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Biomedical subjects

Francisco J Blanco

Publications and source records attributed to Francisco J Blanco.

At least 19 recordsLinked to original sources
PubMed2007

The coiled-coil structure potential of the laminin LCC domain is very fragmented and does not differentiate between natural and non-detected isoforms.

There are 15 known laminins, which differ in the isoforms of the three chains that assemble into the cross-shape molecules that are observed by electron microscopy. The amino acid sequences of the rod-like portion of the long arm have long been recognized as having a potential for coiled-coil structure formation; however, an experimental determination of its structure is hampered by the complexity of laminin, a multidomain, heterotrimeric, and glycosilated 800 kDa molecule. Here, we have investigated the coiled-coil structure potential of laminin to evaluate its distribution along the long arm, the presence of conserved patterns, and differences between natural and non-natural isoforms. With these aims, we have analysed the sequences of each laminin chain in the context of the three-chain assemblies to yield an overall score of coiled-coil potential for the 15 natural laminins and for the other 30 possible but non-detected ones. The potential has been calculated with two different existing methods to exclude algorithm specific biases and with different chain alignments to evaluate the dependency of the results on uncertainties in the specific alignment along the domain. The analysis shows that the distribution of the potential is discontinuous, highly fragmented along the arm, without a common pattern except for a higher potential at the C-terminus, and that natural and non-natural laminins cannot be distinguished based on their coiled-coil potential, indicating that other factors are responsible for the selection of chain assembly.

Animals↗
PubMed2006

Solution structure and NMR characterization of the binding to methylated histone tails of the plant homeodomain finger of the tumour suppressor ING4.

Plant homeodomain (PHD) fingers are frequently present in proteins involved in chromatin remodelling, and some of them bind to histones. The family of proteins inhibitors of growth (ING) contains a PHD finger that bind to histone-3 trimethylated at lysine 4, and those of ING1 and ING2 also act as nuclear phosphoinositide receptors. We have determined the structure of ING4 PHD, and characterised its binding to phosphoinositides and histone methylated tails. In contrast to ING2, ING4 is not a phosphoinositide receptor and binds with similar affinity to the different methylation states of histone-3 at lysine 4.

Histones↗
PubMed2006

A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.

DNA↗
PubMed2006

Structural model of human endoglin, a transmembrane receptor responsible for hereditary hemorrhagic telangiectasia.

Endoglin is a type I membrane protein expressed as a disulphide-linked homodimer on human vascular endothelial cells whose haploinsufficiency is responsible for the dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia (HHT). Structurally, endoglin belongs to the zona pellucida (ZP) family of proteins that share a ZP domain of approximately 260 amino acid residues at their extracellular region. Endoglin is a component of the TGF-beta receptor complex, interacts with the TGF-beta signalling receptors types I and II, and modulates cellular responses to TGF-beta. Here, we have determined for the first time the three-dimensional structure of the approximately 140 kDa extracellular domain of endoglin at 25 A resolution, using single-particle electron microscopy (EM). This reconstruction provides the general architecture of endoglin, which arranges as a dome made of antiparallel oriented monomers enclosing a cavity at one end. A high-resolution structure of endoglin has also been modelled de novo and found to be consistent with the experimental reconstruction. Each subunit comprises three well-defined domains, two of them corresponding to ZP regions, organised into an open U-shaped monomer. This domain arrangement was found to closely resemble the overall structure derived experimentally and the three modelled de novo domains were tentatively assigned to the domains observed in the EM reconstruction. This molecular model was further tested by tagging endoglin's C terminus with an IgG Fc fragment visible after 3D reconstruction of the labelled protein. Combined, these data provide the structural framework to interpret endoglin's functional domains and mutations found in HHT patients.

Amino Acid Sequence↗
PubMed2006

Enhanced antiangiogenic therapy with antibody-collagen XVIII NC1 domain fusion proteins engineered to exploit matrix remodeling events.

Antiangiogenic therapy is nowadays one of the most active fields in cancer research. The first strategies, aimed at inhibiting tumor vascularization, included upregulation of endogenous inhibitors and blocking of the signals delivered by angiogenic factors. But interaction between endothelial cells and their surrounding extracellular matrix also plays a critical role in the modulation of the angiogenic process. This study introduces a new concept to enhance the efficacy of antibody-based antiangiogenic cancer therapy strategies, taking advantage of a key molecular event occurring in the tumor context: the proteolysis of collagen XVIII, which releases the endogenous angiogenesis inhibitor endostatin. By fusing the collagen XVIII NC1 domain to an antiangiogenic single-chain antibody, a multispecific agent was generated, which was efficiently processed by tumor-associated proteinases to produce monomeric endostatin and fully functional trimeric antibody fragments. It was demonstrated that the combined production in the tumor area of complementary antiangiogenic agents from a single molecular entity secreted by gene-modified cells resulted in enhanced antitumor effects. These results indicate that tailoring recombinant antibodies with extracellular matrix-derived scaffolds is an effective approach to convert tumor progression associated processes into molecular clues for improving antibody-based therapies.

