Search PubMed⌕ Search

Biomedical subjects

A Rosato

Publications and source records attributed to A Rosato.

At least 37 records · Page 2Linked to original sources
PubMed1999

Dissecting the immune response to moloney murine sarcoma/leukemia virus-induced tumors by means of a DNA vaccination approach.

The intramuscular inoculation of Moloney murine sarcoma/leukemia (M-MSV/M-MuLV) retroviral complex gives rise to sarcomas that undergo spontaneous regression due to the induction of a strong immune reaction mediated primarily by cytotoxic T lymphocytes (CTL). We used a DNA-based vaccination approach to dissect the CTL response against the Gag and Env proteins of M-MSV/M-MuLV in C57BL/6 (B6) mice and to evaluate whether plasmid DNA-immunized mice would be protected against a subsequent challenge with syngeneic tumor cells expressing the viral antigens. Intramuscular DNA vaccination induced CTL against both Gag and Env proteins. A detailed analysis of epitopes recognized by CTL generated in mice inoculated with the whole virus and with the Gag-expressing plasmid confirmed the presence of an immunodominant peptide in the leader sequence of Gag protein (Gag85-93, CCLCLTVFL) that is identical to that described in B6 mice immunized with Friend MuLV-induced leukemia cells. Moreover, CTL generated by immunization with the Env-encoding plasmid recognized a subdominant Env peptide (Env189-196, SSWDFITV), originally described in the B6.CH-2(bm13) mutant strain. B6 mice immunized with the Gag-expressing plasmid were fully protected against a lethal tumor challenge with M-MuLV-transformed MBL-2 leukemia cells, while vaccination with the Env-expressing plasmid resulted in rejection of the tumor in 44% of the mice and in increased survival of an additional 17% of the animals. Taken together, these results indicate the existence of a hierarchy in the capacity of different structural viral proteins to induce a protective immune response against retrovirus-induced tumors.

Animals↗
PubMed1999

[Anti-beta2 glycoprotein I-beta2 glycoprotein I] immune complexes in patients with antiphospholipid syndrome and other autoimmune diseases.

Antiphospholipid syndrome (APS) is defined by the presence of aPL antibodies in patients with thromboembolic phenomena. Some antiphospholipid (aPL) antibodies, such as those directed against beta2-glycoprotein I (beta2GPI), are associated with thromboembolism, possess Lupus Anticoagulant (LA) activity and recognize their target antigen only when bound to specific surfaces or to phospholipids (PL). To ascertain whether both free and antibody-bound beta2GPI circulate in APS, we set up an ELISA to detect [IgG anti-beta2GPI-beta2GPI] immune complexes. In this system, rabbit anti-human beta2GPI antibodies were adsorbed onto plastic plates, incubated with patient plasma, and bound complexes were detected by means of alkaline phosphatase-labeled goat anti-human IgG; each assay was stopped when positive controls consisting of in vitro generated immune complexes reached an Optical Density (OD) of 0.5 at 405 nm. Plasma from 16 patients with APS showed a mean OD405 of 0.291 (range 0.115-0.558), not statistically different from the mean obtained for 15 age- and sex-matched healthy volunteers (mean OD405 = 0.169, range 0.066-0.264). Surprisingly, levels of immune complexes in 14 patients with other autoimmune diseases and no circulating anti-beta2GPI antibodies were statistically higher (mean OD405 = 0.552, range 0.204-0.991) than those of healthy subjects and patients with APS. These data indicate that while autoantibodies to beta2GPI are mainly unbound in plasma of patients with APS, they are complexed with their antigen in patients with other autoimmune diseases, possibly reflecting a higher binding affinity.

Animals↗
PubMed1998

Solution structure of the oxidized Fe7S8 ferredoxin from the thermophilic bacterium Bacillus schlegelii by 1H NMR spectroscopy.

The solution structure of the paramagnetic seven-iron ferredoxin from Bacillus schlegelii in its oxidized form has been determined by 1H NMR. The protein, which contains 77 amino acids, is thermostable. Seventy-two residues and 79% of all theoretically expected proton resonances have been assigned. The structure has been determined through torsion angle dynamics calculations with the program DYANA, using 966 meaningful NOEs (from a total of 1305), hydrogen bond constraints, and NMR derived dihedral angle constraints for the cluster ligating cysteines, and by using crystallographic information to build up the two clusters. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.68 A for the backbone atoms and of 1.16 A for all heavy atoms. The contributions to the thermal stability of the B. schlegelii ferredoxin are discussed by comparing the present structure to that of the less stable Azotobacter vinelandii ferredoxin I which is the only other available structure of a bacterial seven-iron ferredoxin. It is proposed that the hydrophobic interactions and the hydrogen bond network linking the N-terminus and the C-terminus together and a high number of salt bridges contribute to the stability.

