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Carlo Di Bello

Publications and source records attributed to Carlo Di Bello.

18 recordsLinked to original sources
PubMed2006

Human osteoblast-like cell adhesion on titanium substrates covalently functionalized with synthetic peptides.

Biomaterials to be used for the production of endosseous devices in dental, orthopedic and maxillo-facial applications, might be designed to support, guide and enhance osteoblast adhesion. Cell recruitment onto biomaterial surface is a fundamental step within the complex process responsible for implant osseointegration; this process involves several proteins from the extra cellular matrix (ECM), cytoskeleton and cell membrane. A new strategy to improve endosseous implant integration is based on preparing biomimetic surfaces able to present adhesive factors to cells. Osteoblast adhesion takes place by at least two different mechanisms: the most investigated one implies the interaction with RGD sequences via cell-membrane integrin receptors; a further mechanism concerns the interaction between cell-membrane heparan sulfate proteoglycans and heparin-binding sites of ECM proteins. In the present study two different biomimetic surfaces were obtained by covalently grafting two adhesive peptides on oxidized titanium substrates after silanization: an RGD-containing peptide and a peptide mapped on human vitronectin. The two sequences are known to act via different adhesive mechanisms. The amount of human osteoblasts adhered onto peptide-enriched or not enriched titanium oxidized surfaces and the strength of cell binding were estimated, thus comparing the capacity of the bioactive substrates in promoting cell adhesion.

Aged↗
PubMed2006

The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors.

Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketone-peptides spanning the Lassa virus GPC cleavage site as substrates and inhibitors for the proprotein convertase SKI-1/S1P. The 7-mer MCA (YISRRLL-MCA) and 8-mer MCA (IYISRRLL-MCA) are very efficiently cleaved with respect to both the 6-mer MCA (ISRRLL-MCA) and point mutated fluorogenic analogues, except for the 7-mer mutant Y253F. The importance of the P7 phenylic residue was confirmed by digestions of two 16-mer non-fluorogenic peptidyl substrates that differ by a single point mutation (Y253A). Because NMR analysis of these 16-mer peptides did not reveal significant structural differences at recognition motif RRLL, the P7 Tyr residue is likely important in establishing key interactions within the catalytic pocket of SKI-1. Based on these data, we established through analysis of pro-ATF6 and pro-SREBP-2 cellular processing that decanoylated chloromethylketone 7-mer, 6-mer, and 4-mer peptides containing the core RRLL sequence are irreversible and potent ex vivo SKI-1 inhibitors. Although caution must be exercised in using these inhibitors in in vitro reactions, as they can also inhibit the basic amino acid-specific convertase furin, within cells and when used at concentrations < or = 100 microM these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.

Amino Acid Sequence↗
PubMed2005

Conformational analysis of heparin binding peptides.

A properly engineered biomaterial for dental/orthopaedic applications must induce specific responses from the osteoblasts at the implant site. A most desirable response is an efficient adhesion, as it represents the first phase in the cell/material interaction and the quality of this phase will influence the cell's capacity to organize into a new functional tissue. The four osteoblast-adhesive peptides discussed in this paper are mapped on the 339-364 sequence (339MAPRPSLAKKQRFRHRNRKGYRSQRG364) located in the primary heparin-binding site of human vitronectin (HVP). Adsorbed on a polystyrene scaffold, these peptides display different adhesive activities towards osteoblasts. In this paper we report on the structural analysis in solution of the peptides through NMR and computational techniques. We find that the peptides with the highest adhesive activities display a hydrophobic patch opposite to the charged surface candidate to interact with heparin. These findings suggest that the peptides might adsorb on the polystyrene support in a favourable orientation for their activity. Furthermore, molecular models obtained for the four peptides in solution were used in rigid docking simulations with a heparin model. Assuming that the peptide solution conformations are not very different from the polystyrene-adsorbed structures, the simulations reveal that peptide adhesive activity is also affected by the number of ionic interactions and spacing between charged residues.