Angiogenesis Inhibitors↗
PubMed2006

Folding and aggregation kinetics of a beta-hairpin.

We have investigated the solution structure, equilibrium properties, and folding kinetics of a 17-residue beta-hairpin-forming peptide derived from the protein ubiquitin. NMR experiments show that at 4 degrees C the peptide has a highly populated beta-hairpin conformation. At protein concentrations higher than 0.35 mM, the peptide aggregates. Sedimentation equilibrium measurements show that the aggregate is a trimer, while NMR indicates that the beta-hairpin conformation is maintained in the trimer. The relaxation kinetics in nanosecond laser temperature-jump experiments reveal a concentration-independent microsecond phase, corresponding to beta-hairpin unfolding-refolding, and a concentration-dependent millisecond phase due to oligomerization. Kinetic modeling of the relaxation rates and amplitudes yields the folding and unfolding rates for the monomeric beta-hairpin, as well as assembly and disassembly rates for trimer formation consistent with the equilibrium constant determined by sedimentation equilibrium. When the net charge on the peptides and ionic strength were taken into account, the rate of trimer assembly approaches the Debye-Smoluchowski diffusion limit. At 300 K, the rate of formation of the monomeric hairpin is (17 micros)(-1), compared to rates of (0.8 micros)(-1) to (52 micros)(-1) found for other peptides. After using Kramers theory to correct for the temperature dependence of the pre-exponential factor, the activation energy for hairpin formation is near zero, indicating that the barrier to folding is purely entropic. Comparisons with previously measured rates for a series of hairpins are made to distinguish between zipper and hydrophobic collapse mechanisms. Overall, the experimental data are most consistent with the zipper mechanism in which structure formation is initiated at the turn, the mechanism predicted by the Ising-like statistical mechanical model that was developed to explain the equilibrium and kinetic data for the beta-hairpin from protein GB1. In contrast, the majority of simulation studies favor a hydrophobic collapse mechanism. However, with few exceptions, there is little or no quantitative comparison of the simulation results with experimental data.

Amino Acid Sequence↗
PubMed2006

Decreased metalloproteinase production as a response to mechanical pressure in human cartilage: a mechanism for homeostatic regulation.

Articular cartilage is optimised for bearing mechanical loads. Chondrocytes are the only cells present in mature cartilage and are responsible for the synthesis and integrity of the extracellular matrix. Appropriate joint loads stimulate chondrocytes to maintain healthy cartilage with a concrete protein composition according to loading demands. In contrast, inappropriate loads alter the composition of cartilage, leading to osteoarthritis (OA). Matrix metalloproteinases (MMPs) are involved in degradation of cartilage matrix components and have been implicated in OA, but their role in loading response is unclear. With this study, we aimed to elucidate the role of MMP-1 and MMP-3 in cartilage composition in response to mechanical load and to analyse the differences in aggrecan and type II collagen content in articular cartilage from maximum- and minimum-weight-bearing regions of human healthy and OA hips. In parallel, we analyse the apoptosis of chondrocytes in maximal and minimal load areas. Because human femoral heads are subjected to different loads at defined sites, both areas were obtained from the same hip and subsequently evaluated for differences in aggrecan, type II collagen, MMP-1, and MMP-3 content (enzyme-linked immunosorbent assay) and gene expression (real-time polymerase chain reaction) and for chondrocyte apoptosis (flow cytometry, bcl-2 Western blot, and mitochondrial membrane potential analysis). The results showed that the load reduced the MMP-1 and MMP-3 synthesis (p < 0.05) in healthy but not in OA cartilage. No significant differences between pressure areas were found for aggrecan and type II collagen gene expression levels. However, a trend toward significance, in the aggrecan/collagen II ratio, was found for healthy hips (p = 0.057) upon comparison of pressure areas (loaded areas > non-loaded areas). Moreover, compared with normal cartilage, OA cartilage showed a 10- to 20-fold lower ratio of aggrecan to type II collagen, suggesting that the balance between the major structural proteins is crucial to the integrity and function of the tissue. Alternatively, no differences in apoptosis levels between loading areas were found--evidence that mechanical load regulates cartilage matrix composition but does not affect chondrocyte viability. The results suggest that MMPs play a key role in regulating the balance of structural proteins of the articular cartilage matrix according to local mechanical demands.

