The role of charge transfer in the stability and reactivity of chemical systems from experimental findings.

A variety of phenomena, of apparently different natures, are described within a unifying picture, by properly isolating the role of charge/electron transfer as an interaction component triggering chemical reactivity. This basic quantity is isolated by analyzing, with advanced theoretical methods developed by our group, experimental findings characterized with different techniques, such as double photo-ionization spectra, scattering cross sections and auto-ionization reaction probabilities. Suitable rationalization of such phenomena appears to be crucial for modeling the selectivity of basic elementary processes occurring in systems at increasing complexity of fundamental/applied interest, such as plasmas, flames, interstellar media, planetary atmospheres and biological environments.

The soft X-ray absorption spectrum of the allyl free radical.

The first experimental study of the X-ray absorption spectrum (XAS) of the allyl free radical, CH(2)CHCH(2), is reported. A supersonic He seeded beam of hyperthermal allyl radicals was crossed by a high resolution synchrotron radiation (SR) in the focus of a 3D ion momentum imaging time-of-flight (TOF) spectrometer to investigate the soft X-ray absorption and fragmentation processes. The XAS, recorded as Total-Ion-Yield (TIY), is dominated by C1s electron excitations from either the central carbon atom, C(C), or the two terminal carbon atoms, C(T), to the frontier orbitals, the semi-occupied-molecular-orbital (SOMO) and the lowest-unoccupied-molecular-orbital (LUMO). All of the intense features in the XAS could only be assigned with the aid of ab initio spectral simulation at the Multi-Configuration Self-Consistent-Field (MCSCF) level of theory, this level being required because of the multi-reference nature of the core-excited state wavefunctions of the open shell molecule. The ionization energies (IEs) of the singlet and triplet states of the C1s ionized allyl radical (XPS) were also calculated at the MCSCF level.

Angular and energy distribution of fragment ions in dissociative double photoionization of acetylene molecules at 39 eV.

The two-body dissociation reactions of the dication, C(2)H(2)(2+), produced by 39.0 eV double photoionization of acetylene molecules, have been studied by coupling photoelectron-photoion-photoion coincidence and ion imaging techniques. The results provide the kinetic energy and angular distributions of product ions. The analysis of the results indicates that the dissociation leading to C(2)H(+)+H(+) products occurs through a metastable dication with a lifetime of 108±22 ns, and a kinetic energy release (KER) distribution exhibiting a maximum at ∼4.3 eV with a full width at half maximum (FWHM) of about 60%. The reaction leading to CH(2)(+)+C(+) occurs in a time shorter than the typical rotational period of the acetylene molecules (of the order of 10(-12) s). The KER distribution of product ions for this reaction, exhibits a maximum at ∼4.5 eV with a FWHM of about 28%. The symmetric dissociation, leading to CH(+)+CH(+), exhibits a KER distribution with a maximum at ∼5.2 eV with a FWHM of 44%. For the first two reactions the angular distributions of ion products also indicate that the double photoionization of acetylene occurs when the neutral molecule is mainly oriented perpendicularly to the light polarization vector.

Dissociative double photoionization of singly deuterated benzene molecules in the 26-33 eV energy range.

This work provides new experimental and theoretical results about the formation and dissociation of benzene dication. The experiment has been carried out by using a vacuum ultraviolet radiation from a synchrotron source together with a time-of-flight spectrometer and a position sensitive ion detector. Isotopically labeled benzene molecules with a single deuterium atom have been used in order to study the symmetric dissociation of the benzene dication, not well evident in previous experiments. A threshold of 30.1 ± 0.1 eV has been observed for this dissociation reaction. Moreover, the lifetime of the dissociation of the benzene metastable dication producing CH(3)(+) and C(5)H(3)(+) has been obtained as a function of the photon energy, by the use of a Monte Carlo trajectory analysis of the coincidence distributions. The determined lifetime is independent of the photon energy and has an average value of 0.75 ± 0.22 µs. Theoretical calculations of the energy and structure of dissociation product ions have been also performed to provide crucial information about the dynamics of the charge separation reactions following the photoionization event.

