Biomedical subjects

Paola Grandi

Publications and source records attributed to Paola Grandi.

9 recordsLinked to original sources

PubMed2006

Expression of human immunodeficiency virus type 1 tat from a replication-deficient herpes simplex type 1 vector induces antigen-specific T cell responses.

Herpes simplex type-1 virus (HSV-1) based vectors have been widely used in different gene therapy approaches and also as experimental vaccines against HSV-1 infection. Recent advances in the HSV-1 technology do support the use of replication defective HSV-1 as vaccine vectors for delivery of foreign antigens. We have examined the ability of a recombinant replication-defective HSV-1 vector expressing the HIV-1 Tat protein to induce long-term Tat-specific immune responses in the Balb/c murine model. The results showed that vector administration by the subcutaneous route elicits anti-Tat specific T-cell mediated immune responses in mice characterized by the presence of the Tat-specific cytotoxic activity and production of high levels of IFN-gamma.

AIDS Vaccines↗

PubMed2006

Degradation of fibrillar collagen in a human melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector.

Oncolytic viral therapy provides a promising approach to treat certain human malignancies. These vectors improve on replication-deficient vectors by increasing the viral load within tumors through preferential viral replication within tumor cells. However, the inability to efficiently propagate throughout the entire tumor and infect cells distant from the injection site has limited the capacity of oncolytic viruses to achieve consistent therapeutic responses. Here we show that the spread of the oncolytic herpes simplex virus (HSV) vector MGH2 within the human melanoma Mu89 is limited by the fibrillar collagen in the extracellular matrix. This limitation seems to be size specific as nanoparticles of equivalent size to the virus distribute within tumors to the same extent whereas smaller particles distribute more widely. Due to limited viral penetration, tumor cells in inaccessible regions continue to grow, remaining out of the range of viral infection, and tumor eradication cannot be achieved. Matrix modification with bacterial collagenase coinjection results in a significant improvement in the initial range of viral distribution within the tumor. This results in an extended range of infected tumor cells and improved virus propagation, ultimately leading to enhanced therapeutic outcome. Thus, fibrillar collagen can be a formidable barrier to viral distribution and matrix-modifying treatments can significantly enhance the therapeutic response.

Animals↗

PubMed2006

Proteome survey reveals modularity of the yeast cell machinery.

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.

Genome, Fungal↗

PubMed2004

Jet and accretion-disk emission untangled in 3C 273.

A long-term spectral variability study of 3C 273 on data obtained by BeppoSAX allows us to decouple the beamed nonthermal (jet) and unbeamed thermal (accretion flow) radiation produced in the inner region of a radio-loud active galactic nucleus. Jet power, when compared with unbeamed radiation, increases with energy. The thermal component is generally overwhelmed by the nonthermal radiation, by a factor of 1.2 to 3 in the 2- to 10-keV range and up to a factor of 7 above 20 keV. In only one case, the accretion flow overcomes the jet, allowing the K(alpha) iron line to emerge clearly over the continuum.

Journal Article↗

PubMed2004

Targeting HSV amplicon vectors.

Several techniques have been developed to deliver DNA directly into mammalian cells, spanning in difficulty from simple mixing procedures to complex systems requiring expensive equipment. Viral vectors have proven able to deliver genes into mammalian cells with high efficiency and low toxicity. In particular, herpes simplex virus type-1 (HSV-1) amplicon vectors are well suited for gene transfer studies as they can infect many cell types, both non-dividing and dividing, have a large transgene capacity and are easy to manipulate. For some applications, it may be desirable to target gene delivery to specific cell populations or to transduce normally non-susceptible cells. This can be achieved by modifying one or more of the glycoproteins found in the viral envelope. Glycoprotein C (gC) has a well-characterized heparan sulfate binding domain (HSBD) necessary for HSV binding to cells. Replacing this region with unique ligands can result in less efficient binding to natural target cells and increase binding to cells which express receptors for these ligands. A method to retarget amplicon vectors by replacing gC HSBD with a model ligand, the hexameric histidine-tag, is described, as well as means to evaluate the binding of modified vector as compared to wild-type virus to cells with or without the appropriate receptor, in this case, a his-tag pseudo-receptor. This protocol demonstrates increased binding of modified virus to receptor-positive cells (at levels greater than wild-type) with no loss of infectivity. Retargeted vectors can provide an additional tool for increasing the efficiency of gene delivery to specific cell types.

