Biomedical subjects

Lloyd D Graham

Publications and source records attributed to Lloyd D Graham.

5 recordsLinked to original sources

PubMed2005

The X-ray structure of a hemipteran ecdysone receptor ligand-binding domain: comparison with a lepidopteran ecdysone receptor ligand-binding domain and implications for insecticide design.

The ecdysone receptor is a hormone-dependent transcription factor that plays a central role in regulating the expression of vast networks of genes during development and reproduction in the phylum Arthropoda. The functional receptor is a heterodimer of the two nuclear receptor proteins ecdysone receptor (EcR) and ultraspiracle protein. The receptor is the target of the environmentally friendly bisacylhydrazine insecticides, which are effective against Lepidoptera but not against Hemiptera or several other insect orders. Here we present evidence indicating that much of the selectivity of the bisacylhydrazine insecticides can be studied at the level of their binding to purified ecdysone receptor ligand-binding domain (LBD) heterodimers. We report the crystal structure of the ecdysone receptor LBD heterodimer of the hemipteran Bemisia tabaci (Bt, sweet potato whitefly) in complex with the ecdysone analogue ponasterone A. Although comparison with the corresponding known LBD structure from the lepidopteran Heliothis virescens (Hv) ecdysone receptor revealed the overall mode of ponasterone A binding to be very similar in the two cases, we observed that the BtEcR ecdysteroid-binding pocket is structured differently to that of HvEcR in those parts that are not in contact with ponasterone A. We suggest that these differences in the ligand-binding pocket may provide a molecular basis for the taxonomic order selectivity of bisacylhydrazine insecticides.

Amino Acid Sequence↗

PubMed2003

Isolating substrates for an engineered alpha-lytic protease by phage display.

Panning of a substrate phage library with an alpha-lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered alpha-lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P4 positions, whereas Asp dominated the putative P1 position.

Amino Acid Motifs↗

PubMed2002

Role of insulin-like growth factor binding protein-3 in breast cancer cell growth.

The mitogenic effects of insulin-like growth factors (IGFs) are regulated by a family of insulin-like growth factor binding proteins (IGFBPs). One member of this family, IGFBP-3, mediates the growth-inhibitory and apoptosis-inducing effects of a number of growth factors and hormones such as transforming growth factor-beta, retinoic acid, and 1,25-dihydroxyvitamin D3. IGFBP-3 may act in an IGF-dependent manner by attenuating the interaction of pericellular IGFs with the type-I IGF receptor. It may also act in an IGF-independent manner by initiating intracellular signaling from a cell surface receptor, or by direct nuclear action, or both. The possibility of a membrane-bound receptor is strengthened by recent studies which have identified members of the transforming growth factor-beta receptor family as having a role, either directly or indirectly, in signaling from the cell surface by IGFBP-3. A number of growth factors and hormones stimulate the expression and secretion of cellular IGFBP-3, which then signals from the cell surface to bring about some of the effects attributed to the primary agents. Within the cell, the apoptosis-inducing tumor suppressor, p53, can also induce IGFBP-3 expression and secretion. Since IGFBP-3 upregulates the cell cycle inhibitor, p21(Waf1), and increases the ratio of proapoptotic to antiapoptotic members of the Bcl family, it appears to exert the same effects on major downstream targets of cell signaling as p53 does. The nuclear localization of IGFBP-3 has been described in a number of cell types. IGFBP-3 may act to import IGFs or other nuclear localization signal-deficient signaling molecules into the nucleus. It may also act directly in the nucleus by enhancing the activity of retinoid X receptor-alpha and thereby promote apoptosis. All of the above phenomena will be discussed with particular emphasis on the growth of breast cancer cells.

Breast Neoplasms↗

PubMed2002

Ecdysone-controlled expression of transgenes.

Inducible expression systems show great potential for use in human gene therapy and systems based on insect ecdysone receptors are particularly promising candidates. This article describes such systems and reviews actual and potential uses of ecdysone-controlled transgenes in vitro and in vivo. The ligand specificity of ecdysone receptor-based systems is considered, along with the safety and efficacy of the ecdysteroid and non-steroidal compounds used to activate them.

Animals↗

PubMedDate not supplied

Characterization of a protein-based adhesive elastomer secreted by the Australian frog Notaden bennetti.

When provoked, Notaden bennetti frogs secrete an exudate which rapidly forms a tacky elastic solid ("frog glue"). This protein-based material acts as a promiscuous pressure-sensitive adhesive that functions even in wet conditions. We conducted macroscopic tests in air to assess the tensile strength of moist glue (up to 78 +/- 8 kPa) and the shear strength of dry glue (1.7 +/- 0.3 MPa). We also performed nanomechanical measurements in water to determine the adhesion (1.9-7.2 nN or greater), resilience (43-56%), and elastic modulus (170-1035 kPa) of solid glue collected in different ways. Dry glue contains little carbohydrate and consists mainly of protein. The protein complement is rich in Gly (15.8 mol %), Pro (8.8 mol %), and Glu/Gln (14.1 mol %); it also contains some 4-hydroxyproline (4.6 mol %) but no 5-hydroxylysine or 3,4-dihydroxyphenylalanine (L-Dopa). Denaturing gel electrophoresis of the glue reveals a characteristic pattern of proteins spanning 13-400 kDa. The largest protein (Nb-1R, apparent molecular mass 350-500 kDa) is also the most abundant, and this protein appears to be the key structural component. The solid glue can be dissolved in dilute acids; raising the ionic strength causes the glue components to self-assemble spontaneously into a solid which resembles the starting material. We describe scattering studies on dissolved and solid glue and provide microscopy images of glue surfaces and sections, revealing a porous interior that is consistent with the high water content (85-90 wt %) of moist glue. In addition to compositional similarities with other biological adhesives and well-known elastomeric proteins, the circular dichroism spectrum of dissolved glue is almost identical to that for soluble elastin and electron and scanning probe microscopy images invite comparison with silk fibroins. Covalent cross-linking does not seem to be necessary for the glue to set.

Adhesiveness↗