NMR structural studies on human p190-A RhoGAPFF1 revealed that domain phosphorylation by the PDGF-receptor alpha requires its previous unfolding.

p190-A and -B Rho GAPs (guanosine triphosphatase activating proteins) are the only cytoplasmatic proteins containing FF domains. In p190-A Rho GAP, the region containing the FF domains has been implicated in binding to the transcription factor TFII-I. Moreover, phosphorylation of Tyr308 within the first FF domain inhibits this interaction. Because the structural determinants governing this mechanism remain unknown, we sought to solve the structure of the first FF domain of p190-A Rho GAP (RhoGAPFF1) and to study the potential impact of phosphorylation on the structure. We found that RhoGAPFF1 does not fold with the typical (alpha1-alpha2-3(10)-alpha 3) arrangement of other FF domains. Instead, the NMR data obtained at 285 K show an alpha1-alpha2-alpha 3-alpha 4 topology. In addition, we observed that specific contacts between residues in the first loop and the fourth helix are indispensable for the correct folding and stability of this domain. The structure also revealed that Tyr308 contributes to the domain hydrophobic core. Furthermore, the residues that compose the target motif of the platelet-derived growth factor receptor alpha kinase form part of the alpha 3 helix. We observed that the phosphorylation reaction requires a previous step including domain unfolding, a process that occurs at 310 K. In the absence of phosphorylation, the temperature-dependent RhoGAPFF1 folding/unfolding process is reversible. However, phosphorylation causes an irreversible destabilization of the RhoGAPFF1 structure, which probably accounts for the inhibitory effect that it exerts on the TFII-I interaction. Our results link the ability of a protein domain to be phosphorylated with conformational changes in its three-dimensional structure.

Tratamiento de las fracturas conminutas del calcáneo mediante reconstrucción artrodesis con el Sistema Vira®. Estudio prospectivo de los primeros 50 casos con más de un año de seguimiento / Management of comminuted calcaneal fractures by reconstruction-arthrodesis with the Vira® system A prospective study of the first 50 cases with over one-year follow-up

Objetivo: estudiar la aplicabilidad quirúrgica y los resultados clínicos del Sistema Vira® en el tratamiento de las fracturas graves del calcáneo. Material y método: Se analizaron 50 fracturas agudas de calcáneo, correspondientes a 42 pacientes, tratados con el sistema Vira® en un espacio de tiempo de dos años (edad media: 41 años). Nueve fueron bilaterales y 47 fracturas cerradas. En la clasificación de Sanders, las fracturas fueron de tipo IV y tipo III AB. Efectuamos un estudio radiográfico, clínico y la escala AOFAS, a los 12 meses de la intervención. Resultados: la escala AOFAS a los 12 meses de la intervención obtuvo una puntuación promedio de 76,6 puntos (DS:13,9). El 26% fueron resultados muy buenos, el 62% buenos y el 12% regular-malos. El ángulo de Böhler previo a la intervención era deficiente con mejoría, después de la intervención, significativa (p=0,05) aunque clínicamente poco relevante. Conclusiones: El Sistema Vira representa una opción validada en el tratamiento quirúrgico de las fracturas del calcáneo con buenos resultados clínicos y radiológicos a costa de una mínima agresividad quirúrgica y baja tasa de complicaciones (AU)

Objective: To establish the surgical applicability and clinical results of the Vira® System in the management of severe calcaneal fractures. Material and method: Fifty-three acute calcaneal fractures, corresponding to 44 patients treated with the Vira® System in a time period of two years, were analysed (mean age: 41 years). Nine were bilateral and 50 closed fractures. After Sanders classification most fractures were of type IV and III AB. We performed a radiographic, clinical study, and AOFAS scale, at 12 months of the procedure. Results: the AOFAS scale at 12 months of the procedure obtained a mean score of 76.6 points (SD: 13.9). 26% were very good results, 62% good and 12% fair-poor. The Böhler angle prior to the procedure was defective with significant (p=0.05) improvement, after the procedure, though not clinically relevant. Conclusions: The Vira system is a validated option for the surgical management of calcaneal fractures, with good clinical and radiological results at the expense of a very low surgical aggressiveness and low complication rate (AU)