Dissociative double photoionization of CO2 molecules in the 36-49 eV energy range: angular and energy distribution of ion products.

Dissociative double photoionization of CO(2), producing CO(+) and O(+) ions, has been studied in the 36-49 eV energy range using synchrotron radiation and ion-ion coincidence imaging detection. At low energy, the reaction appears to occur by an indirect mechanism through the formation of CO(+) and an autoionizing state of the oxygen atom. In this energy range the reaction leads to an isotropic distribution of products with respect to the polarization vector of the light. When the photon energy increases, the distribution of products becomes anisotropic, with the two ions preferentially emitted along the direction of the light polarization vector. This implies that the molecule photoionizes when oriented parallel to that direction and also that the CO(2)(2+) dication just formed dissociates in a time shorter than its typical rotational period. At low photon energy, the CO(+) and O(+) product ions separate predominantly with a total kinetic energy between 3 and 4 eV. This mechanism becomes gradually less important when the photon energy increases and, at 49 eV, a process where the two products separate with a kinetic energy between 5 and 6 eV is dominant.

Double photoionization of CO2 molecules in the 34-50 eV energy range.

The double photoionization of CO(2) molecules has been studied in the 34-50 eV photon energy range, by the use of synchrotron radiation and detecting electron-ion and electron-ion-ion coincidences. Three processes have been observed: (i) the formation of the CO(2)(2+) molecular dication, (ii) the production of a metastable (CO(2)(2+))* that dissociates, with an apparent lifetime of 3.1 micros, giving rise to CO(+) and O(+) ions, and (iii) the dissociation leading to the same products, but occurring with a lifetime shorter than 0.05 micros. The relative dependence on the photon energy of the cross section for such processes has been measured. While for the production of the molecular dication a threshold is observed, in agreement with the vertical threshold for double ionization of CO(2), for the dissociative processes the threshold appears to be lower than that value, indicating the presence of an indirect dissociation, probably leading to the formation of CO(+) together with a neutral autoionizing oxygen atom.

Anisotropy of the angular distribution of fragment ions in dissociative double photoionization of N2O molecules in the 30-50 eV energy range.

The double photoionization of N2O molecules by linearly polarized light in the 30-50 eV energy range has been studied by coupling ion imaging technique and electron-ion-ion coincidence. For the two possible dissociative processes, leading to N++NO+ and O++N2+, angular distributions of ionic fragments have been measured, finding an evident anisotropy. This indicates that the molecules ionize when their axis is parallel to the light polarization vector and the fragments are separating in a time shorter than the dication rotational period. The analysis of results provides, in addition to the total kinetic energy of ionic fragments, crucial information about the double photoionization dynamics.

The double photoionization of hydrogen iodide molecules.

The double photoionization of HI molecules has been investigated using vacuum ultraviolet synchrotron radiation in the energy range between 27 and 35 eV. The product ions have been detected by the use of time-of-flight mass spectrometry and the threshold energy for HI2+ and H+ + I+ formation has been determined. These results have been interpreted by the use of a theoretical model which has been previously applied by us to HBr2+ and HCl2+. On the basis of the reliability of such a model, an assessment of the systematic trends of the bond features along the HX2+ (X=F, Cl, Br, I) homologous series is given in this paper. In particular, the increase of the stability of these dications, in their lowest electronic states, when going towards the heavier molecules, has been rationalized considering the systematic variation of the charge transfer coupling between the H-X2+ and the H+-X+ states.

The double photoionization of HCl: an ion-electron coincidence study.

