Lloyd D. Graham

Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells

Perlecan, a major heparan sulfate proteoglycan of vascularized tissues, was immunopurified from media conditioned by human endothelial cells of both arterial and venous origin. The heparan sulfate moiety of perlecan from cultured arterial cells differed in amount and/or composition from that produced by a transformed cell line of venous origin. Both forms of perlecan bound basic fibroblast growth factor with Kd approximately 70 nM. In ELISA experiments, perlecan and its protein core bound to various extracellular matrix components in a manner that was strongly influenced by the format of the assay. Human vascular smooth muscle cells and human endothelial cells adhered to perlecan-coated surfaces, and both cell types adhered better to the venous cell-derived than to the arterial cell-derived perlecan. Removal of the heparan sulfate chains abolished this difference and increased the ability of both types of perlecan to adhere vascular cells. Denaturation of perlecan and its protein core also rendered each of them more adhesive, indicating the presence of conformation-independent adhesion determinants in the polypeptide sequence. Their location was investigated using recombinant perlecan domains. Overall, our results represent the first demonstration of human perlecan acting as an adhesive molecule for human vascular cells and suggest that it may play a role in vascular wound healing.

CRNDE: A Long Non-Coding RNA Involved in CanceR, Neurobiology, and DEvelopment.

CRNDE is the gene symbol for Colorectal Neoplasia Differentially Expressed (non-protein-coding), a long non-coding RNA (lncRNA) gene that expresses multiple splice variants and displays a very tissue-specific pattern of expression. CRNDE was initially identified as a lncRNA whose expression is highly elevated in colorectal cancer, but it is also upregulated in many other solid tumors and in leukemias. Indeed, CRNDE is the most upregulated lncRNA in gliomas and here, as in other cancers, it is associated with a “stemness” signature. CRNDE is expressed in specific regions within the human and mouse brain; the mouse ortholog is high in induced pluripotent stem cells and increases further during neuronal differentiation. We suggest that CRNDE is a multifunctional lncRNA whose different splice forms provide specific functional scaffolds for regulatory complexes, such as the polycomb repressive complex 2 (PRC2) and CoREST chromatin-modifying complexes, which CRNDE helps pilot to target genes.

Ecdysone Receptors: From the Ashburner Model to Structural Biology ∗

In 1974, Ashburner and colleagues postulated a model to explain the control of the puffing sequence on Drosophila polytene chromosomes initiated by the molting hormone 20-hydroxyecdysone. This model inspired a generation of molecular biologists to clone and characterize elements of the model, thereby providing insights into the control of gene networks by steroids, diatomic gases, and other small molecules. It led to the first cloning of the EcR subunit of the heterodimeric EcR-USP ecdysone receptor. X-ray diffraction studies of the ligand-binding domain of the receptor are elucidating the specificity of receptor-ecdysteroid interactions, the selectivity of some environmentally friendly insecticides, the evolution of the EcR-USP heterodimer, and indeed Ashburners classical biochemical evidence for the central role of the ecdysone receptor in his model.

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.

Proteinaceous adhesive secretions from insects, and in particular the egg attachment glue of Opodiphthera sp. Moths

Biochemical and electrophoretic screening of 29 adhesive secretions from Australian insects identified six types that appeared to consist largely of protein. Most were involved in terrestrial egg attachment. Hydrogel glues were subjected to gravimetric analyses and assessed for overall amino acid composition. When 32 proteins in glues from eight insect species were analyzed individually, many proved to be rich in Gly, Ser, and/or Pro, and some contained substantial levels of 4-hydroxyproline. A few proteins were heavily glycosylated. Abundant protein-based secretions were tested as adhesives, mainly by measuring dry shear strength on wood. The strongest (1-2 MPa) was an egg attachment glue produced by saturniid gum moths of the genus Opodiphthera. It was harvested from female colleterial gland reservoirs as a treacle-like liquid that underwent irreversible gelation, and recovered from the capsules of laid eggs as a highly elastic orange-brown hydrogel that could also display high tack. Its protein-based nature was confirmed and explored by spectroscopy, enzymatic degradation, and 2D gel electrophoresis. Its proteins are mostly 80-95 kDa, and sequences (almost all novel) were established for 23 tryptic peptides. Scanning probe microscopy of Opodiphthera hydrogel in water returned median values of 0.83 nN for adhesion, 63 kPa for modulus, and 87% for resilience. Recombinant mimics of this material might be useful as biodegradable commodity adhesives or as specialty biomedical products.

An ortholog of the ecdysone receptor protein (EcR) from the parasitic nematode Haemonchus contortus.

High concentrations (> or =4.2mM) of 20E inhibit the development of Haemonchus contortus eggs to the L3 larval stage. We report the cloning of cDNA encoding an EcR ortholog (HcEcR) from H. contortus mRNA expressed during L3. Phylogenetically, this and the putative EcR from Brugia malayi form a separate branch between arthropod EcRs and liver X receptors. Two isoforms of HcEcR differ in the inclusion/omission of a 3-residue segment in the A/B domain. Single nucleotide polymorphisms at 49 positions can be grouped into two major patterns in the A/BC segment and two in the DE/F segment. Some 35% of the highly conserved ecdysteroid-contacting residues in insect EcRs are also conserved in the HcEcR ligand binding domain, but it contains unusual residue choices at other ligand-contacting positions. Recombinant co-expression of HcEcR DE/F segments with a phthirapteran USP DE/F segment in insect cells resulted in stable proteins which did not heterodimerize or bind [(3)H]ponasterone A.

