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A Rosato

Publications and source records attributed to A Rosato.

At least 19 recordsLinked to original sources
PubMed2006

SPINE bioinformatics and data-management aspects of high-throughput structural biology.

SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.

Computational Biology↗
PubMed2004

Extracts from St John's Wort and their antimicrobial activity.

In an effort to carry out a more in-depth investigation on the antimicrobial properties of H. perforatum, we have assayed different extracts (MeOH; petroleum ether; CHCl(3) and EtOAc) from the aerial parts of the plant against selected microorganisms. Growth inhibition was observed only for Gram-positive bacteria, B. subtilis and B. cereus being the most susceptible to the tested drugs. The Hypericum extract obtained with EtOAc was the most active. The main constituents of this extract, as determined by HPLC analysis, were flavonoids, hypericins and hyperforins. Incubation of the selected microorganisms with the pure chemicals resulted in a significant inhibition of their growth by hypericin, hyperforin and its stable dicyclohexilammonium salt. Flavonoids appeared inactive at all.

Anti-Infective Agents↗
PubMed2003

Effect of antiadhesive agents on peritoneal carcinomatosis in an experimental model.

BACKGROUND: Auto-crosslinked polysaccharide hyaluronan-based solution (Hyalobarrier-gel) prevents postoperative adhesions. However, its effect on tumour growth is still unknown. The aim of the present study was therefore to investigate the impact on survival of intra-abdominally administered Hyalobarrier-gel, native hyaluronan (HA) and hyaluronan/carboxymethylcellulose (HA/CMC), after intraperitoneal tumour implantation. METHODS: After receiving an intraperitoneal inoculum of the human HT29 colorectal cell line, 615 athymic nude mice were assigned randomly to five groups: groups 1 and 2 received Hyalobarrier-gel 20 mg/ml (n = 124) and 40 mg/ml (n = 126) respectively; groups 3 and 4 received HA (n = 120) and HA/CMC film (Seprafilm) (n = 123) respectively. The survival of each treated group was compared with that of group 5, the control, which had no treatment (n = 122). RESULTS: As 34 of the 615 mice were not eligible, 581 animals were considered for the analysis. At 120 days, 136 animals (23.4 per cent) were still alive. At autopsy there was macroscopic absence of tumour in 75 cases (12.9 per cent). No statistically significant differences were found between the treatment and the control groups with respect to postoperative death and absence of tumour implantation. There was no difference in survival rate between the control group and groups treated with Hyalobarrier-gel, HA or HA/CMC. CONCLUSION: Hyalobarrier-gel, HA and HA/CMC had no negative impact on the survival rate in mice that received an intraperitoneal implantation of HT29 colorectal human tumour cells.

Animals↗
PubMed2003

["Switch prophylaxis one a day" with levofloxacin in laparoscopic cholecystectomy].

BACKGROUND: Special attention is paid today to the advisability of carrying out routine antibiotic prophylaxis in laparoscopic cholecystectomy (LC) and to the dosage protocol to be adopted in order to reduce the incidence of infections at the site of the surgical operation which, albeit with lower incidence than in "open" surgery, 5.3% vs 14%, can vanify the advantages of the mini-invasive approach. The demonstrated validity of administering the antibiotic beyond 24 hours after the operation led the authors to verify the clinical effectiveness and tolerance of "switch prophylaxis one a day" (SPOD) with levofloxacin in the prevention of septic complications after LC. METHODS: The experience reported relates to 185 patients suffering from symptomatic and/or complicated lithiasis of the gall-bladder subjected from January 1999 to April 2001 to LC and to antibiotic prophylaxis in accordance with the following dosage protocol: levofloxacin 500 mg i.v. 30 min before operation and 500 mg per os in the 3 days subsequent. RESULTS: The postsurgical evaluation documented the onset of 2 superficial infections in patients in whom LC had been of necessity converted and of a subhepatic abscess in an ASA III patient with acute cholecystitis. Prevention of infections at the surgical site totalled 98.4%. Nausea and slight diarrhoea and an increase in transaminasaemia were observed respectively in 3% and 4% of patients. CONCLUSIONS: The results obtained led the authors to standardise the use of SPOD with levofloxacin in LC operations which, in their opinion represents a rational alternative to the antibiotic prophylaxis regimes most commonly used up to the present (STP and USTP).

