Antonella Bandiera

Inhibition of HMGI-C protein synthesis suppresses retrovirally induced neoplastic transformation of rat thyroid cells.

Elevated expression of the three high-mobility group I (HMGI) proteins (HMGI, HMGY, and HMGI-C) has previously been correlated with the presence of a highly malignant phenotype in epithelial and fibroblastic rat thyroid cells and in experimental thyroid, lung, mammary, and skin carcinomas. Northern (RNA) blot and run-on analyses demonstrated that the induction of HMGI genes in transformed thyroid cells occurs at the transcriptional level. An antisense methodology to block HMGI-C protein synthesis was then used to analyze the role of this protein in the process of thyroid cell transformation. Transfection of an antisense construct for the HMGI-C cDNA into normal thyroid cells, followed by infection with transforming myeloproliferative sarcoma virus or Kirsten murine sarcoma virus, generated cell lines that expressed significant levels of the retroviral transforming oncogenes v-mos or v-ras-Ki and removed the dependency on thyroid-stimulating hormones. However, in contrast with untransfected cells or cells transfected with the sense construct, those containing the antisense construct did not demonstrate the appearance of any malignant phenotypic markers (growth in soft agar and tumorigenicity in athymic mice). A great reduction of the HMGI-C protein levels and the absence of the HMGI(Y) proteins was observed in the HMGI-C antisense-transfected, virally infected cells. Therefore, the HMGI-C protein seems to play a key role in the transformation of these thyroid cells.

Role of the Escherichia coli SbmA in the antimicrobial activity of proline‐rich peptides

In contrast to many antimicrobial peptides, members of the proline‐rich group of antimicrobial peptides inactivate Gram‐negative bacteria by a non‐lytic mechanism. Several lines of evidence indicate that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline‐rich Bac7(1–35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptides mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA‐null mutants, in fact showed resistance to several proline‐rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1–35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline‐rich antimicrobial peptides of eukaryotic origin.

Vertebrate 2xRBD hnRNP proteins: a comparative analysis of genome, mRNA and protein sequences

hnRNP proteins are involved in many cell functions, primarily in pre-mRNA processing. We report here a comparative analysis of the genes of the 2xRBD members of the hnRNP family and of their expression products. Starting from the seven well characterized hnRNP members of human and murine origin (A0, A1, A2/B1, A3, AB, D and DL) and the three MuSashI-like proteins with related RBD tandems (MSI1, MSI2 and DAZAP1), we identified through BLAST search 12 homologous genes in the genome of Danio rerio and 10 in the genome of Takifugu rubripes, which can be divided into three subgroups, each with its highly conserved exon/intron structure, matching perfectly the exon/intron structures found in human and mouse genes. An exception is the gene of hnRNP A0, which is intronless consistently in all the four species. The analysis has been supported also at the level of cDNA and EST databases and extended in this respect to other vertebrate species, namely chicken, Xenopus laevis and Silurana tropicalis. PHYLIP 3.62 package (SEQBOOT, PROTDIST/DNADIST, NEIGHBOR, CONSENSE) was used for all the proteins and their CDSs and human RBDs I and II to infer relevant aspects of the phylogenesis of these proteins. Some clues to the evolution of introns in these genes have come from the analysis of their distribution in homologous genes of other eukaryotes, namely Ciona, Drosophila, Caenorhabditis, Saccharomyces and Arabidopsis.

Cytosine-block telomeric type DNA-binding activity of hnRNP proteins from human cell lines

Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence. All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP). Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif. Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence. In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.

Expression and characterization of human‐elastin‐repeat‐based temperature‐responsive protein polymers for biotechnological purposes

Rapid progress has been made in the design and synthesis of oligomers and polymers that emulate the properties of natural proteins. Molecular bioengineering offers the chance to design and produce artificial polymeric proteins with tailored polymeric properties. The elastin‐like polypeptides are a well‐defined family of polymers with noteworthy characteristic based on the VPGVG repeated motif of bovine elastin. In the human homologue, the most regular sequence is represented by the repetition of the VAPGVG hexapeptidic motif. On the basis of this sequence, a synthetic gene has been designed, cloned and expressed in Escherichia coli to obtain artificial protein polymers. The rapid one‐step in‐frame cloning of any biologically active sequence can be achieved directly in the expression vector, allowing further improvement of the potential of the resulting product.