CAHM, a long non-coding RNA gene hypermethylated in colorectal neoplasia.

The CAHM gene (Colorectal Adenocarcinoma HyperMethylated), previously LOC100526820, is located on chromosome 6, hg19 chr6:163 834 097–163 834 982. It lacks introns, encodes a long non-coding RNA (lncRNA) and is located adjacent to the gene QKI, which encodes an RNA binding protein. Deep bisulphite sequencing of ten colorectal cancer (CRC) and matched normal tissues demonstrated frequent hypermethylation within the CAHM gene in cancer. A quantitative methylation-specific PCR (qMSP) was used to characterize additional tissue samples. With a threshold of 5% methylation, the CAHM assay was positive in 2/26 normal colorectal tissues (8%), 17/21 adenomas (81%), and 56/79 CRC samples (71%). A reverse transcriptase-qPCR assay showed that CAHM RNA levels correlated negatively with CAHM % methylation, and therefore CAHM gene expression is typically decreased in CRC. The CAHM qMSP assay was applied to DNA isolated from plasma specimens from 220 colonoscopy-examined patients. Using a threshold of 3 pg methylated genomic DNA per mL plasma, methylated CAHM sequences were detected in the plasma DNA of 40/73 (55%) of CRC patients compared with 3/73 (4%) from subjects with adenomas and 5/74 (7%) from subjects without neoplasia. Both the frequency of detection and the amount of methylated CAHM DNA released into plasma increased with increasing cancer stage. Methylated CAHM DNA shows promise as a plasma biomarker for use in screening for CRC.

Biocompatibility and modification of the protein‐based adhesive secreted by the Australian frog Notaden bennetti

When provoked, Notaden bennetti frogs secrete a proteinaceous exudate, which rapidly forms a tacky and elastic glue. This material has potential in biomedical applications. Cultured cells attached and proliferated well on glue-coated tissue culture polystyrene, but migrated somewhat slower than on uncoated surfaces. In organ culture, dissolved glue successfully adhered collagen-coated perfluoropolyether lenses to debrided bovine corneas and supported epithelial regrowth. Small pellets of glue implanted subcutaneously into mice were resorbed by surrounding tissues, and all of the animals made a full recovery. An initial but transient skin necrosis at the implant site was probably caused by some of the potentially toxic metabolites present in the frog secretion; these include sterols and carotenoids, as well as fatty alcohols, aldehydes, ketones, acids, and aromatic compounds. Removal of the carotenoid pigments did not significantly alter the glues material properties. In contrast, peroxidase treatment of dissolved glue introduced unnatural crosslinks between molecules of the major protein (Nb-1R) and resulted in the formation of a soft hydrogel, which was very different to the original material.

THE EFFECT OF HUMAN ENDOTHELIAL CELL‐DERIVED PROTEOGLYCANS ON HUMAN SMOOTH MUSCLE CELL GROWTH

Extracellular proteoglycans (PGs) purified from cultured human arterial endothelial cells were tested for their effects on the proliferation of human vascular smooth muscle cells (VSMC). Fractions containing perlecan, the basement membrane heparan sulphate (HS) PG, the large chondrotin sulphate (CS) proteoglycan from connective tissue and other immunoreactive CS did not inhibit the proliferation of human VSMC. Native endothelial extracellular matrix, which was shown to contain the same PGs, demonstrated a pronounced stimulatory effect on the proliferation of human VSMCs. This stimulatory effect was not removed by pre‐incubation of the matrix with 1M NaCl, heparin, platelet extract or plasmin. These experiments demonstrate that PGs produced by human arterial endothelial cells do not inhibit the proliferation of VSMC. These data do not support the hypothesis that human endothelial cells, in vivo control the activation or proliferation of VSMCs directly by the secretion of a non‐proliferative molecule. Instead they support the hypothesis that the endothelial cells counteract intimal hyperplasia of VSMC indirectly by providing a barrier from activating factors in the plasma.

Chapter Four – Structure and Function of Ecdysone Receptors—Interactions with Ecdysteroids and Synthetic Agonists

Abstract The binding of ecdysteroids and the bisacylhydrazine insecticide, tebufenozide, to recombinant ecdysone receptor ligand-binding domains from pest insects points to conserved and variable features of the receptors ligand-binding pocket. Fluorophores conjugated to the terminus of the ecdysteroid alkyl chain have surprisingly little effect on receptor binding, permitting the development of a fluorescence polarization chemical library screen that has led to the discovery of a new class of ecdysone receptor ligands, the methylene lactams. X-ray structures of ecdysone receptor ligand-binding domains have allowed identification of the conserved and variable features within the binding pocket. The structures offer explanations for the lepidopteran selectivity of the bisacylhydrazines, the effect of amino acid replacements on the binding of ecdysteroids and other chemistries, and the preference of a phytophagous pentatomomorphan for makisterone A; indeed, they speak to the control spectra of future ecdysone receptor-targeting insecticides. Possible ligands for nematode ecdysone receptor orthologs are also considered.