Adult↗
PubMed2002

Differential effects of angiostatin, endostatin and interferon-alpha(1) gene transfer on in vivo growth of human breast cancer cells.

The administration of different angiogenesis inhibitors by gene transfer has been shown to result in inhibition of tumor growth in animal tumor models, but the potency of these genes has been only partially evaluated in comparative studies to date. To identify the most effective anti-angiogenic molecule for delivery by retroviral vectors, we investigated the effects of angiostatin, endostatin and interferon(IFN)-alpha(1) gene transfer in in vivo models of breast cancer induced neovascularization and tumor growth. Moloney leukemia virus-based retroviral vectors for expression of murine angiostatin, endostatin and IFN-alpha(1) were generated, characterized, and used to transduce human breast cancer cell lines (MCF7 and MDA-MB435). Secretion of the recombinant proteins was confirmed by biological and Western blotting assays. Their production did not impair in vitro growth of these breast cancer cells nor their viability, and did not interfere with the expression of angiogenic factors. However, primary endothelial cell proliferation and migration in vitro were inhibited by supernatants of the transduced cells containing angiostatin, endostatin, and IFN-alpha(1). Stable gene transfer of the IFN-alpha(1) cDNA by retroviral vectors in both MCF7 and MDA-MB435 cells resulted in a marked and long-lasting inhibition of tumor growth in nude mice that was associated with reduced vascularization. Endostatin reduced the in vivo growth of MDA-MB435, but not MCF7 cells, despite similar levels of in vivo production, and angiostatin did not impair the in vivo growth of either cell line. These findings indicate heterogeneity in the therapeutic efficacy of angiostatic molecules delivered by viral vectors and suggest that gene therapy with IFN-alpha(1) and endostatin might be useful for treatment of breast cancer.

Angiogenesis Inhibitors↗
PubMed2001

Magnetic susceptibility tensor anisotropies for a lanthanide ion series in a fixed protein matrix.

The full series of lanthanide ions (except the radioactive promethium and the S-state gadolinium) has been incorporated into the C-terminal calcium binding site of the dicalcium protein calbindin D(9k). A fairly constant coordination environment is maintained throughout the series. At variance with several lanthanide complexes with small chelating ligands investigated in the past, the large protein moiety provides a large number of NMR signals whose hyperfine shifts can be exclusively ascribed to pseudocontact shifts (PCS). The chemical shifts of 1H and 15N backbone and side chain amide NH groups were accurately measured through HSQC experiments. 1097 PCS were estimated from these by subtracting the diamagnetic contributions measured on HSQC spectra of either the 4f(0) lanthanum(III) or the 4f(14) lutetium(III) derivatives and used to define a quality factor for the structure. The differences in diamagnetic chemical shifts between the two diamagnetic blanks were relatively small, although some were not negligible especially for the nuclei closest to the metal center. These differences were used as a tolerance for the PCS. The magnetic susceptibility tensor anisotropies for each paramagnetic lanthanide ion were obtained as the result of the solution structure determination performed by using the NOEs of the cerium(III) derivative and the PCS of all lanthanides simultaneously. This set of reliable magnetic data permits an experimental assessment of Bleaney's theory relative to the magnetic properties for an extended series of lanthanide complexes in solution. All of the obtained tensors show some rhombicity, as could be expected from the lack of symmetry of the protein environment. The directions of the largest magnetic susceptibility component for Ce, Pr, Nd, Sm, Tb, Dy, and Ho and of the smallest magnetic susceptibility component for Eu, Er, Tm, and Yb were found to be all within 15 degrees from their average (within 20 degrees for Sm), confirming the essential similarity of the coordination environment for all lanthanides. Bleaney's theory is in excellent qualitative agreement with the observed pattern of axial anisotropies. Its quantitative agreement is substantially better than that suggested by previous analyses performed on more limited sets of PCS data for small lanthanide complexes, the so-called crystal field parameter varying only within +/-30% from one lanthanide to another. These variations are even smaller (+/-15%) if a reasonable T(-3) correction is taken into consideration. A knowledge of magnetic susceptibility anisotropy properties of lanthanides is essential in determining the self-orienting properties of lanthanide complexes in solution when immersed in magnetic fields.

Algorithms↗
PubMed2001

Solution structure calculations through self-orientation in a magnetic field of a cerium(III) substituted calcium-binding protein.

