors (GPCR) can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The alpha(1B)adrenoceptor was the first GPCR in which point mutations were shown to trigger receptor activation. This article briefly summarizes some of the findings reported in the last several years on constitutive activity of the alpha(1)adrenoceptor subtypes, the location where mutations have been found in the receptors, the spontaneous activity of native receptors in recombinant as well as physiological systems. In addition, it will highlight how the analysis of the pharmacological and molecular properties of the constitutively active adrenoceptor mutants provided an important contribution to our understanding of the molecular mechanisms underlying the mechanism of receptor activation and inverse agonism.

The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host.

Plasmodium sporozoites make a remarkable journey from the mosquito midgut to the mammalian liver. The sporozoite's major surface protein, circumsporozoite protein (CSP), is a multifunctional protein required for sporozoite development and likely mediates several steps of this journey. In this study, we show that CSP has two conformational states, an adhesive conformation in which the C-terminal cell-adhesive domain is exposed and a nonadhesive conformation in which the N terminus masks this domain. We demonstrate that the cell-adhesive domain functions in sporozoite development and hepatocyte invasion. Between these two events, the sporozoite must travel from the mosquito midgut to the mammalian liver, and N-terminal masking of the cell-adhesive domain maintains the sporozoite in a migratory state. In the mammalian host, proteolytic cleavage of CSP regulates the switch to an adhesive conformation, and the highly conserved region I plays a critical role in this process. If the CSP domain architecture is altered such that the cell-adhesive domain is constitutively exposed, the majority of sporozoites do not reach their target organs, and in the mammalian host, they initiate a blood stage infection directly from the inoculation site. These data provide structure-function information relevant to malaria vaccine development.

MORPHOLOGICAL AND MICROMORPHOLOGICAL CHANGES IN THE STRUCTURE OF A RHODIC HAPLUDOX AS A RESULT OF AGRICULTURAL MANAGEMENT

In evaluation of soil quality for agricultural use, soil structure is one of the most important properties, which is influenced not only by climate, biological activity, and management practices but also by mechanical and physico-chemical forces acting in the soil. The purpose of this study was to evaluate the influence of conventional agricultural management on the structure and microstructure of a Latossolo Vermelho distroférrico típico (Rhodic Hapludox) in an experimental area planted to maize. Soil morphology was described using the crop profile method by identifying the distinct structural volumes called Morphologically Homogeneous Units (MHUs). For comparison, we also described a profile in an adjacent area without agricultural use and under natural regrowth referred to as Memory. We took undisturbed samples from the main MHUs so as to form thin sections and blocks of soil for micromorphological and micromorphometrical analyses. Results from the application of the crop profile method showed the occurrence of the following structural types: loose (L), fragmented (F) and continuous (C) in both profiles analyzed. In the Memory soil profile, the fragmented structures were classified as Fpt_6;∆+tf and Fmt∆_6;, whose micromorphology shows an enaulic-porphyric (porous) relative distribution with a great deal of biological activity as indicated by the presence of vughs and channels. Lower down, from 0.20 to 0.35 m, there was a continuous soil volume (sub-type C∆_6;), with a subangular block microstructure and an enaulic-porphyric relative distribution, though in this case more compact and with aggregate coalescence and less biological activity. The micromorphometrical study of the soil of the Memory Plot showed the predominance of complex pores in NAM (15.03 %), Fmt∆_6; (11.72 %), and Fpt_6;∆+tf (7.73 %), and rounded pores in C∆_6; (8.21 %). In the soil under conventional agricultural management, we observed fragmented structures similar to the Memory Plot from 0.02 to 0.20 m, followed by a volume with a compact continuous structure (C∆_6;), without visible porosity and with few roots. In the MHUs under conventional management, reduction in the packing pores (40 %) was observed, mainly in the continuous units (C). The microstructure had well-defined blocks, with the occurrence of planar pores and less evidence of biological activity. In conclusion, the morphological and micromorphological analyses of the soil profiles studied offered complementary information regarding soil structural quality, especially concerning the changes in pore types as result of mechanical stress undergone by the soil.

Application of the microcanonical multifractal formalism to monofractal systems

The design of appropriate multifractal analysis algorithms, able to correctly characterize the scaling properties of multifractal systems from experimental, discretized data, is a major challenge in the study of such scale invariant systems. In the recent years, a growing interest for the application of the microcanonical formalism has taken place, as it allows a precise localization of the fractal components as well as a statistical characterization of the system. In this paper, we deal with the specific problems arising when systems that are strictly monofractal are analyzed using some standard microcanonical multifractal methods. We discuss the adaptations of these methods needed to give an appropriate treatment of monofractal systems.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Stable alpha-synuclein oligomers strongly inhibit chaperone activity of the Hsp70 system by weak interactions with J-domain co-chaperones.