Amino Acid Sequence↗
PubMed2004

Effect of synthetic peptides on osteoblast adhesion.

The quality of the early cell/material interactions is responsible for the long-term functional properties of any implanted device. Accordingly, "next generation" dental/orthopedic biomaterials should be able to promote osteoblast adhesion thus improving the integration process between surgically placed implants and biological tissues. Recent studies have identified a wide range of biochemical signals that can be exploited to promote adhesion, migration, proliferation and differentiation of cells. The clinical use of natural factors to promote osteoblast adhesion is complicated because those are often insoluble and unstable macromolecules and, in addition, it is difficult to obtain them in high quantities, with good purity grade and at low cost. A valid alternative could be the use of short peptides carrying the minimum active sequence of the natural macromolecular factor. This paper describes the properties of two classes of peptides, promoting different adhesion mechanisms, to enhance rat bone marrow osteoblast adhesion both to polystyrene and to acellular bone matrix.

Amino Acid Sequence↗
PubMed2004

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site 3: role of site-specific mutations.

Proteolytic processing of HIV gp160 to produce gp120 and gp41 is performed by PC enzymes. This process is a prerequisite for the virus infectivity, since both gp120 and gp41 participate in the virus HIV-1 entry mechanism. The structure of the gp120/gp41 junction remains to be elucidated, and the structural features required for molecular recognition between HIV-1 gp160 and proteolytic enzymes have not been clarified. Furin is the best PC candidate for the gp160 proteolytic processing known to date. In previous studies on model peptides, we have shown the relevance of an N-terminal helix for the proper recognition of the gp160 processing site by furin. Here we analyze the effect of point mutations in peptides lacking a regular N-terminal helix. To this end, we present the structure-activity characterization of three peptide analogues of the HIV gp160 processing site that all present mutations in proline at positions P3 and/or P2', while sharing the same N-terminal sequence, containing helix-breaking D-amino acids. Conformational analysis of the peptides was carried out in solution by NMR techniques, and furin's efficiency in cleaving them was measured. Structural findings are presented and discussed in relation to the different exhibited activity.

Catalytic Domain↗
PubMed2004

Anti-HIV activity and conformational studies of peptides derived from the C-terminal sequence of SDF-1.

The entry of the human immunodeficiency virus type 1 (HIV-1) into target cells requires the interaction of viral envelope glycoprotein, gp120, with the human CD4 glycoprotein and a chemokine receptor, usually CCR5 or CXCR4. The natural ligand for CXCR4 is the chemokine SDF-1 that inhibits entry and replication of X4 HIV-1 strains. SDF-1 is produced in two forms, SDF-1alpha (68 residues) and SDF-1beta (72 residues); the difference between them lies in the additional four C-terminal amino acids in the SDF-1beta sequence. Despite the relevance of the N-terminal site in determining the SDF anti HIV-1 activity, SDF-1beta has a stronger activity than SDF-1alpha. Here we demonstrate that a synthetic peptide mapped on the C-terminus of SDF-1beta presents inhibitory activity, whereas an analogue reproducing the C-terminal trait of SDF-1alpha does not show any activity. The opposite biological effect of the two peptides correlates with the type of interaction they each have with heparin and chondroitin sulfate.

Amino Acid Sequence↗
PubMed2004

Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coated Ti and Ti6Al4V disks.

