The TßR-I pre-helix extension is structurally ordered in the unbound form and its flanking prolines are essential for binding.

Transforming growth factor ß isoforms (TGF-ß) are among the most recently evolved members of a signaling superfamily with more than 30 members. TGF-ß play vital roles in regulating cellular growth and differentiation, and they signal through a highly restricted subset of receptors known as TGF-ß type I receptor (TßR-I) and TGF-ß type II receptor (TßR-II). TGF-ß's specificity for TßR-I has been proposed to arise from its pre-helix extension, a five-residue loop that binds in the cleft between TGF-ß and TßR-II. The structure and backbone dynamics of the unbound form of the TßR-I extracellular domain were determined using NMR to investigate the extension's role in binding. This showed that the unbound form is highly similar to the bound form in terms of both the ß-strand framework that defines the three-finger toxin fold and the extension and its characteristic cis-Ile54-Pro55 peptide bond. The NMR data further showed that the extension and two flanking 3(10) helices are rigid on the nanosecond-to-picosecond timescale. The functional significance of several residues within the extension was investigated by binding studies and reporter gene assays in cultured epithelial cells. These demonstrated that the pre-helix extension is essential for binding, with Pro55 and Pro59 each playing a major role. These findings suggest that the pre-helix extension and its flanking prolines evolved to endow the TGF-ß signaling complex with its unique specificity, departing from the ancestral promiscuity of the bone morphogenetic protein subfamily, where the binding interface of the type I receptor is highly flexible.

Iterative structure-based peptide-like inhibitor design against the botulinum neurotoxin serotype A.

The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their K(i) values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2' position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.

A potent peptidomimetic inhibitor of botulinum neurotoxin serotype A has a very different conformation than SNAP-25 substrate.

Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (K(i)=41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the "proton shuttle" E224. This mechanism of inhibition is aided by residue contacts in the conserved S1' pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2' residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.

Assembly of TbetaRI:TbetaRII:TGFbeta ternary complex in vitro with receptor extracellular domains is cooperative and isoform-dependent.

Transforming growth factor-beta (TGFbeta) isoforms initiate signaling by assembling a heterotetrameric complex of paired type I (TbetaRI) and type II (TbetaRII) receptors on the cell surface. Because two of the ligand isoforms (TGFbetas 1, 3) must first bind TbetaRII to recruit TbetaRI into the complex, and a third (TGFbeta2) requires a co-receptor, assembly is known to be sequential, cooperative and isoform-dependent. However the source of the cooperativity leading to recruitment of TbetaRI and the universality of the assembly mechanism with respect to isoforms remain unclear. Here, we show that the extracellular domain of TbetaRI (TbetaRI-ED) binds in vitro with high affinity to complexes of the extracellular domain of TbetaRII (TbetaRII-ED) and TGFbetas 1 or 3, but not to either ligand or receptor alone. Thus, recruitment of TbetaRI requires combined interactions with TbetaRII-ED and ligand, but not membrane attachment of the receptors. Cell-based assays show that TbetaRI-ED, like TbetaRII-ED, acts as an antagonist of TGFbeta signaling, indicating that receptor-receptor interaction is sufficient to compete against endogenous, membrane-localized receptors. On the other hand, neither TbetaRII-ED, nor TbetaRII-ED and TbetaRI-ED combined, form a complex with TGFbeta2, showing that receptor-receptor interaction is insufficient to compensate for weak ligand-receptor interaction. However, TbetaRII-ED does bind with high affinity to TGFbeta2-TM, a TGFbeta2 variant substituted at three positions to mimic TGFbetas 1 and 3 at the TbetaRII binding interface. This proves both necessary and sufficient for recruitment of TbetaRI-ED, suggesting that the three different TGFbeta isoforms induce assembly of the heterotetrameric receptor complex in the same general manner.

Estudio de 90 casos de cáncer del cervix uterino tratados con cirugía radical / Study of 90 cases of cervix cancer treated with radical surgery

En 90m pacientes de la clínica de tumores del Servicio de Ginecologia en el Hospital Universitario de Cali, se practicó anexohisterectomía radical por la técnica de O'KABAYASHI. Todos los casos pertencían al estádio clínico IB. Los pacientes se seleccionaron según el riesgo quirúrgico. Se practicaron los estudios para-clínicos para una cirugía mayor. La preparación de la vagina se hizo con óvulos de Cloranfenicol. Siempre se hizo drenaje de las fosas ilícas por 4 dias, y la sonda vesical permaneció por 8 días; se utilizaron antesépticos urinarios. La mayoria de las pacientes provenían de la zona urbana, de una edad entre 31-50 años (66%). En 51 casos había multiparidad (más de 6 hijos). En 85 casos se encontró Carcinoma Epidermoide. En 5 casos el tumor se asociaba a embarazo del 1o. y 2o.trimestre. El promedio de ganglios extirpados por caso fue 20-30. En sólo un caso de los estadios IB se encontraron ganglios metastásicos. Los accidentes operatórios fueron mínimos. No tuvimos mortalidad operatoria. La principal causa de mortalidad fue la infección urinaria (sonda por 8 días), fístula vesico-vaginal 4 casos. Fístulas recto-vaginal 1 caso. En 64 casos,que tenían más de 5 años de tratamiento, sólo se logró hacer sequimiento adecuado en 32 casos (50%), todos libres de enfermedad en ese período de tiempo. Se presenta el análisis de 90 casos tratados en el Servicio de Ginecología del Hospital Universitario de Cali, Colombia. Estas pacientes provienen de la consulta de Oncología Ginecológica y proceden del área del Departamento del Valle del Cauca y Departamentos vecinos (Cauca-Nariño-Chocó-Risaralda-Quindío). El estudio comprende la década de 1967 - 1976

Enfermedad trofoblástica: Hospital Universitario del Valle 1962-1972 / Trophoblastic disease: Hospital Universitário del Valle 1962-1972

En 248 casos de enfermedad trofoblástica en el Hospital Universitario de Cali-Col> S.A, se da una incidencia de 1 caso por 4.348 embarazos. La mayoria de las pacientes son de nivel socio-económico bajo. Se encontraron 218 molas,12 corioadenomas y 18 coriocarcionomas. El mayoria número de casos estudo entre los 20-30 años. El sintoma predominante fue la hemorragia genital (91%). En 99 radiografías de toráx se encontraron metástasis en 13 casos (13%).El tratamiento fue basado en la evacuación de la Mola,histerectomia y quimioterapia (METHOTREXATE). Sólo 56 casos tuvieron control más de un año. Se logró evidenciar 4 muertes en este grupo de pacientes