Biomedical subjects
Publications and source records attributed to Giuseppe Spoto.
Surface plasmon resonance imaging is used to demonstrate the recognition by the Ricinus communis agglutinin of a new SOD mimic, a bioconjugate of the manganese(II) complex of 1,4,7,10,13-pentaazacyclopentadecane with galactose.
The adsorption of Ar, H2, O2, N2 and CO on (H,Na)-Y zeolite (Si/Al = 2.9, H+/Na+ approximately 5) has been studied at variable-temperature (90-20 K) and sub-atmospheric pressure (0-40 mbar) by FTIR spectroscopy. Unprecedented filling conditions of the zeolite cavities were attained, which allowed the investigation of very weakly adsorbed species and of condensed, liquid-like or solid-like, phases. Two pressure regimes were singled out, characterized by: (i) specific interaction at low pressure of the probe molecules (P) with the internal Brønsted and Lewis sites, and (ii) multilayer adsorption at higher pressure. In the case of CO the perturbation of the protonic sites located inside the sodalite cages was also observed. As the molecule is too large to penetrate the sodalite cage, the perturbation is thought to involve a proton jump tunneling mechanism. The adsorption energy for the (HF)OH...P (P = Ar, H2, O2, N2 and CO) specific interaction involving the high frequency Brønsted acid sites exposed in the supercages was derived following the VTIR (variable temperature infrared spectroscopy) method described by E. Garrone and C. Otero Areán (Chem. Soc. Rev., 2005, 34, 846).
Several different procedures are available for the immobilization of proteins on solid supports, as many advantages derive from this approach, such as the possibility to develop new protein solid-state assays. Enzymes that are anchored on gold surfaces can interact with several different molecules in a tag-free environment, opening the way to surface plasmon resonance (SPR) investigations. Nevertheless, it is often important to know the identity of the affinity-retained analyte, and mass spectrometric analysis, via its unique molecular mass identification, represents a very valuable complementary method. There are many pieces of evidence to suggest that matrix metalloproteinases (MMPs) are involved in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis and cancer, but presumably also exhibiting other functions. The search for new inhibitors of MMPs has prompted research towards the development of new solid-state assays for the rapid evaluation of MMP activity. We have already reported the possibility of measuring the activity of MMP-1 anchored on solid support by coupling SPR with ESI-MS analysis. In this work, we show the in situ atmospheric pressure (AP) MALDI-MS characterization of MMPs anchored on a gold chip with known surface coverage. The study extends the MS analysis to different proteins, and sequence coverage is reported for different digestion and MS procedures.
We have recently highlighted that H-SSZ-13, a highly siliceous zeolite (Si/Al = 11.6) with a chabazitic framework, is the most efficient zeolitic material for hydrogen storage [A. Zecchina, S. Bordiga, J. G. Vitillo, G. Ricchiardi, C. Lamberti, G. Spoto, M. Bjørgen and K. P. Lillerud, J. Am. Chem. Soc., 2005, 127, 6361]. The aim of this new study is thus to clarify both the role played by the acidic strength and by the density of the polarizing centers hosted in the same framework topology in the increase of the adsorptive capabilities of the chabazitic materials towards H2. To achieve this goal, the volumetric experiments of H2 uptake (performed at 77 K) and the transmission IR experiment of H2 adsorption at 15 K have been performed on H-SSZ-13, H-SAPO-34 (the isostructural silico-aluminophosphate material with the same Brønsted site density) and H-CHA (the standard chabazite zeolite: Si/Al = 2.1) materials. We have found that a H2 uptake improvement has been obtained by increasing the acidic strength of the Brønsted sites (moving from H-SAPO-34 to H-SSZ-13). Conversely, the important increase of the Brønsted sites density (moving from H-SSZ-13 to H-CHA) has played a negative role. This unexpected behavior has been explained as follows. The additional Brønsted sites are in mutual interaction via H-bonds inside the small cages of the chabazitic framework and for most of them the energetic cost needed to displace the adjacent OH ligand is higher than the adsorption enthalpy of the OH...H2 adduct. From our work it can be concluded that proton exchanged chabazitic frameworks represent, among zeolites, the most efficient materials for hydrogen storage. We have shown that a proper balance between available space (volume accessible to hydrogen), high contact surface, and specific interaction with strong and isolated polarizing centers are the necessary characteristics requested to design better materials for molecular H2 storage.
Due to its fully reversible nature, H(2) storage by molecular adsorption could represent an advantage with respect to dissociative processes, where kinetic effects during the charging and discharging processes are present. A drawback of this strategy is represented by the extremely weak interactions that require low temperature and high pressure. High surface area materials hosting polarizing sites can represent a viable way toward more favorable working conditions. Of these, in this contribution, we have studied hydrogen adsorption in a series of zeolites using volumetric techniques and infrared spectroscopy at 15 K. We have found that in H-SSZ-13 zeolite the cooperative role played by high surface area, internal wall topology, and presence of high binding energy sites (protons) allows hydrogen to densify inside the nanopores at favorable temperature and pressure conditions.
