The fibulins are a newly abundantly in the
The fibulins are a newly abundantly in the basement membrane of blood vessels. Among them, fibulin-1 is essential for elastin polymerization. Tropoelastin cannot be assembled into mature elastin fibrils without the functional fibulin-1. In adult blood vessels, pronounced fibulin 1 deposition can be found in the matrix that surrounds vascular smooth muscle cells and in the elastic laminae of arteries. Fibulin-1 deficiency in mice causes extensive hemorrhaging and perinatal death. Another study has also shown that patients with unstable angina pectoris and acute myocardial infarction have significantly reduced levels of plasma fibulin-1. These findings have led to the speculation that plasma fibulin-1 may be transferred to or consumed in or around the atherosclerotic lesion. In addition, fibulin-1 has been incorporated into fibrin clots that are associated with atherosclerotic lesions. Findings from DNA microarray studies showed that fibulin-1 is consistently associated with MMP-2 expression in cancer. Thus, altered patterns of fibulin-1 and MMP gene expression suggest a pre-existing structural failure of the aortic wall, which may lead to dissection.
The Affymetrix (Affymetrix, Santa Clara, Calif) platform is very sensitive in detecting signals corresponding to expressed mRNAs that contains 54,675 probe sets that can analyze the expression level of over 47,000 transcripts. Until now, only one German institute study has examined the gene profiles of the dissected and adjacent macroscopically intact aorta using the Atlas human broad array, with 3,537 genes being analyzed.
Introduction The ability to break down proteins and complex carbohydrates, by digestive carbohydrases and proteases in the gut, into free Epidermal Growth Factor (EGF), human recombinant and simple sugars, respectively is essential for growth and development of insects (Bown et al., 2004). In many coleopteran species such as the cowpea bruchid (Callosobruchus maculatus Fabricius) and the western corn rootworm (Diabrotica virgifera virgifera LeConte), cysteine proteases are the major digestive enzymes (Koiwa et al., 2000, Zhu-Salzman et al., 2003). In D. abbreviatus, the presence of both cathepsins (cysteine proteases) and serine proteases have been identified in digestive tract extracts, but only the trypsin-like enzyme was extensively characterized and was shown to be controlled by AeaTMOF (Yan et al., 1999). Cathepsins belong to the papain family of proteins that include lysosomal proteins (Cristofoletti et al., 2005) that are involved in cellular protein catabolism (Kamboj et al., 1993, Stachowiak et al., 2004). Coleopterans and hemipterans utilize cathepsins to digest their food in the digestive tract lumen (Terra, 1990). Cathepsins are synthesized as pre-pro-enzymes and are secreted into the lumen by the gut epithelial cells. The pre-region serves as a signal peptide allowing the enzyme to enter the cellular secretory pathway targeting it for secretion. The pro-region is essential for the proper folding and for inhibiting proteolytic activity until it is cleaved in the gut’s lumen by autocatalysis generating a mature active protein. D. abbreviatus midgut is partitioned into anterior and posterior regions, similar to the aphid, Aphis gossypii Glover (Deraison et al., 2004), and the weevil Otiorhynchus sulcatus Fabricius (Edwards et al., 2010). Although no direct pH measurements have been done to show the pH of these regions, in the water weevil Lissorhoptrus brevirostris, chymotrypsin like activity in the gut was the highest in the middle gut and posterior gut sections strongly indicating that in these sections the pH is basic, a requirement for chymotrypsin activity (Hernández et al., 2003). These observations suggest that acidic cathepsins would not be able to function in the posterior region of the gut. All cathepsins are characterized by a cysteine residue at the active site (Turk et al., 2000) with a broad specificity for cleaving peptide bonds. Additionally, cathepsins are classified based on characteristics such as their activities with unique substrates and specific inhibitors, and pH optimum for activity (Kirschke et al., 1977, Turk et al., 2000, Rawlings et al., 2012). In vitro enzymatic assays used to characterize cathepsins feature small peptide substrates consisting of a sequence of amino acid residues linked to a measurable chromogenic or fluorogenic leaving group (Tchoupe et al., 1991). The preferred amino acid sequences of substrates for various proteases including cathepsin L are determined via Positional Scanning Synthetic Combinatorial Libraries (Cotrin et al., 2004, Choe et al., 2006). Cathepsin L has been shown to have substrate tolerance for hydrophobic residues in the P2 and P3 positions (Otto and Schirmeister, 1997) with a preference for leucine or arginine at the P2 position (Clara et al., 2011). A protease database (MEROPS) groups various proteases according to their chemical properties including the sequence of amino acids in suitably cleaved substrates (Rawlings et al., 2012). Using information garnered from the MEROPS database, and from the characterization studies of the recombinant cathepsin L1 (Da-CTSL1) we show for the first time that Da-CTSL1 is a unique basic enzyme that could function in the presumed basic posterior region of D. abbreviatus gut.