Amino Acid Sequence↗
PubMed1998

DNA immunization of mice against SIVmac239 Gag and Env using Rev-independent expression plasmids.

Simian immunodeficiency virus (SIV) structural gene expression, including gag and env, strictly depends on the interaction of the viral posttranscriptional regulator Rev with its target RNA, the Rev-responsive element (RRE). A small RNA element, termed the constitutive transport element (CTE), located in the 3' portion of simian retrovirus 1 (SRV-1) mRNA, can efficiently substitute for the human immunodeficiency virus (HIV) Rev-RRE interaction, and thus render HIV expression and replication Rev independent. We tested the ability of the SRV-1 CTE to drive the expression of SIVmac239 env and gag from subgenomic constructs designed for possible use in vaccine trials. In vitro expression studies showed that when the SRV-1 sequence is coupled to the SIV gag and env mRNAs, it functions in an orientation-dependent fashion, and leads to strong expression of SIV Gag and Env in human and monkey cell lines; levels of CTE-mediated protein expression were similar to those obtained with a functional Rev-RRE system. On the other hand, in murine fibroblast-like cells, SIV Gag and Env were expressed from constructs at relatively high levels even in the absence of Rev-RRE; nevertheless, their expression was increased by the presence of the SRV-1 CTE. As reported previously for HIV, the murine cell lines appeared to be defective for Rev-RRE activity, and required overexpression of Rev to induce a Rev response. Intramuscular injection of the gag-CTE and env-CTE constructs in BALB/c mice resulted in the expression of the corresponding mRNAs, and the production of anti-Gag and anti-Env antibodies, thus suggesting that these vectors might be used for genetic immunization approaches.

Animals↗
PubMed1998

Therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased anti-tumour cytotoxic T-lymphocyte activity.

In syngeneic mice, the H5V polyoma middle-T oncogene-transformed endothelioma cell line induces Kaposi's sarcoma-like cavernous haemangiomas that regress transiently, probably because of an anti-tumour immune response, but eventually grow progressively and kill the host. To evaluate the generation of tumour-specific cytotoxic T lymphocytes (CTLs), spleen cells of tumour-bearing mice were restimulated with irradiated H5V cells in mixed leucocyte-tumour cell cultures. Tumour-specific CTLs were demonstrable only when low numbers of H5V stimulator cells were used (<1 H5V cell per 50 splenocytes). We found that H5V cells secrete immunosuppressive mediators because CTL generation was blocked when H5V cells culture supernatants were added to allogeneic mixed leucocyte cultures. As numerous tumour-derived immunosuppressive mediators may interfere with interleukin 12 (IL-12) production, we tested whether IL-12 treatment of the tumour-bearing mice would augment their immune response and thus suppress tumour growth. Indeed, IL-12 inhibited tumour growth and prevented mortality, but did not increase anti-H5V CTL generation either in vitro or in vivo. Moreover, the anti-tumour activity in IL-12-treated mice was abrogated by anti-interferon (IFN)-gamma monoclonal antibody (MAb) co-administration. These results strongly suggest that the anti-tumour effect of IL-12 is principally mediated by IFN-gamma release that in turn blocks H5V cell proliferation and induces the release of factors that suppress angiogenesis.

Animals↗
PubMed1998

Differential effect on TCR:CD3 stimulation of a 90-kD glycoprotein (gp90/Mac-2BP), a member of the scavenger receptor cysteine-rich domain protein family.