Within the frame of a research aimed at characterizing paramagnetic metal ions capable of inducing self-orientation of metalloproteins in solution, we have studied the complex of the 75-amino-acid calcium-binding protein calbindin D(9k) with one Ce(III) ion (CaCeCb). Backbone (15)N-(1)H (1)J values have been determined for CaCeCb at two different magnetic fields. The above values showed a distinct dependence on the magnetic field, which is caused by the partial orientation of the molecule in solution. The difference in the values at the two magnetic fields provides structural constraints, which have been used to refine the structure of CaCeCb. The refined structure showed an improvement in terms of the number of residues falling in favored regions of the Ramachandran plot. The comparison of the molecular magnetic susceptibility tensor, obtained from the (15)N-(1)H (1)J values, with the magnetic susceptibility tensor of the metal, obtained from pseudocontact shifts, showed that the orientation of the molecule in solution is mainly determined by the Ce(III) ion. This paper shows that Ce(III), like low-spin Fe(III) in hemoproteins, is sufficiently magnetically anisotropic to induce self-orientation to an extent which can be exploited for solution structure determination.

Algorithms↗
PubMed2001

Some patients with antiphospholipid syndrome express hitherto undescribed antibodies to cardiolipin-binding proteins.

Contrary to infective anticardiolipin (aCL) antibodies, autoimmune aCL antibodies react with phospholipids (PL) mainly via binding to the plasma glycoprotein cofactor beta2-Glycoprotein I (beta2GPI). While there is a well-documented link between the risk of thrombosis and the presence of beta2GPI-dependent anticardiolipin antibodies, the pathological impact of other antiphospholipid antibodies is less clear. By means of cardiolipin affinity-chromatography, we isolated and identified 3 CL-binding proteins, complement component C4, complement factor H and a kallikrein-sensitive glycoprotein, and tested for the presence of autoantibodies against these proteins in patients with antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and other autoimmune diseases. High titers of autoantibodies to C4 as compared to age- and sex-matched healthy controls were present in 3 of 26 patients with APS, and weak titers were found in 2 of 26 patients with SLE and in none of 26 patients with other autoimmune diseases. Autoantibodies to complement factor H were found in 4 APS, 3 SLE and none of the other autoimmune patients. Autoantibodies to kallikrein-sensitive glycoprotein were detected in 6 APS patients, 1 SLE patient, and 1 patient with another autoimmune disease. A close relationship between these antibodies was found, suggesting their origin from a common macromolecular complex. However, no relationship with anti-beta2GPI antibodies was found, with the three patients with higher levels of autoantibodies having a low titer of anti-beta2GPI antibodies. In conclusion, some patients with APS harbor circulating antibodies to other CL-binding proteins which might be useful to further characterize these patients.

Adolescent↗
PubMed2000

Backbone dynamics of human Cu,Zn superoxide dismutase and of its monomeric F50E/G51E/E133Q mutant: the influence of dimerization on mobility and function.

The backbone assignment of reduced human dimeric Cu,Zn superoxide dismutase (SOD) was performed on a sample 100% enriched in (15)N, (13)C and 70% enriched in (2)H. (15)N T(1), T(2), and T(1)(rho) and (15)N-(1)H NOE assignment was performed at 600 MHz proton frequency on both wild-type SOD and the monomeric F50E/G51E/E133Q mutant. This allowed a comparison of the mobility in the subnanosecond and in the millisecond to microsecond time scales of the two systems. Both proteins are rather rigid, although some breathing of the beta sheets is detected in the wild type dimer. The monomer displays large mobility in the loops in the first part of the sequence, in loop IVa where point mutations have been introduced and at the C-terminus. The dimeric wild type is rigidified at loop IVa and at the C-terminus. Only loop VII shows a higher mobility in the dimer (besides some individual NH moieties). Conformational equilibria are displayed in the monomeric form around cysteines 57 and 146, thus explaining the disorder of arginine 143 which is the most important residue in guiding O(2)(-) toward the copper ion. The larger mobility in the wild type form with respect to the monomer in the picosecond to nanosecond time scale of helix alpha1 and loop VIIb, which provides the correct electrostatic driving force for O(2)(-) in the active channel, has been discussed in terms of favoring the activity of SOD.

Amino Acid Substitution↗
PubMed2000

In vivo antitumor activity and host toxicity of methoxymorpholinyl doxorubicin: role of cytochrome P450 3A.