Diseño y desarrollo de un sistema de osteosíntesis para la reconstrucción-artrodesis mínimamente invasiva de fracturas intraarticulares de calcáneo / Design and development of an osteosynthesis system for minimally invasive reconstruction-arthrodesis of calcaneal intra-articular fractures

Objetivo. Desarrollar una instrumentación que restaure la forma del calcáneo fracturado y facilite la artrodesis primaria de la articulación subastragalina mínimamente invasiva en las fracturas intraarticulares graves del calcáneo. Material y método. Se estudió un nuevo sistema de osteosíntesis para las fracturas de calcáneo en tres fases consecutivas: a) diseño del sistema, b) ensayos biomecánicos y c) prueba de los prototipos en el cadáver. Resultados. Los fundamentos del diseño original han sido validados con mínimos cambios. Desde el punto de vista biomecánico el implante desarrollado ha superado las pruebas de resistencia y fatiga y ha demostrado un buen manejo quirúrgico en el cadáver. Conclusiones. El implante diseñado cumple sus objetivos, se adapta a las condiciones mecánicas y dimensionales del calcáneo

Purpose. To develop instruments that can restore the shape of a fractured calcaneus and facilitate minimally invasive primary arthrodesis of the subtalar joint in severe calcaneal intraarticular fractures. Materials and methods. We studied a new osteosynthesis system for calcaneal fractures in three consecutive phases: a) system design, b) biomechanical tests and c) prototype testing in cadavers. Results. The basic characteristics of the original design were validated with minimal changes. From the biomechanical point of view the implant developed has passed strength and fatigue tests and has shown itself to provide good surgical management in cadavers. Conclusions. The implant designed achieves its purpose, and adapts to the mechanical conditions and dimensions of the calcaneal bone (AU)

Tumor adenomatoide de útero / Uterine adenomatoid tumor

El tumor adenomatoide es una neoplasia infrecuente. Su localización preferente es el aparato genital, tanto masculino como femenino, y sólo en raras ocasiones se describe en otros lugares como la glándula suprarrenal o el peritoneo. En el tracto genital femenino se localiza en trompa de Falopio, útero y ovario. Es una neoplasia de carácter benigno, caracterizada por una proliferación de estructuras pseudoglandulares inmersas en un tejido estromal reactivo. Su histogénesis ha sido ampliamente debatida y se acepta su origen mesotelial en razón de que ofrece características inmunohistoquímicas y ultraestructurales compartidas con los mesoteliomas. El tumor adenomatoide uterino se presenta como una masa única y asintomática. Se descubre como hallazgo casual o bajo la presunción de un leiomioma uterino por su similitud en las técnicas de imagen. Existen casos con un patrón de crecimiento pseudoinvasivo que obligan al diagnóstico diferencial con el adenocarcinoma. En estos casos los estudios inmunohistoquímicos adquieren una especial importancia de cara a realizar un diagnóstico correcto que evite un tratamiento excesivamente agresivo

Adenomatoid tumor is an uncommon neoplasm. It is preferentially found in the genital systems of both males and females and only rarely reported in other sites, such us the adrenal gland or peritoneum. In the female genital tract they are found in the fallopian tube, uterus and ovarian hilus. It is a benign neoplasm characterized by a proliferation of gland-like structures arranged in stromal tissue. Its histogenesis has been debated for a long time and it is commonly accepted that they have a mesotelial cell origin, as they possess similar inmunohistochemical and ultraestructural features of mesotheliomas. Adenomatoid tumor of the uterus is generally presented as an asymptomatic unique mass. They are diagnosed as an incidental finding o under the supposition of a leiomioma because of their similar imaging findings. There are cases with a pattern of spread simulating invasion which must be differentiated of adenocarcinoma. In these cases the inmunohistochemical studies have the importance to offer a correct diagnostic in order to avoid an extremely aggressive treatment