We studied the effects of a 90-kD glycoprotein (gp90/Mac-2BP) belonging to the scavenger receptor family, present in normal serum and at increased levels in inflammatory disease and cancer patients, on some T cell function parameters. Whereas the lymphocyte proliferative response to non-specific mitogens such as phytohaemagglutinin (PHA) and concanavalin A (Con A), but not pokeweed mitogen (PWM), was strongly reduced, probably due to the lectin-binding properties of gp90/Mac-2BP, the response to T cell receptor (TCR) agonists such as superantigens and allogeneic cells was potentiated. When lymphocytes were stimulated with different anti-TCR:CD3 MoAbs, both in soluble and solid-phase form, gp90/Mac-2BP was able to down-regulate the proliferative response to anti-CD3 MoAb, whereas the response to anti-TCR alphabeta MoAb was enhanced. A similar differential effect was observed when a MoAb against CD5 (another member of the scavenger receptor superfamily) was added to anti-CD3 or anti-TCR-stimulated cells; anti-CD5 MoAb strongly down-modulated the CD3-mediated response, whereas its presence in culture was associated with potentiation of the response to TCR alphabeta agonists. gp90/Mac-2BP was able per se to up-regulate Ca2+ levels in freshly isolated lymphocytes; moreover, its presence in culture was associated with increased Ca2+ mobilization following stimulation with anti-TCR alphabeta, but not anti-CD3 MoAb. These data indicate that gp90/Mac-2BP could be able to influence some immune responses, possibly through multiple homologous interactions with other members of the scavenger receptor family; moreover, our findings suggest that signalling through the different components of the TCR:CD3 complex may follow distinct activation pathways into the cells.

3T3 Cells↗
PubMed1998

A new ketolide, HMR 3004, active against streptococci inducibly resistant to erythromycin.

HMR 3004 is a new hydrazono ketolide characterized by a 3-keto function instead of the cladinose moiety. The effect of this antimicrobial agent on inducible and constitutive macrolide-lincosamide-streptogramin B (MLSB) resistance was tested in a lacZ reporter system under control of several ermAM-like attenuator variants. For one constitutively resistant Streptococcus agalactiae strain, three inducibly resistant Streptococcus pneumoniae strains, and one inducibly resistant Enterococcus faecalis strain, the attenuators fused with lacZ were cloned into the shuttle plasmid pJIM2246 and the plasmid was introduced into Staphylococcus aureus RN4220. For the wild-type attenuators, HMR 3004 was a very weak inducer, unlike its cladinose counterpart RU 6652 and erythromycin. As expected, for the fusion originating from the constitutively resistant S. agalactiae strain, the level of uninduced beta-galactosidase synthesis was high. For one S. pneumoniae attenuator, mutations in the 3' end of the attenuator that weakened the stem-loop structure that sequesters the ribosome-binding site and start codon for ermAM methylase could explain the high level of uninduced beta-galactosidase produced. For streptococci, the activity of HMR 3004 correlated with the basal level of beta-galactosidase synthesized. The weak inducer activity of HMR 3004 explained its activity against inducibly MLSB-resistant S. pneumoniae but did not correlate with the moderate activity of the antibiotic against inducibly resistant E. faecalis.

Anti-Bacterial Agents↗
PubMed1997

Solution structure of reduced microsomal rat cytochrome b5.

The solution structure of the major form of the reduced soluble fragment of rat microsomal cytochrome b5 has been solved through 1H-NMR spectroscopy. The protein contains 98 amino acids. Proton assignment was available for residues 1-94, except 90 [Guiles, R. D., Basus, V. J., Kuntz, I. D. & Waskell, L. (1992) Biochemistry 31, 11,365-11,375] and has been confirmed. From 1722 NOEs, of which 1203 were found to be meaningful, a family of 40 energy-minimized structures has been obtained with average backbone rmsd (for residues 5-89) of 0.078 +/- 0.018 nm and average target function of 0.0045 nm2, no distance violations being larger than 0.029 nm. The structure has been compared with the X-ray structure of the oxidized rat mitochondrial isoenzyme and with that of the highly similar bovine microsomal isoenzyme in the oxidized form. The analysis of the elements of secondary structure is instructive in terms of their stability and of their occurrence in related structures, and of the capability of NMR and X-ray spectroscopy to observe them. Some detailed structural variations are noticed among the solved structures of the various isoenzymes and between solid and solution. The structural features in solution of the residues proposed to be involved in protein-protein recognition are found to be largely conserved with respect to the solid state.

Amino Acid Sequence↗
PubMed1997

Solution structure of oxidized horse heart cytochrome c.