Methoxymorpholinyl doxorubicin (MMDX; PNU 152243) is a promising doxorubicin derivative currently undergoing clinical evaluation. Previous in vitro studies suggested that the compound undergoes hepatic biotransformation by cytochrome P450 (CYP) 3A into a more cytotoxic metabolite(s). The present study examined the role of CYP3A-mediated metabolism in the in vivo antitumor activity and host toxicity of MMDX in the mouse model and investigated the potential for increasing the therapeutic effectiveness of the drug by inducing its hepatic CYP-catalyzed activation. We found that MMDX cytotoxicity for cultured M5076 tumor cells was potentiated 22-fold by preincubating the drug with NADPH-supplemented liver microsomes from untreated C57BL/6 female mice. A greater (50-fold) potentiation of MMDX cytotoxicity was observed after its preincubation with liver microsomes isolated from animals pretreated with the prototypical CYP3A inducer pregnenolone-16alpha-carbonitrile. In contrast, in vivo administration of the selective CYP3A inhibitor troleandomycin (TAO) reduced both potentiation of MMDX cytotoxicity and the rate of CYP3A-catalyzed N-demethylation of erythromycin by isolated liver microsomes (55.5 and 49% reduction, respectively). In vivo antitumor activity experiments revealed that TAO completely suppressed the ability of 90 microg/kg MMDX i.v., a dose close to the LD10, to delay growth of s.c. M5076 tumors in C57BL/6 mice and to prolong survival of DBA/2 mice with disseminated L1210 leukemia. Moreover, TAO administration markedly inhibited the therapeutic efficacy of 90 microg/kg MMDX i.v. in mice bearing experimental M5076 liver metastases; a complete loss of MMDX activity was observed in liver metastases-bearing animals receiving 40 microg/kg MMDX i.v. plus TAO. However, pregnenolone-16alpha-carbonitrile pretreatment failed to enhance MMDX activity in mice bearing either s.c. M5076 tumors or experimental M5076 liver metastases. Additional experiments carried out in healthy C57BL/6 mice showed that TAO markedly inhibited MMDX-induced myelosuppression and protected the animals against lethal doses of MMDX. Taken together, these findings demonstrate that an active metabolite(s) of MMDX synthesized via CYP3A contributes significantly to its in vivo antitumor activity and host toxicity.

Animals↗
PubMed2000

Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.

Cytochrome b5 is heterogeneous in solution because of the presence of two isomers (A and B), differing in the rotation of the heme plane around the axis defined by the alpha and gamma meso protons. For rabbit cytochrome b5, the A/B ratio is 5 : 1. The solution structure of the major form of the oxidized soluble fragment of rabbit microsomal cytochrome b5 (94 amino acids) is here solved through NMR spectroscopy. From 1908 NOEs, of which 1469 were meaningful, there were 246 pseudocontact shifts and 18 3J couplings, a family of 40 energy-minimized conformers were obtained with average backbone rmsd (for residues 4-84) of 0.060 +/- 0.016 nm and average target function of 0.0078 nm2, no distance violations being larger than 0.03 nm. The structure was compared with the solution structures of the A (major) and B (minor) isomers of the rat cytochrome in the oxidized form. The A/B ratio for the rat cytochrome is 1.5 : 1, despite the very high sequence similarity (93%) to the rabbit protein. This comparison has provided insights into the factors determining the distribution in solution of the two isomers differing with respect to heme orientation. It appears that residues 23 and 74 are both important in determining this distribution, through interaction of their side chains with the prosthetic group. Hydrophobic and steric interactions are the key factors in determining the relative stability of one isomer with respect to the other.

Amino Acid Sequence↗
PubMed1999

The solution structure of oxidized Escherichia coli cytochrome b562.

The solution structure of the oxidized, paramagnetic form of cytochrome b562 from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)JHNHalpha values, and 339 pseudocontact shifts. The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha2 and alpha3, as found in the solid state. The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues. The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller. This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b562 of states with S > 1/2. The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained. Helix alpha4 seems to be the most sensitive to the chemical environment in terms of structure and mobility. The pKa values affecting the hyperfine-shifted signals are also discussed. Quite intriguing is the comparison of the structure of cytochrome b562 with the available structures of cytochromes c' which display a similar folding motif and similar pKa values but very little sequence similarity.

Amino Acid Sequence↗
PubMed1999

Structural and dynamical properties of a partially unfolded Fe4S4 protein: role of the cofactor in protein folding.

