An uncharacterized gene locus (Chr16:hCG_1815491), now named colorectal neoplasia differentially expressed (gene symbol CRNDE), is activated early in colorectal neoplasia. The locus is unrelated to any known protein-coding gene. Microarray analysis of 454 tissue specimens (discovery) and 68 previously untested specimens (validation) showed elevated expression of CRNDE in >90% of colorectal adenomas and adenocarcinomas. These findings were confirmed and extended by exon microarray studies and RT-PCR assays. CRNDE transcription start sites were identified in CaCo2 and HCT116 cells by 5′-RACE. The major transcript isoforms in colorectal cancer (CRC) cell lines and colorectal tissue are CRNDE-a, -b, -d, -e, -f, -h, and -j. Except for CRNDE-d, the known CRNDE splice variants are upregulated in neoplastic colorectal tissue; expression levels for CRNDE-h alone demonstrate a sensitivity of 95% and specificity of 96% for adenoma versus normal tissue. A quantitative RT-PCR assay measuring CRNDE-h RNA levels in plasma was (with a threshold of 2 -ΔCt = 2.8) positive for 13 of 15 CRC patients (87%) but only 1 of 15 healthy individuals (7%). We conclude that individual CRNDE transcripts show promise as tissue and plasma biomarkers, potentially exhibiting high sensitivity and specificity for colorectal adenomas and cancers.

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.

Colorectal neoplasia differentially expressed (CRNDE) is a novel gene that is activated early in colorectal cancer but whose regulation and functions are unknown. CRNDE transcripts are recognized as long non-coding RNAs (lncRNAs), which potentially interact with chromatin-modifying complexes to regulate gene expression via epigenetic changes. Complex alternative splicing results in numerous transcripts from this gene, and we have identified novel transcripts containing a highly-conserved sequence within intron 4 ("gVC-In4"). In colorectal cancer cells, we demonstrate that treatment with insulin and insulin-like growth factors (IGF) repressed CRNDE nuclear transcripts, including those encompassing gVC-In4. These repressive effects were negated by use of inhibitors against either the PI3K/Akt/mTOR pathway or Raf/MAPK pathway, suggesting CRNDE is a downstream target of both signaling cascades. Expression array analyses revealed that siRNA-mediated knockdown of gVC-In4 transcripts affected the expression of many genes, which showed correlation with insulin/IGF signaling pathway components and responses, including glucose and lipid metabolism. Some of the genes are identical to those affected by insulin treatment in the same cell line. The results suggest that CRNDE expression promotes the metabolic changes by which cancer cells switch to aerobic glycolysis (Warburg effect). This is the first report of a lncRNA regulated by insulin/IGFs, and our findings indicate a role for CRNDE nuclear transcripts in regulating cellular metabolism which may correlate with their upregulation in colorectal cancer.

No abstract

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 Ashburner's classical biochemical evidence for the central role of the ecdysone receptor in his model.

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.

Lipoamide and a peptide, Thr-Val-Glu-Gly-Asp-Lys-Ala-Ser-Met-Glu lipoylated on the N6-amino group of the lysine residue, were tested as substrates for reductive acetylation by the pyruvate decarboxylase (E1p) component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The peptide has the same amino acid sequence as that surrounding the three lipoyllysine residues in the lipoate acetyltransferase (E2p) component of the native enzyme complex. Lipoamide was shown to be a very poor substrate, with a Km much higher than 4 mM and a value of kcat/Km of 1.5 M-1.s-1. Under similar conditions, the three E2p lipoyl domains, excised from the pyruvate dehydrogenase complex by treatment with Staphylococcus aureus V8 proteinase, could be reductively acetylated by E1p much more readily, with a typical Km of approximately 26 microM and a typical kcat of approximately 0.8 s-1. The value of kcat/Km for the lipoyl domains, approximately 3.0 x 10(4) M-1.s-1, is about 20,000 times higher than that for lipoamide as a substrate. This indicates the great improvement in the effectiveness of lipoic acid as a substrate for E1p that accompanies the attachment of the lipoyl group to a protein domain. The free E2o lipoyl domain was similarly found to be capable of being reductively succinylated by the 2-oxoglutarate decarboxylase (E1o) component of the 2-oxoglutarate dehydrogenase complex of E. coli. The 2-oxo acid dehydrogenase complexes are specific for their particular 2-oxo acid substrates. The specificity of the E1 components was found to extend also to the lipoyl domains.(ABSTRACT TRUNCATED AT 250 WORDS)

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.