α-Synuclein aggregation and accumulation in Lewy bodies are implicated in progressive loss of dopaminergic neurons in Parkinson disease and related disorders. In neurons, the Hsp70s and their Hsp40-like J-domain co-chaperones are the only known components of chaperone network that can use ATP to convert cytotoxic protein aggregates into harmless natively refolded polypeptides. Here we developed a protocol for preparing a homogeneous population of highly stable ^6;-sheet enriched toroid-shaped α-Syn oligomers with a diameter typical of toxic pore-forming oligomers. These oligomers were partially resistant to in vitro unfolding by the bacterial Hsp70 chaperone system (DnaK, DnaJ, GrpE). Moreover, both bacterial and human Hsp70/Hsp40 unfolding/refolding activities of model chaperone substrates were strongly inhibited by the oligomers but, remarkably, not by unstructured α-Syn monomers even in large excess. The oligomers acted as a specific competitive inhibitor of the J-domain co-chaperones, indicating that J-domain co-chaperones may preferably bind to exposed bulky misfolded structures in misfolded proteins and, thus, complement Hsp70s that bind to extended segments. Together, our findings suggest that inhibition of the Hsp70/Hsp40 chaperone system by α-Syn oligomers may contribute to the disruption of protein homeostasis in dopaminergic neurons, leading to apoptosis and tissue loss in Parkinson disease and related neurodegenerative diseases.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Microglial responsiveness as a sensitive marker for trimethyltin (TMT) neurotoxicity.

Activation of microglia is a well-documented phenomenon associated with diverse pathological conditions of the central nervous system. In order to investigate the involvement of microglial cells in the neurotoxic action of the heavy metal compound trimethyltin, three-dimensional brain cell cultures were treated during an early developmental period, using concentrations at or below the limit of cytotoxicity. Microglial cells were studied by cytochemical staining, using horseradish peroxidase-conjugated B4 isolectin (GSI-B4). In parallel, neurotoxic effects were assessed by determining the content of synaptophysin and synapsin I, both in the total homogenates and in the synaptosomal fraction of the cultures. Changes in the content of the specific growth cone protein, GAP-43, were also analyzed. It was found that low, non-cytotoxic concentrations of TMT (10(-9) to 10(-8) M) caused a significant increase in the number and/or the clustering of microglial cells. A decrease in the synaptic protein (synapsin I, synaptophysin) content was detected at 10(-8) M of TMT in synaptosomal fractions, whereas in the total homogenates, changes in synaptic proteins and GAP-43 were observed only at the cytotoxic TMT concentration (10(-6) M). Although it remains to be shown whether the microglial response is caused by direct or indirect action of TMT, the present findings show that microglial responsiveness can be detected prior to any sign of neuronal degeneration, and may serve as a sensitive indicator for heavy metal neurotoxicity in the brain.

EADock: docking of small molecules into protein active sites with a multiobjective evolutionary optimization.

In recent years, protein-ligand docking has become a powerful tool for drug development. Although several approaches suitable for high throughput screening are available, there is a need for methods able to identify binding modes with high accuracy. This accuracy is essential to reliably compute the binding free energy of the ligand. Such methods are needed when the binding mode of lead compounds is not determined experimentally but is needed for structure-based lead optimization. We present here a new docking software, called EADock, that aims at this goal. It uses an hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 A around the center of mass of the ligand position in the crystal structure, and on the contrary to other benchmarks, our algorithm was fed with optimized ligand positions up to 10 A root mean square deviation (RMSD) from the crystal structure, excluding the latter. This validation illustrates the efficiency of our sampling strategy, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures could be explained by the presence of crystal contacts in the experimental structure. Finally, the ability of EADock to accurately predict binding modes on a real application was illustrated by the successful docking of the RGD cyclic pentapeptide on the alphaVbeta3 integrin, starting far away from the binding pocket.

Identification of SPLUNC1's ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airways.

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (W64;2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the ^6;-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 _6;m, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.

The contact region between three domains of the extracellular loop of ASIC1a is critical for channel function.

Acid-sensing ion channels are members of the epithelial Na(+) channel/degenerin family. They are neuronal nonvoltage-gated Na(+) channels that are activated by extracellular acidification. In this study, we investigated the role of a highly conserved region of the extracellular part of ASIC1a that forms the contact between the finger domain, the adjacent beta-ball, and the upper palm domain in ASIC1a. The finger domain contributes to the pH-dependent gating and is linked via this contact zone to the rest of the protein. We found that mutation to Cys of residues in this region led to decreased channel expression and current amplitudes. Exposure of the engineered Cys residues to Cd(2+) or to charged methane thiosulfonate sulfhydryl reagents further reduced current amplitudes. This current inhibition was not due to changes in acid-sensing ion channel pH dependence or unitary conductance and was likely due to a decrease of the probability of channel opening. For some mutants, the effect of sulfhydryl reagents depended on the pH of exposure in the range 7.4 to 6.8, suggesting that this zone undergoes conformational changes during inactivation. Our study identifies a region in ASIC1a whose integrity is required for normal channel function.

Structural assessment of single amino acid mutations: application to TP53 function.

