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Calcitonin (CT) is a 32-amino-acid calciotropic peptide hormone which acts on target cells via a G protein-coupled seven-transmembrane receptor (CTR). In this study, we report the design, synthesis and characterization of four potent bioactive and photoreactive CT analogs, each of which contains a single benzophenone moiety inserted at different and discrete locations within the CT molecule. Replacement of all Lys residues in salmon CT (sCT) with Arg, followed by replacement of hydrophobic residues with a Lys(epsilon-p-benzoylbenzoyl) residue [Lys(epsilon-pBz2)] was found to preserve high biological activity. We substituted Val8, Leu16 and Leu19 by Lys(epsilon-pBz2), and acylated the N-terminus by a pBz2 moiety, thus distributing the photoaffinity moiety in the different analogs across a large portion of the CT sequence. With both transfected and endogenous CTRs from several species, all four benzophenone-containing analogs were shown to be virtually indistinguishable from the parent sCT analog in both receptor binding properties and stimulation of cAMP accumulation. Upon photolysis, in the presence of CTR, the radioiodinated photoreactive CT analog ([Arg11,18,Lys19(epsilon-pBz2)]sCT (K19)) covalently labels a membrane component of approximately 70 kDa. Receptor cross-linking is inhibited specifically in the presence of excess sCT. We also examined the interaction of these CT analogs with a hemagglutinin (HA) epitope-tagged CTR. The HA-CTR displayed CT binding and CT-dependent cAMP stimulation identical with native CTR. Both K19 and another bioactive analog (-Arg11,18, Lys8(epsilon-pBz2)]sCT (K8)) specifically photoaffinity cross-link to the HA-CTR. These benzophenone-containing CT analogs should facilitate studies of hormone-receptor interactions and allow the direct identification of a CT binding domain(s) within the receptor by the analysis of photochemically cross-linked conjugates.

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A calcitonin receptor complementary DNA (cDNA) was cloned by expression of a cDNA library from a porcine kidney epithelial cell line in COS cells. The 482-amino acid receptor has high affinity for salmon calcitonin (dissociation constant Kd approximately 6 nM) and is functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP). The receptor shows no sequence similarity to other reported G protein-coupled receptors but is homologous to the parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor, indicating that the receptors for these hormones, which regulate calcium homeostasis, represent a new family of G protein-coupled receptors.

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We have developed an in vitro cell culture model to examine the interaction between connective tissue cells and orthopaedic implant biomaterials. Human connective tissue cells grown on different materials exhibit distinct responses in terms of attachment, morphology, proliferative capacity and matrix biosynthesis. Our results closely complement in vivo observations concerning biocompatibility and demonstrate the usefulness of this in vitro system for evaluating biomaterials. More importantly, this model can be used to define the specific cellular and biochemical processes that are responsible for the local tissue responses to orthopaedic implant materials.

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From the first millennium B.C. through the 9th-century A.D. Classic Maya collapse, nonurban populations grew exponentially, doubling every 408 years, in the twin-lake (Yaxha-Sacnab) basin that contained the Classic urban center of Yaxha. Pollen data show that forests were essentially cleared by Early Classic time. Sharply accelerated slopewash and colluviation, amplified in the Yaxha subbasin by urban construction, transferred nutrients plus calcareous, silty clay to both lakes. Except for the urban silt, colluvium appearing as lake sediments has a mean total phosphorus concentration close to that of basin soils. From this fact, from abundance and distribution of soil phosphorus, and from continuing post-Maya influxes (80 to 86 milligrams of phosphorus per square meter each year), which have no other apparent source, we conclude that riparian soils are anthrosols and that the mechanism of long-term phosphorus loading in lakes is mass transport of soil. Per capita deliveries of phosphorus match physiological outputs, approximately 0.5 kilogram of phosphorus per capita per year. Smaller apparent deliveries reflect the nonphosphatic composition of urban silt; larger societal outputs, expressing excess phosphorus from deforestation and from food waste and mortuary disposal, are probable but cannot be evaluated from our data. Eutrophication is not demonstrable and was probably impeded, even in less-impacted lakes, by suspended Maya silt. Environmental strain, the product of accelerating agroengineering demand and sequestering of nutrients in colluvium, developed too slowly to act as a servomechanism, damping population growth, at least until Late Classic time.