A fundamental goal in the field of implantology is the design of specific devices able to induce a controlled and rapid "osseointegration". This result has been achieved by means of surface modifications aimed at optimizing implant-to-bone contact; furthermore, bone cell adhesion on implant surface has been directly improved by the application of biomolecules that stimulate new tissue formation, thus controlling interactions between biological environment and implanted materials. Actually, methods for biochemical factor delivery at the interface between implant surface and biological tissues are under investigation; a reliable technique is represented by the inclusion of biologically active molecules into biocompatible and biodegradable materials used for coating implant surface. This paper focuses the application of three polymeric materials already acknowledged in the clinical practice, i.e. poly-L-lactic acid (PLLA), poly-DL-lactic acid (PDLA), and sodium alginate hydrogel. They have been used to coat Ti (Ti2) and Ti6Al4V (Ti5) disks; their characteristics have been determined and their performances compared, with specific regard to the ability in allowing osteoblast adhesion in vitro. Moreover, profilometry data analysis permitted to identify a specific roughness parameter (peak density) which mainly controls the amount of osteoblast adhesion.

Alginates↗
PubMed2004

Surface treatments and roughness properties of Ti-based biomaterials.

Nowadays, the use of implanted devices is a well-acknowledged practice in the field of orthopaedic and dental surgery. Scientific research and clinical experience suggest that the successful exploitation of these devices mainly depends on osseointegration, considered as both anatomical congruency and load-bearing capacity. Indeed, the osseointegration process is influenced by a wide range of factors: anatomical location, implant size and design, surgical procedure, loading effects, biological fluids, age and sex, and, in particular, surface characteristics. For this reason, several attempts have been aimed at modifying implant surface composition and morphology to optimise implant-to-bone contact and improve integration. Preliminary interactions between implanted materials and biological environment are deemed to be governed by the surface properties; they control the amount and quality of cell adhesion on the surface and, consequently, cell/tissue growth. Thus, surface properties govern new bone tissue formation and implant osseointegration. This paper reviews the state of art in the field of physical, chemical and biochemical treatments commonly used on Ti-based biomaterials for the production of biomedical devices. In particular, roughness characteristics due to physical and chemical techniques are investigated; the development of biologically active surfaces by means of biochemical functionalisation is also considered.

Adsorption↗
PubMed2004

Synthetic peptides for AIDS research.

In spite of the relevance of the results obtained through the clinical application of chemotherapeutic agents (reverse transcriptase and proteinase inhibitors) that are able to prolong the life span of affected people, acquired immunodeficiency syndrome (AIDS) remains a serious and lethal disease. AIDS is caused by a type 1 human immunodeficiency virus (HIV-1) and formation of a complex among the gp120, CD4 and CCR5/CXCR4 surface proteins represents a key-step in the infection. The use of synthetic peptides reproducing reduced sequences of these proteins has contributed to increase the knowledge of the mechanism that determines the penetration of the HIV viruses into the target-cells. In addition, short peptides with minimum structural requirements for anti-HIV activity hold greater potential as lead compounds for rational drug design than macromolecular proteins. In this context, our studies concern: the role of gp120 V3 loop in CD4 binding, the importance of the N-terminal sequence of HIV CCR5 coreceptor, the potential inhibitory properties of sequences patterned on CXCR4 natural ligand (SDF-1) and the role of secondary structure in determining gp160 enzymatic processing into gp120.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Is the V3 loop involved in HIV binding to CD4?

The entry of the human immunodeficiency virus into cells requires the interaction of the viral envelope glycoprotein gp120 with CD4 and a chemokine receptor. The gp120 binding site has been previously mapped to the Ig-CDR2-like region of CD4 first domain. A second area of this domain (Ig-CDR3-like region) is involved in gp120-CD4 interactions, but its gp120 counterpart remained so far unknown. Using a photoaffinity labeling experiment, we demonstrate that a peptide, mapping the (307-330)m region of HIV-MN-gp120 V3 loop, binds a sequence including a part of the Ig-CDR3-like region. These results may contribute to explain the complex mechanism of human immunodeficiency virus penetration, helping the development of new therapeutic agents.

Amino Acid Sequence↗
PubMed2003

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site, 2: relevance of an N-terminal helix.

