RNA helicases catalyze the unwinding of double-stranded RNA structures to perform numerous genetic processes. These enzymes are characterized by the presence of a conserved helicase domain with ...specific helicases motifs whose amino acid sequence allows the differentiation between DEAD and DExH-box RNA helicase families. Taking advantage of the availability of the complete genome sequence of
Entamoeba histolytica, the protozoan responsible for human amoebiasis, we have performed a genomic survey for DEAD and DExH-box RNA helicases encoding genes in this organism. By extensive
in silico analysis, we identified 20 EhDEAD and 13 EhDExH-box RNA helicases, which contain almost all the conserved helicase motifs. Additionally, several EhDEAD and EhDExH proteins present specific N- and C-terminal domains that could be related to subcellular localization or function. Phylogenetic studies and sequences analysis suggested that this large EhDEAD/DExH-box RNA helicases family has been generated by gene or internal regions duplication, mutation events, introns formation and motif deletions. Interestingly,
EhDexh1 and EhDeaxh10 genes seem to be formed by gene fusion of two ancestral bacterial genes, a mechanism that appears to be evolutionary conserved in the eukaryotic lineage of orthologous proteins. Finally, RT-PCR assays, microarrays and proteomics data analysis showed that several
EhDead are differentially expressed in relation to distinct culture conditions. These computational and experimental data provide new information on the evolution of EhDEAD/EhDExH-box RNA helicases and their potential relevance for RNA metabolism in
E. histolytica.
A group of cDNAs isolated from a subtractive ciliary body library of a normal human eye donor revealed 100% identity with
TIGR a candidate gene responsible for juvenile open angle glaucoma Science ...275 (1997) 668–670. Several structural features of the deduced human protein have been noted: a cleavable N-terminal signal peptide, a periodic repetition at the N-terminus of leucine and arginine residues at every seventh and eleven position respectively in helix conformation (leucine zipper-like motif) exhibiting homology with myosin, and with olfactomedin in the C-terminus. The mRNA for
TIGR is abundantly expressed in the ciliary body, iris, heart and skeletal muscle.
Mutations in the MYOC/TIGR gene are responsible for autosomal dominant primary open angle glaucoma (POAG). Almost all mutations responsible for POAG have been detected in the coding region (in ...particular at exon 3). By using the techniques of PCR, SSCP, automated sequencing and restriction analysis, we have studied 79 patients suffering from glaucoma. We have found five patients with sequence variants in the consensus region of the promoter. These sequence variants might be involved in the altered association between the consensus region and the corresponding transcription factor. This possibility might be favouring the association of other transcription factors, which would operate as activators or inhibitors of the transcription, altering the MYOC/TIGR expression.
The proteins processed by the secretory pathway (secretome) are critical players in the development of multi‐cellular eukaryotic organisms but have yet to be comprehensively studied at the genomic ...level. In this study, we use the Target P algorithm to predict human (13–20% of proteins found in individual datasets) and Fugu (14%) secretomes based on analysis of their nearly complete proteomes. We combine internal processing with prediction software to automate secreted protein identification and overcome one of the major challenges associated with EST data: identification of the minority of clones that encode N‐terminally‐complete proteins. We discuss the use of these methods to predict secreted proteins in EST‐based consensus sequence sets, and we validate these predictions using an assay for cell‐free cotranslational translocation. Analysis of TIGR Porcine Gene Index 4.0 as a test dataset resulted in the identification of 352 N‐terminally‐complete, putative secreted proteins. In functional agreement with our predictions, 34 of 40 (85%) of these cDNAs were verified to be cotranslationally translocated in an in vitro translation system. The methods developed here are specifically designed to accept partial open reading frames and improve secreted protein predictions in eukaryotic transcriptomes, and are valuable for the analysis and annotation of eukaryotic EST databases.
TIGR/MYOC, a novel 504 amino acids (aa) protein of unknown function, has recently been linked to glaucoma. The protein is both intra- and extracellular and most known mutations map to its C-terminus, ...an olfactomedin-like domain. To investigate the properties of a TIGR/MYOC peptide lacking this important domain, we constructed a replication-deficient adenovirus with the first 344 aa and over-expressed the truncated protein in primary human trabecular meshwork cells and perfused human anterior segment cultures. The truncated mutant contains the entire N-terminus plus 98 aa of the olfactomedin-like domain. We found that the delivered truncated mutant accumulates inside the cell, reduces secretion of endogenous TIGR/MYOC and induces an increase in outflow facility at 48 h post-infection. Based on these findings, we hypothesize that TIGR/MYOC might have a dual role in trabecular meshwork function. This dual role might be that of an intracellular modulator of vesicular transport as well as that of a secreted protein involved in extracellular matrix conformation. Both functions could have a direct effect in maintaining aqueous humor outflow facility.
Mutations in TIGR/MYOC (myocilin), a secretory protein of unknown function, have been recently linked to glaucoma. Most known mutations map to the C-terminus, an olfactomedin-like domain. We have ...previously shown that, in contrast to the wild-type, a truncated form of myocilin lacking the olfactomedin domain is not secreted. In this study, we present evidence that the mutant protein is not correctly processed in the endoplasmic reticulum (ER) and accumulates into insoluble aggregates. In addition, we show that the presence of increasing amounts of mutant protein induces a fraction of the soluble, native myocilin to move to the insoluble fraction. Given the importance of such protein aggregates in the etiology of several aging-related diseases, we propose that olfactomedin-defective mutants might contribute to the pathology of glaucoma through a mechanism involving intracellular accumulation of misfolded proteins.
Mutations in the the glaucoma gene GCL1A , also known as trabecular meshwork glucocorticoid response (TIGR) or myocilin (Myoc), have been shown to be associated with juvenile-onset primary open-angle ...glaucoma. Very little is known about the pattern of expression of the TIGR gene in human ocular tissues. In-situ hybridization experiments demonstrated the localization of TIGR mRNA in cells throughout the iris, ciliary muscle, and the filtering portion of the trabecular meshwork of normal eye donors. The expression of TIGR protein was investigated by Western blot using an epitope-directed antibody to the carboxy terminus region of TIGR. This antibody was able to distinguish a recombinant TIGR fusion protein from a truncated TIGR form containing the naturally occurring Gln 368 ?stop mutation. In tissue extracts from the iris, ciliary body, and trabecular meshwork, the antibody recognized a major protein band of 57-kDa molecular mass.
Titanium–graphite hybrid composite laminates exhibit a coupled damage growth mode of facesheet cracking and delamination. Part I of this work modeled the growth of the coupled damage mode. Fatigue ...experiments were conducted on single edge notch tension specimens to measure the crack growth rate. This paper compares the model predictions with experimental data. The three-dimensional finite element model successfully captured the damage growth behavior for two of the lay-ups (Ti/0/90/0
2
s and Ti/90/0/90
2
s) in the experimental program. However, in a third lay-up, Ti/0/90/±30
s, the underlying damage modes were found to be sufficiently different than the other two lay-ups and the model did not capture the steady-state growth behavior. The effects of temperature and specimen size were also investigated for TiGr laminates. Except for the effects of the load ratio, elevated temperatures did not affect the crack growth rate significantly. For wider specimens, the steady-state fatigue crack growth behavior was similar to the narrow specimens, indicating that the steady-state facesheet crack growth behavior is independent of specimen size.
We describe Quality of Information (QoI) in terms of Type Theory in order to: clarify the meaning of QoI and desired capabilities of "quality aware" systems; to show how any quality metric can be ...expressed as a function of data and data provenance which negates the need for specific quality annotations; and to indicate how data processing may automatically be applied to deliver high quality information.
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