Aged↗
PubMed2006

Hereditary hemorrhagic telangiectasia, a vascular dysplasia affecting the TGF-beta signaling pathway.

Hereditary hemorrhagic telangiectasia (HHT) is caused by mutations in endoglin (ENG; HHT1) or ACVRL1/ALK1 (HHT2) genes and is an autosomal dominant vascular dysplasia. Clinically, HHT is characterized by epistaxis, telangiectases and arteriovenous malformations in some internal organs such as the lung, brain or liver. Endoglin and ALK1 proteins are specific endothelial receptors of the transforming growth factor (TGF)-beta superfamily that are essential for vascular integrity. Genetic studies in mice and humans have revealed the pivotal role of TGF-beta signaling during angiogenesis. Through binding to the TGF-beta type II receptor, TGF-beta can activate two distinct type I receptors (ALK1 and ALK5) in endothelial cells, each one leading to opposite effects on endothelial cell proliferation and migration. The recent isolation and characterization of circulating endothelial cells from HHT patients has revealed a decreased endoglin expression, impaired ALK1- and ALK5-dependent TGF-beta signaling, disorganized cytoskeleton and the failure to form cord-like structures which may lead to the fragility of small vessels with bleeding characteristic of HHT vascular dysplasia or to disrupted and abnormal angiogenesis after injuries and may explain the clinical symptoms associated with this disease.

Activin Receptors, Type I↗
PubMed2005

Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets.

The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.

Amino Acid Sequence↗
PubMed2005

Multiple parosteal lipoma associated to polyarthritis.

Parosteal lipoma is a benign adipose tissue tumor situated directly in the bone cortex. All cases previously reported in the literature were described as solitary lesions. We describe a patient who presented with polyarthritis associated with multiple parosteal lipomatous involvement. A tissue sample from the distal portion of the forearm confirmed the presence of cumulative fat tissue with nodes of esteatonecrosis. To the best of our knowledge this is the first case of multiple parosteal lipoma associated to polyarthritis.

Adipose Tissue↗
PubMed2005

Interaction and functional interplay between endoglin and ALK-1, two components of the endothelial transforming growth factor-beta receptor complex.

Transforming growth factor-beta (TGF-beta) signaling in endothelial cells is able to modulate angiogenesis and vascular remodeling, although the underlying molecular mechanisms remain poorly understood. Endoglin and ALK-1 are components of the TGF-beta receptor complex, predominantly expressed in endothelial cells, and mutations in either endoglin or ALK-1 genes are responsible for the vascular dysplasia known as hereditary hemorrhagic telangiectasia. Here we find that the extracellular and cytoplasmic domains of the auxiliary TGF-beta receptor endoglin interact with ALK-1 (a type I TGF-beta receptor). In addition, endoglin potentiates TGF-beta/ALK1 signaling, with the extracellular domain of endoglin contributing to this functional cooperation between endoglin and ALK-1. By contrast, endoglin appears to interfere with TGF-beta/ALK-5 signaling. These results suggest that the functional association of endoglin with ALK-1 is critical for the endothelial responses to TGF-beta.

Activin Receptors, Type I↗
PubMed2005

Proteomic characterization of human normal articular chondrocytes: a novel tool for the study of osteoarthritis and other rheumatic diseases.

Articular cartilage is composed of cells and an extracellular matrix. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. There is currently a great lack of knowledge about the chondrocyte proteome. To solve this deficiency, we have obtained the first reference map of the human normal articular chondrocyte. Cells were isolated from cartilages obtained from autopsies without history of joint disease. Cultured cells were used to obtain protein extracts which were resolved by 2-DE and visualized by silver nitrate or CBB staining. Almost 200 spots were excised from the gels and analyzed using MALDI-TOF or MALDI-TOF/TOF MS. The analysis leads to the identification of 136 spots that represent 93 different proteins. A significant proportion of proteins are involved in cell organization (26%), energy (16%), protein fate (14%), metabolism (12%), and cell stress (12%). From all the identified proteins, annexins, vimentin, transgelin, destrin, cathepsin D, heat shock protein 47, and mitochondrial superoxide dismutase were more abundant in chondrocytes than in other types of mesenchymal cells such as Jurkat-T cells. As metabolic program of chondrocytes is altered in osteoarthritis and other rheumatic diseases, this proteomic map is an important tool for future studies on these pathologies.