Cáncer de mama hereditario / Breast cancer heriditary

El cáncer de mama es la neoplasia más frecuente entre las mujeres. Aproximadamente del 5% al 10% de todos los cánceres de mama y el 10% de los cánceres de ovario son de origen hereditario. Las mutaciones germinales de al menos dos genes supresores, BCRA 1 y BRCA 2, se asocian al 85% de todos los cánceres hereditarios. En este artículo, revisamos la importancia de identificar a estas mujeres tanto para la práctica clínica, como para la investigación de los genes implicados en el proceso tumoral y la transmisión del factor genético a través de varias generaciones con el fin de lograr diagnósticos precoces y mayores posibilidades de curación

Breast cancer is the most frequent neoplasia among women. Approximately 5% to 10% of all breast carcinomas and 10% of ovarian carcinomas are of hereditary origin. Those germinal mutations of the least two suppressing tumoral genes, BRCA 1 and BRCA 2, have been associated at about 85% of all hereditary cancers. In this text review the importance to identify these women for clinical practise, to investigate the genes involved in this tumoral process and the transmission of the genetical factor for several generations, to obtain and earlier diagnosis and more possibilities of cure

Cáncer de mama y gestación / Breast cancer and gestation

El cáncer de mama asociado a la gestación se define como el cáncer de mama diagnosticado durante la gestación o en los 12 meses tras el parto. La incidencia del cáncer de mama asociado a la gestación está entre el 0,9 y el 3,9%. El pronóstico no es significativamente diferente del cáncer de mama no asociado a la gestación, excepto en los casos en que el retraso en el diagnóstico se asocia con estadios más avanzados. En estadios tempranos, la cirugía del cáncer se recomienda como tratamiento primario; la RT está contraindicada durante la gestación y la QT, si se considera necesario, puede ser aplicada después del primer trimestre. Este artículo revisa lo publicado sobre el cáncer de mama asociado a la gestación, y presenta un caso clínico, su diagnóstico, su tratamiento y su pronóstico (AU)

Breast cancer associated to pregnancy is defined as breast cancer diagnosed during pregnancy or during the 12 months postpartum. The incidence of breast cancer associated to pregnancy is between 0.7 and 3.9%. The prognosis is no different from non pregnancy associated breast cancer, except in cases where a delay in diagnosis is associated with more advanced disease. In early stages, cancer surgery is recornmended as the primary treatment; radiotherapy is contraindicated throughout pregnancy; and chemotherapy, if considered necessary, may be given after the first trimester. This article reviews the literature of breast cancer based on a clinical case, its diagnosis, treatment and prognosis (AU)

Solution structures of the YAP65 WW domain and the variant L30 K in complex with the peptides GTPPPPYTVG, N-(n-octyl)-GPPPY and PLPPY and the application of peptide libraries reveal a minimal binding epitope.

The single mutation L30 K in the Hu-Yap65 WW domain increased the stability of the complex with the peptide GTPPPPYTVG (K(d)=40(+/-5) microM). Here we report the refined solution structure of this complex by NMR spectroscopy and further derived structure-activity relationships by using ligand peptide libraries with truncated sequences and a substitution analysis that yielded acetyl-PPPPY as the smallest high-affinity binding peptide (K(d)=60 microM). The structures of two new complexes with weaker binding ligands chosen based on these results (N-(n-octyl)-GPPPYNH(2) and Ac-PLPPY) comprising the wild-type WW domain of Hu-Yap65 were determined. Comparison of the structures of the three complexes were useful for identifying the molecular basis of high-affinity: hydrophobic and specific interactions between the side-chains of Y28 and W39 and P5' and P4', respectively, and hydrogen bonds between T37 (donnor) and P5' (acceptor) and between W39 (donnor) and T2' (acceptor) stabilize the complex.The structure of the complex L30 K Hu-Yap65 WW domain/GTPPPPYTVG is compared to the published crystal structure of the dystrophin WW domain bound to a segment of the beta-dystroglycan protein and to the solution structure of the first Nedd4 WW domain and its prolin-rich ligand, suggesting that WW sequences bind proline-rich peptides in an evolutionary conserved fashion. The position equivalent to T22 in the Hu-Yap65 WW domain sequence is seen as responsible for differentiation in the binding mode among the WW domains of group I.

