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  • It was thus found that E plays an


    It was thus found that E1 plays an important role in G. intestinalis biology. Inhibiting E1 expression by RNA antisense in trophozoites was lethal while E1 overexpression induced a five-fold increase in cyst formation during encystation. The changes in E1 location during this process and its localization in the cyst wall might show a role for E1 and ubiquitination in related cellular process. All the results presented here strongly indicated that E1, and ubiquitination itself, is essential for Giardia development and differentiation. The specific proteolytic process observed in E1 might be a clue in the evolution of this family of enzymes.
    Acknowledgements This work was supported by the Universidad Nacional de Colombia\'s research division (DIB) projects 8003322 and 8003244, the Colombian Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) project 486-80 and the Argentinian Ministerio de Ciencia y Tecnología (MinCyT) project CO/08/10. C.A.N has been awarded a scholarship from the “Universidad Nacional de Colombia” Outstanding Graduate Students Program.
    Introduction Ciona intestinalis is known to be the closest living invertebrate relative of vertebrates. C. intestinalis is a good experimental model for evolutionary and functional studies of the endocrine system owing to its relatedness to the vertebrate system. This marine organism has been proposed as a good laboratory model owing to its phylogenetic position in the complete genome project. Moreover, studies suggest that some of the antimicrobial peptides obtained from this marine organism could be biologically useful [1,2]. C. intestinalis grows, reproduces, and spreads quickly [3], competing with another suspension feeders for food or heavily grazing phytoplankton [4]. Its growth has been documented in Hong Kong, New Zealand, Japan, Canada, Spain, South Africa, and Chile [5,6]. C. intestinalis has a maturation period of approximately 2–3 months and high reproductive output [7]. Thus, C. intestinalis is listed as a source organism for material supply. Marine organisms are a highly promising resource for bioactive substances and natural products. The bioactive of peptides is dependent on their amino (±)-Bay K 8644 weight composition and sequence levels. Nowadays, peptides from marine organisms have opened a new potential model for drug development. The discovery of novel natural products that induce osteoblast differentiation is a key goal of therapy to bone disease. Thus, we consider the possibility that bioactive calcitonin from C. intestinalis may promote osteoblast differentiation. Osteoporosis or porous bone is the common metabolic bone disorder. It has characterized by a systemic reduction of bone mineral, bone volume, and strength [8]. Osteoporosis is a bone disease in which bones become fragile by a systemic reduction of bone mass and strength and also is controlled by an imbalance between resorption and formation of the bone [[9], [10], [11]]. Bone remodeling is controlled by a balance between bone resorption and formation. This balance depends on the interaction between osteoblasts, which induce new bone formation, and osteoclasts that induce bone resorption [12]. During osteoblast differentiation, transcription factors such as osterix and Runx2 stimulate the activation of alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OSC), which lead to bone formation [13]. The bone morphogenetic protein (BMP) was known as the growth factor superfamily, which can stimulate osteoblast differentiation in MC3T3-E1 cells. Among these BMPs, bone morphogenetic protein 2 (BMP 2) is an important in the stimulation bone formation in vitro and in vivo [9,14,15]. BMP2 also has the ability to stimulate cyclooxygenase-2 (COX-2) expression, which regulates osteoblastic differentiation in vitro [14]. In osteoblast precursor cells, BMP 2 stimulates osteogenic action by activating Smad1/5/8, which controls the expression of differentiation markers such as bone sialoprotein, ALP, OPN, and OSC [16]. Osteogenesis is a complex process that involves both Smad1/5/8 pathway and mitogen-activated protein kinase (MAPK). The p38 and extracellular signal-regulated kinases (ERK) are important mediators in the MAPK pathway and contribute to the induction of cell differentiation [[16], [17], [18]]. For this study, we chose the pre-osteoblasts cells (MC3T3-El), which is commonly used as a model system to study bone formation. These cells display an orderly development in a culture characterized by the proliferation and differentiation of MC3T3-El cells, followed by proliferation, matrix maturation, and matrix mineralization phases, and display characteristics such as increased ALP activity, matrix accumulation, OSC and OPN expression, as well as eventual mineralization. Peptide-derived has potential activity as antioxidants, antihypertensives, and anticancer drugs and is widely used for bone regeneration. One of them is calcitonin, gene structures of the second calcitonin gene-related peptide from human, salmon, and C. intestinalis was identified the structure [[19], [20], [21]]. However, only calcitonin from human and salmon has been resulted (±)-Bay K 8644 weight to inhibit the bone resorption, and increase the growth of bone and cartilage [19,21]. Sekiguchi et al. [20] demonstrated that calcitonin from C. intestinalis is expressed more than calcitonin-vertebrate in organs. In addition, the calcitonin gene from C. intestinalis is composed of four exons and three introns, which was found to specifically inhibit osteoclasts by suppressing tartrate-resistant acid phosphatase activity [20]. However, little is known about the influence and mechanism of sea squirt C. intestinalis calcitonin-like peptide (CCLP) on pre-osteoblast MC3T3-E1 cells. The goal of this study shows the effects of CCLP inducing osteoblast proliferation and differentiation in MC3T3-E1 cells.