The solution structure of oxidized horse heart cytochrome c was obtained at pH 7.0 in 100 mM phosphate buffer from 2278 NOEs and 241 pseudocontact shift constraints. The final structure was refined through restrained energy minimization. A 35-member family, with RMSD values with respect to the average structure of 0.70 +/- 0.11 A and 1.21 +/- 0.14 A for the backbone and all heavy atoms, respectively, and with an average penalty function of 130 +/- 4.0 kJ/mol and 84 +/- 3.7 kJ/mol for NOE and pseudocontact shift constraints, respectively (corresponding to a target function of 0.9 A2 and 0.2 A2), was obtained. The solution structure is somewhat different from that recently reported (Qi et al., 1996) and appears to be similar to the X-ray structure of the same oxidation state (Bushnell et al., 1990). A noticeable difference is a rotation of 17 +/- 8 degrees of the imidazole plane between solid and solution structure. Detailed and accurate structural determinations are important within the frame of the current debate of the structural rearrangements occurring upon oxidation or reduction. From the obtained magnetic susceptibility tensor a separation of the hyperfine shifts into their contact and pseudocontact contributions is derived and compared to that of the analogous isoenzyme from S. cerevisiae and to previous results.

Amino Acid Sequence↗
PubMed1997

CTL response and protection against P815 tumor challenge in mice immunized with DNA expressing the tumor-specific antigen P815A.

A DNA immunization approach was used to induce an immune response against the tumor-specific antigen P815A in DBA/2 mice. The P1A gene, which encodes the P815A antigen, was modified by the addition of a short sequence coding for a tag epitope recognized by the monoclonal antibody AU1, and cloned into the eukaryotic expression vector pBKCMV, resulting in plasmid pBKCMV-P1A. L1210 cells stably transfected with pBKCMV-P1A expressed P1A mRNA and were lysed by the syngeneic P815A-specific cytotoxic clone CTL-P1:5, thus confirming that the tag-modified P1A protein underwent correct processing and presentation. A single intramuscular injection of 100 microg of pBKCMV-P1A induced the expression of P1A mRNA for at least 4 months. Eighty percent of DBA/2 mice injected three times with 100 microg of pBKCMV-P1A generated cytotoxic T lymphocytes (CTL) that lysed P815 tumor cells, whereas mock-inoculated animals failed to show any cytotoxicity. Moreover, experiments designed to evaluate the protection of pBKCMV-P1A-immunized mice against a lethal challenge with P815 tumor cells showed that 6 of 10 immunized mice rejected the tumor, and 2 mice showed prolonged survival compared to control animals.

Animals↗
PubMed1997

The immunogenicity of experimental tumors is strongly biased by the expression of dominant viral cytotoxic T-lymphocyte epitopes.

The immunogenic Friend-Moloney-Rauscher (FMR) virus-induced tumors have been used extensively to clarify the cellular and molecular mechanisms responsible for tumor rejection and to develop immunotherapeutic strategies. We characterize here the trimolecular complex MHC class I-antigenic determinant-T cell receptor involved in the induction of a protective CTL response against the RMA thymoma. This complex is mainly composed by the D(b) molecule interacting with a Rauscher virus antigen (Ag) determinant and the Vbeta5+ T cell receptor. We also show that the chemically induced EL-4 thymoma acquires the susceptibility to recognition by anti-RMA CTLs and the ability to elicit a protective anti-RMA CTL response only upon infection by a virus of the FMR family and that RMA and FMR virus infected EL-4 cells share tumor-associated Ag. The data strongly support the hypothesis that the high immunogenicity of virus-induced or infected tumors is determined by the expression of immunodominant virus-encoded Ag. The demonstration of a different outcome in the immune responses elicited in the presence or in the absence of viral Ag further open the contention of the molecular requirements for immunogenicity and should stimulate a more careful revision of unexpected cross-reactivity among tumors.

Animals↗
PubMed1997

Inflammatory polyarthropathy and bone remodeling in HTLV-I Tax-transgenic mice.

Transgenic mice carrying the tax gene of human T-cell lymphotropic virus type I displayed a high prevalence of arthropathy. The percentage of affected animals increased with age, reaching a peak of 43% at 20 months. Southern analysis of deoxyribonucleic acid (DNA) from tissue samples indicated that disease development was related to tax copy number. Histopathologic evaluation of the ankle joints disclosed deep erosion of the synovial lining, fibrous tissue proliferation together with angiogenesis, mononuclear cell infiltration, and activation of osteoclasts. Radiologic examination confirmed joint involvement and revealed bone architecture modifications. The phenotype exhibited by the affected animals closely resembles that of seronegative arthritis in humans, and may indicate tax protein as a causal agent of arthropathy observed in HTLV-I infected individuals.