Synthesis and properties of oligonucleotides containing 8-bromo-2'-deoxyguanosine.

The preparation of oligonucleotides containing 8-bromo-2'-deoxyguanosine is described. Substitution of G by 8-bromoguanine on an alternating CG decamer stabilizes the Z-form in such a way that the B-form was not observed. Melting temperatures showed that duplexes in which 8-bromo-2'-deoxyguanosine paired with natural bases were much less stable.

Structural analysis of WW domains and design of a WW prototype.

Two new NMR structures of WW domains, the mouse formin binding protein and a putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain, only 35 amino acids in length, defines the smallest monomeric triple-stranded antiparallel beta-sheet protein domain that is stable in the absence of disulfide bonds, tightly bound ions or ligands. The structural roles of conserved residues have been studied using site-directed mutagenesis of both wild type domains. Crucial interactions responsible for the stability of the WW structure have been identified. Based on a network of highly conserved long range interactions across the beta-sheet structure that supports the WW fold and on a systematic analysis of conserved residues in the WW family, we have designed a folded prototype WW sequence.

A tale of two secondary structure elements: when a beta-hairpin becomes an alpha-helix.

In this work, we have analyzed the relative importance of secondary versus tertiary interactions in stabilizing and guiding protein folding. For this purpose, we have designed four different mutants to replace the alpha-helix of the GB1 domain by a sequence with strong beta-hairpin propensity in isolation. In particular, we have chosen the sequence of the second beta-hairpin of the GB1 domain, which populates the native conformation in aqueous solution to a significant extent. The resulting protein has roughly 30 % of its sequence duplicated and maintains the 3D-structure of the wild-type protein, but with lower stability (up to -5 kcal/mol). The loss of intrinsic helix stability accounts for about 80 % of the decrease in free energy, illustrating the importance of local interactions in protein stability. Interestingly enough, all the mutant proteins, included the one with the duplicated beta-hairpin sequence, fold with similar rates as the GB1 domain. Essentially, it is the nature of the rate-limiting step in the folding reaction that determines whether a particular interaction will speed up, or not, the folding rates. While local contacts are important in determining protein stability, residues involved in tertiary contacts in combination with the topology of the native fold, seem to be responsible for the specificity of protein structures. Proteins with non-native secondary structure tendencies can adopt stable folds and be as efficient in folding as those proteins with native-like propensities.

A novel NMR experiment for the sequential assignment of proline residues and proline stretches in 13C/15N-labeled proteins.

A new pulse sequence is described for the sequential assignment of proline residues in 13C/15N-labeled proteins by correlating C delta and C alpha chemical shifts of proline residues with the H alpha chemical shift of the preceding residue. Notably, the experiment can provide the sequential connectivities in poly-proline stretches, which cannot be determined using standard triple resonance experiments. Excellent solvent suppression is achieved by coherence selection via a heteronuclear gradient echo. The new pulse sequence has been successfully applied to the 11 kDa HRDC domain.

Theoretical calculations, synthesis and base pairing properties of oligonucleotides containing 8-amino-2'-deoxyadenosine.

Theoretical calculations on double and triple helices containing 8-amino-2'-deoxyadenosine were made to analyze the possible differences in base pairing properties between 8-aminoadenine and adenine. These calculations indicate a strong preferential stabilization of the triplex over the duplex when adenine is replaced by 8-aminoadenine. In addition, a protected phosphoramidite derivative of 8-amino-2'-deoxyadenosine was prepared for the introduction of 8-aminoadenine into synthetic oligonucleotides using the phosphite-triester approach. DNA triple helical structures are normally observed at acidic pH. However, when oligonucleotides carrying 8-aminoadenine are used, very stable triple helical structures can be observed even at neutral pH. Biological applications of triple helices could benefit from the use of 8-aminoadenine derivatives.

The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins.