The double photoionization of HCl molecules by synchrotron radiation has been studied in the energy range between 30 and 50 eV. The HCl(2+) and Cl(2+) product ions have been detected by a photoelectron-photoion-coincidence technique, while the H(+)+Cl(+) formation, which follows the double ionization of HCl, has been studied by photoelectron-photoion-photoion coincidence. The photon energy threshold for the production of HCl(2+) ions has been found to be 35.4+/-0.6 eV, while for the dissociative channel leading to H(+)+Cl(+), it has been measured a threshold at 36.4+/-0.6 eV and a change in the slope of the cross-section energy dependence at 38.7+/-0.7 eV. The production of H+Cl(2+) occurs with a threshold photon energy of 42.8+/-1.1 eV. These results appear to be in a good agreement with previous data by different experimental techniques and recent theoretical calculations performed by our laboratory.

Risk factors for uncontrolled hypertension in Italy.

To identify factors related to poor control of blood pressure in primary care, we designed a retrospective case-control analysis of clinical and demographic data recorded in the General Practitioners (GP) database. Study data were provided on a voluntary basis by 21 GPs from a practice-based network in primary care. The study included 2519 hypertensive patients enrolled between January 1 and December 31, 2000. The interventions were antihypertensive medication, and the main outcome measures were control of systolic and diastolic blood pressure (BP). The independent variables considered were: age of patient and GP; patient gender, body mass index, history of smoking, diabetes mellitus, or cholesterol tests; family history of hypertension; previous visits for cardiologic, nephrologic, or vascular surgery evaluation; prior hospitalizations for myocardial infarction or heart failure, and number of admissions for surgery; length of patient follow-up, type of antihypertensive medication, mean daily dosage, adherence to the drug regimen, and number of other medications currently being taken by the patient. Blood pressure was uncontrolled (>140/90 mmHg) in 1525 (60%) of the 2519 hypertensive patients enrolled. The presence of diabetes mellitus, increasing patient age, and increasing GP age significantly increased the risk of uncontrolled BP. Factors significantly associated with a reduced risk of uncontrolled BP were the number of other medications currently being taken by the patient and a prior history of MI. We conclude that the failure of antihypertensive medication to adequately control BP is determined by both the patient's characteristics and factors related to the patient-doctor relationship. Successful treatment of hypertension requires patient adherence to the regimen that has been agreed on by the patient and the physician.

DNA repair enzymes and cytotoxic effects of temozolomide: comparative studies between tumor cells and normal cells of the immune system.

O6-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair system (MRS) play a crucial role in the susceptibility of tumor cells to the cytotoxic effects of agents that generate O6-methylguanine in DNA, including the triazene compound temozolomide (TMZ). Studies performed with peripheral blood mononuclear cells (MNC) showed that TMZ was scarcely active on lymphocyte functions not dependent on cell proliferation (e.g. NK activity and cytokine-mediated induction of CD1b molecule in adherent MNC). In contrast, TMZ depressed proliferation and lymphokine activated killer (LAK) cell generation in response to IL-2. In this case, a reasonably good inverse relationship was found between OGAT levels of MNC and their susceptibility to TMZ. This study also analyzed the ratio of the toxic effect of TMZ on MNC and on tumor cells (i.e. "Tumor-Immune Function Toxicity Index", TIFTI). A particularly favorable TIFTI can be obtained when OGAT levels are extremely high in MNC and markedly low in tumor cells. This holds true for MRS-proficient neoplastic cells, but not for MRS-deficient tumors. In conclusion, strategies aimed at modulating OGAT and MRS may improve the clinical response to TMZ. However, the use of OGAT inhibitors to potentiate the antitumor activity of TMZ might result in a concomitant increase of the immunosuppressive effects of the drug, thus reducing the relative TIFTI.

hMSH3 overexpression and cellular response to cytotoxic anticancer agents.