Transglutaminase-catalyzed preparation of human elastin-like polypeptide-based three-dimensional matrices for cell encapsulation.

Elastin is a specialized component of the extracellular matrix and constitutes an interesting model for bio-inspired material design with a high potential for applications in biomedical and biotechnological fields. In this work, we have focused on the set up of a simple, enzymatic method for the preparation of three-dimensional matrices based on an human elastin-like biomimetic polypeptide. The matrices were described and assayed as culture substrate for cell seeding and growth. An advantage of the here-presented method is the use of mild conditions that preserve cell viability. This can be a powerful tool for many applications that require direct embedding of cells in the matrix.

Comparison of thermal behavior of two recombinantly expressed human elastin-like polypeptides for cell culture applications.

Two synthetic genes that code for artificial proteins have been constructed that were modeled on the most regularly repeated hydrophobic domain of human tropoelastin. We compare the physicochemical properties of the recombinant products that differ in their primary structure; the alanine/lysine-rich cross-linking domains, which are highly conserved in mammalian tropoelastin, were either present or absent in the recombinant products. Both biopolymers showed thermoresponsive properties, and variations were observed that were dependent on solution conditions. Cell compatibility was assayed using the biopolymers as coating agents in culture experiments with a neuroblastoma cell line; cell adhesion and proliferation effects were evaluated. The cells were found to retain their neural differentiation potential. The data presented in our work support the usefulness of these versatile biopolymers for a variety of applications related to biotechnology and biomedicine.

Human recombinant elastin-like protein coatings for muscle cell proliferation and differentiation

Recombinant proteins represent a new and promising class of polymeric materials in the field of biomaterials research. An important model for biomaterial design is elastin, the protein accounting for the elasticity of several tissues. Human elastin-like polypeptides (HELPs) have been developed as recombinant versions of elastin with the purpose of enhancing some peculiar characteristics of the native protein, like self-assembling. In this paper, we report on a comparative study of rat myoblasts response to coatings based on two different HELP macromolecules, with respect to control cultures on bare cell culture polystyrene and on a standard collagen coating. Cell behavior was analyzed in terms of adhesion, proliferation and differentiation. The collected data strongly suggest the use of HELPs as excellent biomaterials for tissue engineering and regenerative medicine applications.

Human Elastin-Based Recombinant Biopolymers Improve Mesenchymal Stem Cell Differentiation

Elastin-based polypeptides are a class of smart biopolymers representing an important model in the design of biomaterials. The combination of biomimetic materials with cells that have great plasticity provides a promising strategy for the realization of highly engineered cell-based constructs for regenerative medicine and tissue repair applications. Two recombinant biopolymers inspired by human elastin are assessed as coating agents to prepare biomimetic surfaces for cell culture. These substrates are assayed for hBM MSC culture. The coated surfaces are also characterized with AFM to evaluate the topographical features of the deposited biopolymers. The results suggest that the elastin-derived biomimetic surfaces play a stimulatory role on osteogenic differentiation of MSCs.

ASSEMBLY AND OPTIMIZATION OF EXPRESSION OF SYNTHETIC GENES DERIVED FROM THE HUMAN ELASTIN REPEATED MOTIF

Naturally occurring proteins often possess interesting properties that make them attractive for the realization of innovative biomaterials. Repetitive artificial polypeptides have been modeled on the repeated domains of mammalian elastin, retaining and even enhancing their thermally responsive behavior. These protein polymers have been produced by recombinant biotechnology and due to their smart properties show a huge potential for a wide range of biomedical and biotechnological applications. For this reason, production of large quantities of highly purified material is a crucial step. We focused our attention on elastin-derived polypeptides based on the hexapeptidic motif typical of human elastin. Synthetic genes were assembled starting from a monomeric unit, and the different conditions were assayed to optimize the yield of the artificial polypeptides. Optimization of Escherichia coli strain and of the extraction procedure led to significant improvement in expression and recovery of the recombinant products. Electron micrographs of expressing bacteria under optimized conditions showed the accumulation of the recombinant product in the induced cells.