Adolescent↗
PubMed2004

Mitochondrial dysfunction in osteoarthritis.

In osteoarthritis (OA) a time or age dependent process leads to aberrant cartilage structure which is characterized by reduced number of chondrocytes, loss of existing cartilage extracellular matrix, the production of matrix with abnormal composition and pathologic matrix calcification. Because chondrocyte matrix synthesis and mineralization are modulated by the balance between ATP generation and consumption, the mechanism by which chondrocytes generate energy have been a topic of interest. The analysis of mitochondrial respiratory chain (MRC) activity in OA chondrocytes shows a significant decrease in complexes II and III compared to normal chondrocytes. On the other hand, mitochondrial mass is increased in OA, as demonstrated by a significant rise in CS activity. Furthermore, OA cells show a reduction in the mitochondrial membrane potential (deltapsim) as demonstrated by using the fluorescent probe JC-1. OA cartilage contains high number of apoptotic chondrocytes, and mitochondria play a key role in apoptosis. Interestingly, OA cartilages show markedly elevated Bcl-2 and caspasa-3 expression. This expression is also correlated with chondrocyte apoptosis and OA lesions. The pathogenesis of OA includes elaboration of increased amounts of NO as a consequence of up-regulation of chondrocyte-inducible NO synthase induced by IL-1, TNF-alpha and other factors. NO reduces chondrocyte survival and induces cell death with morphologic changes characteristic of chondrocyte apoptosis. NO reduces the activity of complex IV and decreases the deltapsim as measured as the ratio of red/green fluorescence. Furthermore, NO induces the mRNA expression of caspase-3 and -7, and it reduces the expression of mRNA bcl-2 and the bcl-2 protein synthesis. Some studies suggest that the chondrocyte mitochondria are specialized for calcium transport and are important in the calcification of the extracellular matrix. Mineral formation has been demonstrated in matrix vesicles (MV) and within mitochondria. Direct suppression of mitochondrial respiration promoted MV-mediated mineralization in chondrocytes. Regulation of MRC may be one of the signaling pathways by which NO modulates articular cartilage matrix biosynthesis and pathologic mineralization. After age 40, the incidence of OA in humans increases progressively with increasing age. Studies show a trend to statistic significance between the age and the reduction of complex I activity of human normal chondrocytes. However, the study of relation between age and deltapsim in normal chondrocytes do not demonstrate any significant correlation. It has been reported that as the number of population doublings increased, mitochondrial DNA was degraded and the number of mitochondria per chondrocyte decline. One approach for determining the role of mitochondria in OA is to determine the effects of the MRC inhibition and to compare them with the findings in OA. Inhibition of MRC with antimycin prevents the normal ability of TGFbeta to increase excretion of Pi, thereby worsening deposition of pathologic HA crystals. In chondrocytes, the inhibition of complex IV with NaN3 modified both the deltapsim and the survival of cells inducing apoptosis. Inhibition of complex I with rotenone increases the expression and synthesis of Bcl-2 and Cox-2, both effects are similar effects to produced by IL-1 in human chondrocytes.

Journal Article↗
PubMed2004

Solution structure of the hypothetical protein Mth677 from Methanobacterium thermoautotrophicum: a novel alpha+beta fold.

The structure of Mth677, a hypothetical protein from Methanobacterium thermoautotrophicum (Mth), has been determined by using heteronuclear nuclear magnetic resonance (NMR) methods on a double-labeled (15)N-(13)C sample. Mth677 adopts a novel alpha+beta fold, consisting of two alpha-helices (one N terminal and one C terminal) packed on the same side of a central beta-hairpin. This structure is likely shared by its three orthologs, detected in three other Archaebacteria. There are no clear features in the sequences of these proteins or in the genome organization of Mth to make a reliable functional assignment to this protein. However, the structural similarity to Escherichia coli MinE, the protein which controls that division occurs at the midcell site, lends support to the proposal that Mth677 might be, in Mth, the counterpart of the topological specificity domain of MinE in E. coli.

Amino Acid Sequence↗
PubMed2003

A novel cell binding site in the coiled-coil domain of laminin involved in capillary morphogenesis.