Proteolytic activation of the HIV-1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C-terminus of the sequence R508-E-K-R511 (site 1), in spite of the presence of another consensus sequence, Lys500-Ala-Lys-Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498-Gly516, we previously suggested a possible role of an N-terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23-residue peptide h-REKR, designed to exhibit a large N-terminal helix, followed by the gp160 native sequence, Arg508-Gly516. h-REKR is digested by furin with high efficiency, comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98 % trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N-terminal segment. Such a long helix does not seem to affect the loop conformation of the C-terminal site 1-containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.

Amino Acid Sequence↗
PubMed2003

Pattern identification and classification in gene expression data using an autoassociative neural network model.

The application of DNA microarray technology for analysis of gene expression creates enormous opportunities to accelerate the pace in understanding living systems and identification of target genes and pathways for drug development and therapeutic intervention. Parallel monitoring of the expression profiles of thousands of genes seems particularly promising for a deeper understanding of cancer biology and the identification of molecular signatures supporting the histological classification schemes of neoplastic specimens. However, the increasing volume of data generated by microarray experiments poses the challenge of developing equally efficient methods and analysis procedures to extract, interpret, and upgrade the information content of these databases. Herein, a computational procedure for pattern identification, feature extraction, and classification of gene expression data through the analysis of an autoassociative neural network model is described. The identified patterns and features contain critical information about gene-phenotype relationships observed during changes in cell physiology. They represent a rational and dimensionally reduced base for understanding the basic biology of the onset of diseases, defining targets of therapeutic intervention, and developing diagnostic tools for the identification and classification of pathological states. The proposed method has been tested on two different microarray datasets-Golub's analysis of acute human leukemia [Golub et al. (1999) Science 286:531-537], and the human colon adenocarcinoma study presented by Alon et al. [1999; Proc Natl Acad Sci USA 97:10101-10106]. The analysis of the neural network internal structure allows the identification of specific phenotype markers and the extraction of peculiar associations among genes and physiological states. At the same time, the neural network outputs provide assignment to multiple classes, such as different pathological conditions or tissue samples, for previously unseen instances.

Adenocarcinoma↗
PubMed2003

Plasminogen activator inhibitor-2: a molecular biomarker for head and neck cancer progression.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy in which the early diagnosis of premalignant lesions is known to directly correlate with increased survival. However, only a portion of biopsies showing dysplasia will progress to cancer, and there are no currently accepted criteria for predicting which lesions will progress. Therefore, diagnostic protocols that can identify the lesions that are likely to become HNSCC are required. RNA was isolated from normal keratinocytes, the immortalized but nontumorigenic HaCat cell line, and the tumor cell lines SCC-4, SCC-9, SCC-25, and OSCC-3. The RNA was then labeled and used to probe nylon microarray filters that contained a total of 9184 genes (5295 named and 3889 Expression Sequence Tags). Genes whose expression demonstrated a 3-fold or greater change were considered significant. Comparison of expression profiles from normal, HaCat, and four tumor cell lines revealed changes in gene expression in a total of 508 genes. Of these, 16 genes showed a consistent loss of expression when comparing normal to immortalized keratinocytes. In addition, 10 genes demonstrated a consistent loss of expression in the tumor cell lines only. In this latter group of genes, plasminogen activator inhibitor-2 (PAI-2), a gene whose expression has been linked to cell invasion, was additionally investigated. Altered expression of PAI-2 in the different cultured cells was validated via real-time quantitative-PCR. In addition, immunohistochemical evaluation of biopsy samples revealed a high expression of PAI-2 in both normal and dysplastic epithelium with a marked decrease of expression in areas of the biopsies containing HNSCC. These data demonstrate that genomic profiling can then be used to identify potential genotypic/phenotypic biomarkers that may predict which dysplastic lesions are most likely to progress to HNSCC.

Biomarkers, Tumor↗
PubMed2002

Novel osteoblast-adhesive peptides for dental/orthopedic biomaterials.