Mutations or transcriptional silencing of mismatch repair genes have been linked with tumour cell resistance to O(6)-guanine methylating agents, 6-thioguanine, cisplatin, doxorubicin and etoposide. Recently, it has been demonstrated that overexpression of the MSH3 protein is associated with depletion of the mismatch binding factor MutSalpha, and then with a marked reduction in the efficiency of base/base mismatch repair. In the present study we evaluated sensitivity of the HL-60 cell line and its methotrexate-resistant subline HL-60R, which overexpresses the hMSH3 gene, to a panel of chemotherapeutic agents. Cell growth inhibition induced by temozolomide, 6-thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine was significantly lower in the hMSH3-overexpressing HL-60R cell line as compared with the HL-60 parental line. Moreover, HL-60R cells were more resistant than HL-60 cells to chromosome aberrations induced by either N-methyl-N'-nitro-N-nitrosoguanidine or temozolomide, and to apoptosis triggered by the latter drug. Both cell lines were equally susceptible to growth inhibition induced by cisplatin, etoposide or doxorubicin. In addition, HL-60 and HL-60R cells showed comparable sensitivity to the clastogenic and apoptotic effects of cisplatin and etoposide. These results further confirm that loss of base/base mismatch repair is the most important molecular mechanism involved in cell resistance to O(6)-guanine methylating agents and 6-thioguanine. However, the status of the mismatch repair system could still influence tumour cell sensitivity to cisplatin, etoposide and doxorubicin, depending on the specific component of the system that is lost, and on the genetic background of the cell.

Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor.

The vascular endothelial growth factor is produced by a large variety of human tumors, including melanoma, in which it appears to play an important role in the process of tumor-induced angiogenesis. Little information is available on the role of placenta growth factor, a member of the vascular endothelial growth factor family of cytokines, in tumor angiogenesis, even though placenta growth factor/vascular endothelial growth factor heterodimers have been recently isolated from tumor cells. To investigate the role of placenta growth factor and vascular endothelial growth factor homodimers and heterodimers in melanoma angiogenesis and growth, 19 human melanoma cell lines derived from primary or metastatic tumors were characterized for the expression of these cytokines and their receptors. Release of placenta growth factor and vascular endothelial growth factor polypeptides into the supernatant of human melanoma cells was demonstrated. Reverse transcriptase polymerase chain reaction analysis showed the presence of mRNAs encoding at least three different vascular endothelial growth factor isoforms (VEGF(121), VEGF(165), and VEGF(189)) and transcripts for two placenta growth factor isoforms (PlGF-1 and PlGF-2) in human melanoma cells. In addition, placenta growth factor expression in human melanoma in vivo was detected by immunohistochemical staining of tumor specimens. Both primary and metastatic melanoma cells were found to express the mRNAs encoding for vascular endothelial growth factor and placenta growth factor receptors (KDR, Flt-1, neuropilin-1, and neuropilin-2), and exposure of melanoma cells to these cytokines resulted in a specific proliferative response, supporting the hypothesis of a role of these angiogenic factors in melanoma growth. J Invest Dermatol 115:1000-1007 2000

Effect of prostaglandin A1 on proliferation and telomerase activity of human melanoma cells in vitro.

Previous studies have shown that cyclopentenone prostaglandins are endowed with antitumour activity in various murine and human tumour models. In the present investigation four human melanoma cell lines were treated with graded concentrations (4-16microg/ml) of prostaglandin A1 (PGA1) for 24 or 48 h in vitro. At the end of the treatment, cell proliferation (measured in terms of DNA synthesis) and telomerase activity were determined. The results showed that PGA1 induced concentration-dependent inhibition of DNA synthesis at 48 h but not at 24 h in SK-MEL-28 cells. In contrast, marked inhibition of telomerase activity was detected after only 24 h of PGA1 treatment. Moreover, after 48h of treatment with the agent, inhibition of telomerase was more pronounced than inhibition of cell proliferation. Additional studies performed with three freshly generated melanoma cell lines confirmed that PGA1 produced early inhibition of cell growth accompanied by marked impairment of telomerase activity. These results suggest that PGA1 could be of potential value as antitumour agent, on the basis of two distinct mechanisms: direct cytostatic/cytotoxic effects on melanoma cells, and inhibitory activity on a tumour-associated enzymatic function (i.e. telomerase) that is responsible for cancer cell immortality.

Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties.

The gene coding for Pseudomonas aeruginosa cytochrome c-551 was expressed in Pseudomonas putida under aerobic conditions, using two different expression vectors; the more efficient proved to be pNM185, induced by m-toluate. Mature holo-(cytochrome c-551) was produced in high yield by this expression system, and was purified to homogeneity. Comparison of the recombinant wild-type protein with that purified from Ps. aeruginosa showed no differences in structural and functional properties. Trp56, an internal residue in cytochrome c-551, is located at hydrogen-bonding distance from haem propionate-17, together with Arg47. Ionization of propionate-17 was related to the observed pH-dependence of redox potential. The role of Trp56 in determining the redox properties of Ps. aeruginosa cytochrome c-551 was assessed by site-directed mutagenesis, by substitution with Tyr (W56Y) and Phe (W56F). The W56Y mutant is similar to the wild-type cytochrome. On the other hand, the W56F mutant, although similar to the wild-type protein in spectral properties and electron donation to azurin, is characterized by a weakening of the Fe-Met61 bond, as shown in the oxidized protein by the loss of the 695 nm band approx. 2 pH units below the wild-type. Moreover, in W56F, the midpoint potential and its pH-dependence are both different from the wild-type. These results are consistent with the hypothesis that hydrogen-bonding to haem propionate-17 is important in modulation of the redox properties of Ps. aeruginosa cytochrome c-551.

Antioxidant status in various tissues of the mouse after fasting and swimming stress.

We studied the effect of fasting and swimming stress on a number of non-enzymatic and enzymatic antioxidant factors in various mouse tissues in order to see if their action was synergic. We examined levels of reduced (GSH), oxidized (GSSG) and total glutathione, total SH groups (TSH), sum of GSH and protein sulphydryl groups of cytosolic fractions, and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase in adductor muscle, heart and liver. We also studied blood levels of GSH and glutathione bound to protein by mixed disulphides (GSSP). The case series consisted of four groups of animals (n = 10 for each group), namely no swimming and no fast, no swimming and fast, swimming and no fast, and swimming and fast. Fasting (18 h) resulted in a significant GSH depletion in all of the organs studied (-39% in the liver, -30% in the adductor muscle, -21% in the heart); GSSG increased significantly in the heart (+19%). Swimming to exhaustion, which lasted 3.95 (0.18) min [mean (SD), n = 10] with no significant difference between fast and no fast, resulted in a significant GSH depletion, to a percentage lower than that observed after fasting, in the adductor muscle and heart (-12% and -11%, respectively). In the blood of swimming mice, significant increases in GSH (+10%) and GSSG (+21%) levels were observed, whereas GSSP decreased (-15%). Enzyme activities after swimming were modified in only a few cases, and in a complex way. The findings of GSH depletion and a decrease in SOD activity in the adductor muscle seems to confirm the sensitivity of this organ to an overproduction of reactive oxygen species. At the same time, the GSSP decrease observed in blood was a new and unexpected finding, one that indicates a very prompt adaptation of red cells to increased oxidant states.

Pseudomonas aeruginosa nitrite reductase (or cytochrome oxidase): an overview.

The biochemistry and molecular biology of nitrite reductase, a key enzyme in the dissimilatory denitrification pathway of Ps aeruginosa which reduces nitrite to NO, is reviewed in this paper. The enzyme is a non-covalent homodimer, each subunit containing one heme c and one heme d1. The reaction mechanisms of nitrite and oxygen reduction are discussed in detail, as well as the interaction of the enzyme with its macromolecular substrates, azurin and cytochrome c551. Special attention is paid to new structural information, such as the chemistry of the d1 prosthetic group and the primary sequence of the gene and the protein. Finally, results on the expression both in Ps aeruginosa and in heterologous systems are presented.