BACKGROUND: The HRDC (helicase and RNaseD C-terminal) domain is found at the C terminus of many RecQ helicases, including the human Werner and Bloom syndrome proteins. RecQ helicases have been shown to unwind DNA in an ATP-dependent manner. However, the specific functional roles of these proteins in DNA recombination and replication are not known. An HRDC domain exists in both of the human RecQ homologues that are implicated in human disease and may have an important role in their function. RESULTS: We have determined the three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy. The structure resembles auxiliary domains in bacterial DNA helicases and other proteins that interact with nucleic acids. We show that a positively charged region on the surface of the Sgs1p HRDC domain can interact with DNA. Structural similarities to bacterial DNA helicases suggest that the HRDC domain functions as an auxiliary domain in RecQ helicases. Homology models of the Werner and Bloom HRDC domains show different surface properties when compared with Sgs1p. CONCLUSIONS: The HRDC domain represents a structural scaffold that resembles auxiliary domains in proteins that are involved in nucleic acid metabolism. In Sgs1p, the HRDC domain could modulate the helicase function via auxiliary contacts to DNA. However, in the Werner and Bloom syndrome helicases the HRDC domain may have a role in their functional differences by mediating diverse molecular interactions.

Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels.

Syntrophins are modular proteins belonging to the dystrophin associated glycoprotein complex and are thought to be involved in the regulation of the muscular system. Screening of peptide libraries revealed selectivity of the synotrophin PDZ domain toward the motif R/K/Q-E-S/T-X-V-COO- found to be highly conserved in the alpha-subunit C-terminus of vertebrate voltage gated sodium channels (VGSCs). The solution structure of the domain in complex with the peptide G-V-K-E-S-L-V shows specific interactions between the conserved residues in the peptide and syntrophin-characteristic residues in the domain. We propose that syntrophins localize VGSCs to the dystrophin network through its PDZ domain.

Automated NOESY interpretation with ambiguous distance restraints: the refined NMR solution structure of the pleckstrin homology domain from beta-spectrin.

We have used a novel, largely automated, calculation method to refine the NMR solution structure of the pleckstrin homology domain of beta-spectrin. The method is called ARIA for Ambiguous Restraints for Iterative Assignment. The starting point for ARIA is an almost complete assignment of the proton chemical shifts, and a list of partially assigned NOEs, mostly sequential and secondary structure NOEs. The restraint list is then augmented by automatically interpreting peak lists generated by automated peak-picking. The central task of ARIA is the assignment of ambiguous NOEs during the structure calculation using a combination of ambiguous distance restraints and an iterative assignment strategy. In addition, ARIA calibrates ambiguous NOEs to derive distance restraints, merges overlapping data sets to remove duplicate information, and uses empirical rules to identify erroneous peaks. While the distance restraints for the structure calculations were exclusively extracted from homonuclear 2D experiments, ARIA is especially suited for the analysis of multidimensional spectra. Applied to the pleckstrin homology domain, ARIA generated structures of good quality, and of sufficiently high accuracy to solve the X-ray crystal structure of the same domain by molecular replacement. The comparison of the free NMR solution structure to the X-ray structure, which is complexed to D-myo-inositol-1,4,5-triphosphate, shows that the ligand primarily induces a disorder-order transition in the binding loops, which are disordered in the NMR ensemble but well ordered in the crystal. The structural core of the protein is unaffected, as evidenced by a backbone root-mean-square difference between the average NMR coordinates and the X-ray crystal structure for the secondary structure elements of less than 0.6 A.

Structure of the WW domain of a kinase-associated protein complexed with a proline-rich peptide.

The WW domain is a new protein module with two highly conserved tryptophans that binds proline-rich peptide motifs in vitro. It is present in a number of signalling and regulatory proteins, often in several copies. Here we investigate the solution structure of the WW domain of human YAP65 (for Yes kinase-associated protein) in complex with proline-rich peptides containing the core motif PPxY. The structure of the domain with the bound peptide GTPPPPYTVG is a slightly curved, three-stranded, antiparallel beta-sheet. Two prolines pack against the first tryptophan, forming a hydrophobic buckle on the convex side of the sheet. The concave side has three exposed hydrophobic residues (tyrosine, tryptophan and leucine) which form the binding site for the ligand. A non-conserved isoleucine in the amino-terminal flanking region covers a hydrophobic patch and stabilizes the WW domain of human YAP65 in vitro. The structure of the WW domain differs from that of the SH3 domain and reveals a new design for a protein module that uses stacked aromatic surface residues to arrange a binding site for proline-rich peptides.