Next generation dental/orthopedic biomaterials must be designed to enhance and support osteoblast adhesion. The osteoblasts use different ways to adhere, that is, integrin- and proteoglycan-mediated mechanisms. The present study reports on the synthesis and osteoblast-adhesive properties of peptides carrying RGD motifs and of sequences mapped on human vitronectin. Our data suggest that osteoblast adhesion on polystyrene plates modified with a linear peptide, in which the GRGDSP sequence is repeated four times, was significantly higher when compared to the adhesion obtained using branched peptides, interestingly containing the same motif. Osteoblast adhesion assays on acellular bone matrix using this active peptide gave very promising results. We also demonstrated that a novel peptide, carrying the X-B-B-B-X-B-B-X motif (where B is a basic amino acid and X is a nonbasic residue), promotes proteoglycan-mediated osteoblast adhesion more efficiently with respect to the KRSR sequence that was recently proposed as heparan-sulfate binding peptide.

Amino Acid Sequence↗
PubMed2002

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site.

The selective proteolytic activation of the HIV-1 envelope glycoprotein gp160 by furin and other precursor convertases (PCs) occurs at the carboxyl side of the sequence Arg508-Glu-Lys-Arg511 (site 1), in spite of the presence of another consensus sequence: Lys500-Ala-Lys-Arg503 (site 2). We report on the solution structural analysis of a 19-residue synthetic peptide, p498, which spans the two gp160-processing sites 1 and 2, and is properly digested by furin at site 1. A molecular model is obtained for p498, by means of molecular dynamics simulations, from NMR data collected in trifluoroethanol/water. The peptide N-terminal side presents a 9-residue helical segment, enclosing the processing site 2; the C-terminal segment can be described as a loop exposing the processing site 1. A hypothesis for the docking of p498 onto the catalytic domain of human furin, modeled by homology and fitting previous site-directed mutagenesis studies, is also presented. p498 site 1 is shown to have easy access to the furin catalytic site, unlike the nonphysiological site 2. Finally, on the basis of available data, we suggest a possible structural motif required for the gp160-PCs recognition.

Amino Acid Sequence↗
PubMedDate not supplied

Synthetic peptides for study of human immunodeficiency virus infection.

The formation of a complex among gp120, CD4, and CCR5/CXCR4 represents a key step in human immunodeficiency virus (HIV) infection. The use of synthetic peptides reproducing sequences of these surface proteins has increased knowledge about the interactions that determine the penetration of HIV viruses into target cells. The final aim of such investigations is the design of molecules able to inhibit the initial step of infection and the development of high-sensitivity in vitro assays for detection of HIV. In particular, the studies presented herein concern the role of the gp120 V3 loop in the CD4 binding, the importance of the N-terminal sequence of HIV-coreceptor CCR5, the sequences patterned on CXCR4 natural ligand (stromal-derived factor 1 [SDF-1]) as inhibitory peptides, and the importance of substrate secondary structure in determining the enzymatic processing of gp120 precursor (gp160).

Amino Acid Sequence↗
PubMedDate not supplied

Fermentation diagnosis by multivariate statistical analysis.

During the course of fermentation, online measuring procedures able to estimate the performance of the current operation are highly desired. Unfortunately, the poor mechanistic understanding of most biologic systems hampers attempts at direct online evaluation of the bioprocess, which is further complicated by the lack of appropriate online sensors and the long lag time associated with offline assays. Quite often available data lack sufficient detail to be directly used, and after a cursory evaluation are stored away. However, these historic databases of process measurements may still retain some useful information. A multivariate statistical procedure has been applied for analyzing the measurement profiles acquired during the monitoring of several fed-batch fermentations for the production of erythromycin. Multivariate principal component analysis has been used to extract information from the multivariate historic database by projecting the process variables onto a low-dimensional space defined by the principal components. Thus, each fermentation is identified by a temporal profile in the principal component plane. The projections represent monitoring charts, consistent with the concept of statistical process control, which are useful for tracking the progress of each fermentation batch and identifying anomalous behaviors (process diagnosis and fault detection).

Biotechnology↗