Heteronuclear multidimensional NMR spectroscopy was used to investigate in detail the structural and dynamical properties of a partially unfolded intermediate of the reduced high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum present in 4 M guanidinium chloride solution. After an extensive assignment of 15N and 1H resonances, NOE data, proton longitudinal relaxation times, and 3JHNHalpha coupling constants as well as 15N relaxation parameters (T1, T2, T1rho, and 1H-15N NOE) were obtained and used to build a structural model of the intermediate. The Fe4S4 cluster of the HiPIP plays a decisive role in determining the resulting structure, which is random in the N-terminal half of the protein and partially organized in the loops between the cysteines bound to the cluster. Consistent with the structural data, the backbone mobility is typical of folded proteins in the regions where there are elements of structure and increases with the structural indetermination.

Iron-Sulfur Proteins↗
PubMed1999

DNA-based vaccination against tumors expressing the P1A antigen.

Based on experience acquired in the last few years, we describe some technical steps and provide suggestions on how to induce an immune response against tumors expressing the weakly immunogenic antigen P1A by means of a DNA-based vaccination approach. P1A is the product of a normal mouse gene, which shares many characteristics with already identified human tumor-associated antigens, and therefore represents a useful experimental model to evaluate the efficacy of new vaccination strategies potentially applicable to the field of human tumors. Information gained with this model has been applied with success in other experimental settings, and thus we think that the procedure described herein may constitute a valid platform that can be implemented and further refined.

Animals↗
PubMed1999

Mitochondrial cytochromes c: a comparative analysis.

The structures of 113 eukaryotic cytochrome c proteins of known sequence have been modeled in the oxidized state based on the existing crystallographic and NMR structures. The secondary structural elements and the overall three-dimensional structure were found to be maintained throughout the super-family, despite variability in the sequence of individual proteins. The iron axial ligands and their reciprocal orientation were found to be nearly universally conserved. Residues constituting the hydrophobic core of the protein are also very highly conserved or conservatively substituted. Certain surface-exposed charged as well as hydrophobic groups have also been found to be conserved to the same degree as core residues. Patterns of conservation of exposed residues identify regions of the protein that are likely to be critical for its function in electron transfer.

Amino Acid Sequence↗
PubMed1999

Delivery of methoxymorpholinyl doxorubicin by interleukin 2-activated NK cells: effect in mice bearing hepatic metastases.

The possibility of using interleukin 2 (IL-2)-activated natural killer cells (A-NK) to carry methoxymorpholinyl doxorubicin (MMDX; PNU 152243) to liver-infiltrating tumours was explored in mice bearing 2-day established M5076 reticulum cell sarcoma hepatic metastases. In vitro, MMDX was 5.5-fold more potent than doxorubicin against M5076 tumour cells. MMDX uptake by A-NK cells correlated linearly with drug concentration in the incubation medium [correlation coefficient (r) = 0.999]; furthermore, as MMDX incorporation was readily reproducible in different experiments, the amount of drug delivered by A-NK cells could be modulated. In vivo experiments showed that intravenous (i.v.) injection of MMDX-loaded A-NK cells exerted a greater therapeutic effect than equivalent or even higher doses of free drug. The increase in lifespan (ILS) following A-NK cell delivery of 53 microg kg(-1) MMDX, a dosage that is ineffective when administered in free form, was similar to that observed in response to 92 microg kg(-1) free drug, a dosage close to the 10% lethal dose (ILS 42% vs. 38% respectively). These results correlated with pharmacokinetic studies showing that MMDX encapsulation in A-NK cells strongly modifies its organ distribution and targets it to tissues in which IL-2 activated lymphocytes are preferentially entrapped after i.v. injection.

Adolescent↗
PubMed1999

Inducible or constitutive expression of resistance in clinical isolates of streptococci and enterococci cross-resistant to erythromycin and lincomycin.

Thirty-five of 40 clinical isolates of enterococci and streptococci cross-resistant to erythromycin and lincomycin and harbouring erm genes were inducibly resistant to these drugs, suggesting that ribosomal methylation is predominantly inducibly expressed in these bacterial genera. Regulatory regions located upstream of the erm genes of four inducible and three constitutive strains were amplified and sequenced. Expression of constitutive resistance in two strains of Streptococcus pneumoniae and Enterococcus faecalis could be accounted for by a large deletion or a DNA duplication within the regulatory regions, respectively.

Anti-Bacterial Agents↗