Structure of the binding site for inositol phosphates in a PH domain.

Phosphatidylinositol bisphosphate has been found to bind specifically to pleckstrin homology (PH) domains that are commonly present in signalling proteins but also found in cytoskeleton. We have studied the complexes of the beta-spectrin PH domain and soluble inositol phosphates using both circular dichroism and nuclear magnetic resonance spectroscopy, and X-ray crystallography. The specific binding site is located in the centre of a positively charged surface patch of the domain. The presence of 4,5-bisphosphate group on the inositol ring is critical for binding. In the crystal structure that has been determined at 2.0 A resolution, inositol-1,4,5-trisphosphate is bound with salt bridges and hydrogen bonds through these phosphate groups whereas the 1-phosphate group is mostly solvent-exposed and the inositol ring has virtually no interactions with the protein. We propose a model in which PH domains are involved in reversible anchoring of proteins to membranes via their specific binding to phosphoinositides. They could also participate in a response to a second messenger such as inositol trisphosphate, organizing cross-roads in cellular signalling.

Structure of the dsRNA binding domain of E. coli RNase III.

The double-stranded RNA binding domain (dsRBD) is a approximately 70 residue motif found in a variety of modular proteins exhibiting diverse functions, yet always in association with dsRNA. We report here the structure of the dsRBD from RNase III, an enzyme present in most, perhaps all, living cells. It is involved in processing transcripts, such as rRNA precursors, by cleavage at short hairpin sequences. The RNase III protein consists of two modules, a approximately 150 residue N-terminal catalytic domain and a approximately 70 residue C-terminal recognition module, homologous with other dsRBDs. The structure of the dsRBD expressed in Escherichia coli has been investigated by homonuclear NMR techniques and solved with the aid of a novel calculation strategy. It was found to have an alpha-beta-beta-beta-alpha topology in which a three-stranded anti-parallel beta-sheet packs on one side against the two helices. Examination of 44 aligned dsRBD sequences reveals several conserved, positively charged residues. These residues map to the N-terminus of the second helix and a nearby loop, leading to a model for the possible contacts between the domain and dsRNA.

Structure of the pleckstrin homology domain from beta-spectrin.

The 'pleckstrin homology' or PH domain is a 100-residue protein module. It is present in many kinases, different isoforms of phospholipase C, GTPase-activating proteins and nucleotide-exchange factors. Its function is not known, but many proteins that contain a PH domain interact with GTP-binding proteins. The PH domain in beta-adrenergic receptor kinase may be involved in binding to the beta gamma subunits of a trimeric G-protein. We report here the three-dimensional structure of the PH domain of the cytoskeletal protein spectrin using homonuclear nuclear magnetic resonance. The core of the molecule is an antiparallel beta-sheet consisting of seven strands. The C terminus is folded into a long alpha-helix, and another helix is present in one of the surface loops. The molecule is electrostatically polarized and contains a pocket which may be involved in the binding of a ligand. There is a distant relationship to the peptidyl-prolyl-cis-trans-isomerase FKBP in which this pocket is involved in the binding of the macrocyclic compound FK506 (refs 8-11).

Prolonged suxamethonium-induced neuromuscular blockade associated with organophosphate poisoning.

We report a patient, exposed to Malathion during agricultural work, who suffered a prolonged apnoea after the administration of suxamethonium. He had a normal phenotype (E1u E1u), but an extremely low plasma cholinesterase activity. The diagnosis was made by assaying cholinesterase activity and analysing the enzymic components by electrophoresis on polyacrylamide gel slabs. The results indicated that the apnoea was a result of the low activity of plasma cholinesterase induced by Malathion.