Age Factors↗
PubMed1997

The human T-cell lymphotropic virus type 1 Tof protein contains a bipartite nuclear localization signal that is able to functionally replace the amino-terminal domain of Rex.

The X region of human T-cell lymphotropic virus type 1 (HTLV-1) encodes two nucleolar/nuclear proteins, the posttranscriptional regulator of mRNA expression Rex and a protein of unknown function named Tof. To gain insight into the possible biological role of Tof, we investigated the mechanism governing its intracellular trafficking and identified its nucleolar/nuclear localization signal (NLS). Mutational analysis of Tof revealed that its NLS was located between amino acids 71 and 98 and contained two arginine-rich domains that functioned in an interdependent manner. Studies of Tof-Rex hybrid proteins showed that the Tof NLS could functionally replace the NLS of Rex at the level of nuclear targeting. As the NLS of Rex is known to mediate its interaction with its RNA target, the Rex-responsive element (RXRE), we tested whether the NLS of Tof could replace that of Rex in mediating activation of a RXRE-containing mRNA. Results showed that the NLS of Tof was indeed able to mediate activation of RXRE-containing mRNAs, suggesting that Tof itself may function as a regulator of RNA expression and utilization. A comparison of their compartmentalization in response to actinomycin D treatment indicated that Tof did not share Rex's shuttling pathway. Expression of Tof from its natural multiply spliced mRNA required the presence of Rex, suggesting that Tof may regulate viral or cellular mRNA expression during the later stages of viral replication.

Amino Acid Sequence↗
PubMed1997

Paramagnetic relaxation as a tool for solution structure determination: Clostridium pasteurianum ferredoxin as an example.

The possibility of using the relaxation properties of nuclei for solution structure determination of paramagnetic metalloproteins is critically evaluated. First of all, it is theoretically and experimentally demonstrated that magnetization recovery in nonselective inversion recovery experiments can be approximated to an exponential in both diamagnetic and paramagnetic systems. This permits the estimate of the contribution of paramagnetic relaxation when dominant or sizable. Then, it is shown that the averaging of paramagnetic relaxation rates due to cross relaxation is often tolerably small with respect to the use of paramagnetic relaxation rates as constraints for structural determination. Finally, a protocol is proposed to use such paramagnetic relaxation rates, which depend on the sixth power of the metal to resonating nucleus distance, as constraints for solution structure determination of proteins. As an example, the available solution structure of the oxidized ferredoxin from Clostridium pasteurianum has been significantly improved in resolution especially in the proximity of the metal ions by using 69 new constraints based on paramagnetic relaxation.

Clostridium↗
PubMed1996

The solution structure refinement of the paramagnetic reduced high-potential iron-sulfur protein I from Ectothiorhodospira halophila by using stable isotope labeling and nuclear relaxation.

The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe-4S]2+ polymetallic center has been fully labeled with 15N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eur. J. Biochem. 225, 715-725]. We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E. COSY and 15N-1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (rho para) has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and rho para data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the rho para constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH alpha and 37 3JHNC' (providing 45 phi dihedral angle constraints) 54 3JH alpha H beta and 31 3JNH beta (providing 26 chi 1 dihedral angle constraints), 4 chi 2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho para constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.

Amino Acid Sequence↗
PubMed1996

CD45 regulates apoptosis induced by extracellular adenosine triphosphate and cytotoxic T lymphocytes.

Several lines of evidence implicate protein tyrosine phosphatases (PTP) in the regulation of apoptotic cell death. We have evaluated the role of CD45, the major PTP of hematopoietic cells, in apoptosis induced by extracellular ATP (ATPe) and cytotoxic T lymphocytes (CTL). We observed that two CD45- clones obtained by mutagenesis of the Fas- cell line L1210, exhibit a higher susceptibility to apoptosis induced by ATPe, which was also evident in Ca(2+)-free conditions, when compared to the parental cell line or CD45+ variants. The CD45- cells were also more susceptible to death mediated by an alloreactive CTL clone. When the cytotoxic assay was performed in the presence of EGTA, a Ca2+ chelator, which prevents cytotoxic granule exocytosis and perforin polymerization on target cell membranes, only the CD45- target cells were killed by the CTL clone. These results suggest that a cytotoxic pathway other than the secretory or Fas-dependent pathways was responsible for the enhanced susceptibility of CD45- cells to death, and therefore provide further evidence for the role of ATPe as a possible mediator of Ca(2+)-independent target cell destruction by CTL.

Adenosine Triphosphate↗