Animals↗

PubMed2004

HSV-1 virions engineered for specific binding to cell surface receptors.

Expression of specific peptide epitopes on the surface of virions has significant potential for studying viral biology and designing vectors for targeted gene therapy. In this study, an HSV-1 amplicon plasmid expressing a modified glycoprotein C (gC), in which the heparan sulfate binding domain was replaced with a His-tag, was used in generating HSV-1 virions. Western blot analysis demonstrated the presence of modified gC in the purified virions. The amplicon vectors were packaged using a gC-, lacZ+ helper virus to generate a mixture of high-titer helper virus (lacZ+) and amplicon vectors (GFP+), which expressed modified gC in the virion envelope. His-tagged virions bound to 293 6H cells expressing a cell surface pseudo-His-tag receptor four-fold more efficiently than to parental 293 cells and also proved more effective than wild-type virus in binding to both cell types. Binding resulted in productive infection by the modified virions with expression of reporter genes and cytopathic effect comparable to those of wild-type virions. Thus, not only can HSV-1 tropism be manipulated to recognize a non-herpes simplex binding receptor, but it is also possible to increase the infective capacity of the vectors beyond that of the wild-type virus via specific ligand receptor combinations.

Animals↗

PubMed2003

HSV-1 amplicon peptide display vector.

There are significant uses for expressing foreign peptide epitopes in viral surface attachment proteins in terms of investigating viral targeting, biology, and immunology. HSV-1 attachment, followed by fusion and entry, is mediated in large part by the binding of viral surface glycoproteins to cell surface receptors, primarily through heparan sulfate (HS) glycosaminoglycan residues. We constructed a HSV-1 amplicon plasmid (pCONGA) carrying the gC primary attachment protein gene with unique restriction sites flanking the HS binding domain (HSBD) (residues 33-176) to allow rapid, high efficiency substitution with foreign peptide domains. To test this system, a His tag with an additional unique restriction site (for selection and assay digests) was recombined into the pCONGA HSBD site to create pCONGAH. Infection of pCONGAH transfected Vero cells with HSV-1 helper virus (gCdelta2-3 or hrR3) produced His-modified gC as demonstrated by western blot analysis with co-localization of anti-gC and anti-His tag antibodies to a protein of appropriate molecular weight (50 kd). As CONGA and CONGAH amplicons carry a GFP transgene and the gCdelta2-3 and hrR3 viruses carry a lacZ transgene, vector stocks produced from 1 x 10(5) Vero cells could be titered for competent vector on cell monolayers and were demonstrated to contain 2 x 10(5) amplicon vector transducing units (t.u.)/ml and 1 x 10(7) virus t.u./ml. As the amplicon plasmids also contain the neomycin resistance gene (neo(r)), long term vector producer cell lines were created using G418 selection. This amplicon system provides means to rapidly and efficiently generate HSV-1 amplicon and viral vector expressing surface attachment proteins modified with different peptide epitopes for investigational and therapeutic uses, with the advantages of an amplicon plasmid that can be used with interchangeable helper virus vectors, is designed specifically for easy manipulation, and carries GFP and neo(r) transgenes for marker and selection functions.

Animals↗

PubMed2002

Functional organization of the yeast proteome by systematic analysis of protein complexes.

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

Cells, Cultured↗

PubMed2002

90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors.

We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.

Blotting, Northern↗