Recently, we reported the isolation and characterization of an anti-laminin antibody that modulates the extracellular matrix-dependent morphogenesis of endothelial cells. Here we use this antibody to precisely map the binding site responsible for mediating this biologically important interaction. By using a phage display-assisted mapping strategy to preserve protein structure, we demonstrate for the first time that the coiled-coil region of laminin contains a cell binding site. The adhesion motif is formed by residues contributed by both alpha and gamma chains, and is located in the middle part of the rod-like portion in a highly flexible area, which corresponds to a protease-susceptible site. Based on this information, a peptide mimotope was used to characterize the cognate receptor. Although we can not rule out the implication of other receptors, our results demonstrate that the laminin helical rod active site interacts with alpha2beta1 integrin on the surface of endothelial cells. These findings provide new insight into the complex mechanisms regulating capillary morphogenesis.

Amino Acid Sequence↗
PubMed2003

Mitochondrial respiratory activity is altered in osteoarthritic human articular chondrocytes.

OBJECTIVE: Osteoarthritis (OA) is a degenerative rheumatic disease that is associated with extracellular matrix degradation and chondrocyte apoptosis in the articular cartilage. The role of mitochondria in degenerative diseases is widely recognized. We undertook this study to evaluate mitochondrial function in normal and OA chondrocytes and to examine age-related changes in mitochondria. METHODS: Mitochondrial function was evaluated by analyzing respiratory chain enzyme complexes and citrate synthase (CS) activities as well as changes in mitochondrial membrane potential (Delta Psi m). The activities of mitochondrial respiratory chain complexes (complex I: rotenone-sensitive NADH-coenzyme Q(1) reductase; complex II: succinate dehydrogenase; complex III: antimycin-sensitive ubiquinol cytochrome c reductase; and complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from OA and normal cartilage. Delta Psi m was measured by JC-1 using flow cytometry. Statistical analysis was performed using the Mann-Whitney U test and Student's t-test as well as several models of multiple linear regression. RESULTS: OA articular chondrocytes had reduced activities of complexes II and III compared with cells from normal cartilage. However, the mitochondrial mass was increased in OA. Cultures of OA chondrocytes contained a higher proportion of cells with de-energized mitochondria. We found no relationship between mitochondrial function and donor age either in normal or in OA chondrocytes. CONCLUSION: These findings suggest the involvement of mitochondrial function in the pathophysiology of OA. Cartilage degradation by OA and cartilage aging may be two different processes.

Adult↗
PubMedDate not supplied

Xeno-implantation of pig chondrocytes into rabbit to treat localized articular cartilage defects: an animal model.

Articular cartilage has only a limited ability to regenerate. The transplantation of autologous chondrocytes is currently used to treat focal defects in human articular cartilage, although use of organs, tissues, or cells from different species is being investigated as an alternative treatment. The object of this study was to use xeno-transplantation of cultured pig chondrocytes for the repair of rabbit chondral defects, and to analyze the significance of tissue rejection in this animal model. Partial chondral defects, including removal of cartilage tissue and a part of the subchondral bone, were created in the lateral femoral condyles of 30 adult New Zealand White rabbits. A periosteal flap was sutured to the native cartilage with the cambium layer facing the defect. As a control, culture medium was injected into the defect void of one group of rabbits while in a treatment group, chondrocytes, isolated from normal femoral pig cartilage, were injected into the defect void. All rabbits were killed by 24 weeks. Macroscopic changes of the cartilage were analyzed using Mankin's score. The distal femoral portion was studied histologically using hematoxylin and eosin, alcian blue, toluidine blue, and Mason's trichrome. Pig cells and pig genetic material were detected in the neo-synthesized tissue by immunohistochemical detection of SLA-II-DQ and polymerase chain reaction analysis of the gene SLA-II-DQB. The synovial membrane was studied histologically by hematoxylin and eosin staining. In the control group, on average, less than 25 percent of the chondral defect was filled. The repair tissue had an irregular surface with few cells similar to chondrocytes or fibroblasts and a minimal formation of extracellular matrix. In the treatment group, the chondral defect was approximately 90 percent filled with good integration between the neo-synthesized cartilage and the native cartilage. The repair tissue had a smooth surface with cells similar to chondrocytes and a hyaline-like extracellular matrix. The neo-synthesized cartilage was morphologically similar to hyaline cartilage. Importantly, there were no signs of graft-vs.-host rejections or infiltration by immune cells. In the neo-synthesized tissue, pig genetic material was detected in 27 +/- 5 percent of all cells. These cells containing pig genetic material were distributed throughout the neo-synthesized cartilage. We conclude that the xeno-transplantation of chondrocytes could be an alternative method for the repair of articular cartilage defects.

Animals↗