Single amino acid substitution is the type of protein alteration most related to human diseases. Current studies seek primarily to distinguish neutral mutations from harmful ones. Very few methods offer an explanation of the final prediction result in terms of the probable structural or functional effect on the protein. In this study, we describe the use of three novel parameters to identify experimentally-verified critical residues of the TP53 protein (p53). The first two parameters make use of a surface clustering method to calculate the protein surface area of highly conserved regions or regions with high nonlocal atomic interaction energy (ANOLEA) score. These parameters help identify important functional regions on the surface of a protein. The last parameter involves the use of a new method for pseudobinding free-energy estimation to specifically probe the importance of residue side-chains to the stability of protein fold. A decision tree was designed to optimally combine these three parameters. The result was compared to the functional data stored in the International Agency for Research on Cancer (IARC) TP53 mutation database. The final prediction achieved a prediction accuracy of 70% and a Matthews correlation coefficient of 0.45. It also showed a high specificity of 91.8%. Mutations in the 85 correctly identified important residues represented 81.7% of the total mutations recorded in the database. In addition, the method was able to correctly assign a probable functional or structural role to the residues. Such information could be critical for the interpretation and prediction of the effect of missense mutations, as it not only provided the fundamental explanation of the observed effect, but also helped design the most appropriate laboratory experiment to verify the prediction results.

A novel population of human melanoma-specific CD8 T cells recognizes Melan-AMART-1 immunodominant nonapeptide but not the corresponding decapeptide.

HLA-A2-restricted cytolytic T cells specific for the immunodominant human tumor Ag Melan-A(MART-1) can kill most HLA-matched melanoma cells, through recognition of two naturally occurring antigenic variants, i.e., Melan-A nonamer AAGIGILTV and decamer EAAGIGILTV peptides. Several previous studies have suggested a high degree of TCR cross-reactivity to the two peptides. In this study, we describe for the first time that some T cell clones are exclusively nonamer specific, because they are not labeled by A2/decamer-tetramers and do not recognize the decamer when presented endogenously. Functional assays with peptides gave misleading results, possibly because decamers were cleaved by exopeptidases. Interestingly, nonapeptide-specific T cell clones were rarely Valpha2.1 positive (only 1 of 19 clones), in contrast to the known strong bias for Valpha2.1-positive TCRs found in decamer-specific clones (59 of 69 clones). Molecular modeling revealed that nonapeptide-specific TCRs formed unfavorable interactions with the decapeptide, whereas decapeptide-specific TCRs productively created a hydrogen bond between CDR1alpha and glutamic acid (E) of the decapeptide. Ex vivo analysis of T cells from melanoma metastases demonstrated that both nonamer and decamer-specific T cells were enriched to substantial frequencies in vivo, and representative clones showed efficient tumor cell recognition and killing. We conclude that the two peptides should be regarded as distinct epitopes when analyzing tumor immunity and developing immunotherapy against melanoma.

EuroPhenome: a repository for high-throughput mouse phenotyping data.

The broad aim of biomedical science in the postgenomic era is to link genomic and phenotype information to allow deeper understanding of the processes leading from genomic changes to altered phenotype and disease. The EuroPhenome project (http://www.EuroPhenome.org) is a comprehensive resource for raw and annotated high-throughput phenotyping data arising from projects such as EUMODIC. EUMODIC is gathering data from the EMPReSSslim pipeline (http://www.empress.har.mrc.ac.uk/) which is performed on inbred mouse strains and knock-out lines arising from the EUCOMM project. The EuroPhenome interface allows the user to access the data via the phenotype or genotype. It also allows the user to access the data in a variety of ways, including graphical display, statistical analysis and access to the raw data via web services. The raw phenotyping data captured in EuroPhenome is annotated by an annotation pipeline which automatically identifies statistically different mutants from the appropriate baseline and assigns ontology terms for that specific test. Mutant phenotypes can be quickly identified using two EuroPhenome tools: PhenoMap, a graphical representation of statistically relevant phenotypes, and mining for a mutant using ontology terms. To assist with data definition and cross-database comparisons, phenotype data is annotated using combinations of terms from biological ontologies.

Heterogeneity and event dependence in the analysis of sickness absence

Sickness absence (SA) is an important social, economic and public health issue. Identifying and understanding the determinants, whether biological, regulatory or, health services-related, of variability in SA duration is essential for better management of SA. The conditional frailty model (CFM) is useful when repeated SA events occur within the same individual, as it allows simultaneous analysis of event dependence and heterogeneity due to unknown, unmeasured, or unmeasurable factors. However, its use may encounter computational limitations when applied to very large data sets, as may frequently occur in the analysis of SA duration. To overcome the computational issue, we propose a Poisson-based conditional frailty model (CFPM) for repeated SA events that accounts for both event dependence and heterogeneity. To demonstrate the usefulness of the model proposed in the SA duration context, we used data from all non-work-related SA episodes that occurred in Catalonia (Spain) in 2007, initiated by either a diagnosis of neoplasm or mental and behavioral disorders. As expected, the CFPM results were very similar to those of the CFM for both diagnosis groups. The CPU time for the CFPM was substantially shorter than the CFM. The CFPM is an suitable alternative to the CFM in survival analysis with recurrent events,especially with large databases.