Paramagnetic centers at the surface of ionic oxides in the form of trapped electrons can be generated by exposure of the material to alkali metal or hydrogen atoms or of molecular hydrogen under UV irradiation. For many years, it has been assumed that the resulting paramagnetic centers consist of oxygen vacancies filled by one electron. High-resolution electron spin resonance spectra and ab initio quantum chemical calculations show that the paramagnetic centers consist of (H(+))(e(-)) electron pairs formed at morphological irregularities of the surface. At least three different kinds of (H(+))(e(-)) centers, [A], [B], and [C], have been identified with abundances of 80%, 10%, and 8%, respectively. In this work, we compare a wide set of measured and computed g-factors and hyperfine coupling constants of the unpaired electron with the surrounding (25)Mg, (17)O, and (1)H nuclei and we propose a general assignment of the centers. (H(+))(e(-)) pairs formed at Mg(4c) ions at steps and edges account for species [A], centers formed at Mg(4c) ions at reverse corners correspond to species [B], and species [C] originates from (H(+))(e(-)) pairs formed at Mg(3c) ions at corners and kinks.
The present work reports the synthesis of trans-2-amino-5(6)-fluoro-6(5)-hydroxy-1-phenyl-2,3-dihydro-1H-indenes (4a-f, 5a-f) as a continuation of our studies to better understand the significance of the halo substituent in the trans-1-phenyl-2-aminoindane series and to extend knowledge of the monophenolic ligands of DA receptors. The affinity of the new compounds and related methoxylated precursors (10-15 and 18-23) was estimated in vitro by displacement of [(3)H]SCH23390 (for D(1)-like receptors) or [(3)H]YM-09-151-2 (for D(2)-like receptors) from homogenates of porcine striatal membranes. The results indicate that unsubstituted amines 4a, 5a, 10, and 11 are poorly effective at DA receptors. The introduction of two n-propyl groups on the nitrogen atom (compounds 14, 15, 4c, and 5c) and N-allyl-N-methyl- or N-methyl-N-propyl- substitution (compounds 20-23, 4e, 4f, 5e, 5f) increased the D(2)-like affinities and selectivity. The D(2)-like agonistic activity of selected compounds 15, 20, 21, 4e, 5c, and 5e was proved by evaluating their effects on the cyclic guanosine monophosphate (cGMP) content in rat neostriatal membranes. All tested compounds displayed a potential dopamine D(2)-like agonist profile decreasing basal levels of cGMP. The selective D(2)-like agonism of compounds 20 and 5e was proved by their effects on basal striatal adenylyl cyclase activity.
Matrix metalloproteinases (MMPs) are a family of Zn-dependent endo-peptidases known for their ability to cleave several components of the extracellular matrix, but which can also cleave many non-matrix proteins. There are many evidences that MMPs are involved in physiological and pathological processes, and a huge effort has been put in the development of possible inhibitors that could reduce the activity of MMPs, as it is clear that the ability to monitor and control such activity plays a pivotal role in the search for potential drugs aimed at finding a cure for several diseases such as pulmonary emphysema, rheumatoid arthritis, fibrotic disorders and cancer.A powerful method currently available to study enzyme-inhibitor interactions is based on the use of the surface plasmon resonance (SPR) technique. When MMP interactions are studied, a procedure by which inhibitors are normally anchored on sensor chips and SPR technique is used in order to study their interaction with MMPs molecules is usually followed. This is because it is currently believed that MMPs cannot be anchored on the sensor-chip surface without losing their activity. However, this approach gives rise to problems, as the anchoring of low-molecular-weight inhibitors on gold surfaces easily affects their ability to interact with MMPs. For this reason, the anchoring of MMPs is highly desirable.A new experimental protocol that couples the Fourier transform-SPR (FT-SPR) technique with electrospray ionization-mass spectroscopy (ESI-MS) is described here for the evaluation of the activity of MMP-1 catalytic domain (cdMMP-1) anchored on gold surfaces. The cdMMP-1 surface coverage is calculated by using FT-SPR and the enzyme activity is estimated by ESI-MS. The proposed method is label-free.
BACKGROUND: During orthodontic tooth movement, the early response of periodontal tissues to mechanical stress is an acute inflammatory one. This study uses a longitudinal design to examine lactate dehydrogenase (LDH) activity in gingival crevicular fluid (GCF) to determine if GCF LDH can be used as a diagnostic aid in monitoring tooth movement and tissue response during orthodontic treatment. METHODS: Seventeen patients (mean age: 16.1 years) participated in the study. Each patient was undergoing treatment for distal movement, and an upper first molar served as the test tooth (TT), while the contralateral (CT) and antagonist (AT) teeth were used as controls. The CT was included in the orthodontic appliance, but was not subjected to the distal movement; the AT was free from any orthodontic appliance. The GCF around the experimental teeth was harvested from both mesial and distal tooth sites immediately before appliance activation, and on days 7, 14, and 21. Clinical gingival conditions were also recorded. RESULTS: Gingival crevicular fluid LDH activity was significantly elevated in all sites of the TT and CT, as compared to the AT, where LDH activity remained at the baseline level throughout the study. Enzyme activity levels were also greater in the TT than in the CT, and in the compression sites. CONCLUSIONS: Our results suggest that GCF LDH levels reflect the biological activity that takes place in the periodontium during orthodontic movement, and therefore they can be used as a diagnostic tool for monitoring for correct orthodontic tooth movement in clinical practice.
Cu(I) ions in Cu-ZSM-5 form Cu+(H2) complexes, stable at room temperature and sub-atmospheric H2 pressure, which do not have any homogeneous analogue except for matrix-isolated [Cu(eta2-H2)Cl]. Comparison with the unstable Na+(H2) adducts formed in the parent Na-ZSM-5 zeolite allow the conclusion that the Cu(I)/H2 bond is governed by sigma-pi overlap forces.
Dipyridyl pendants, suitably attached onto a calix[4]arene fixed in its 1,3-alternate conformation, form stable complexes with copper(ii) in acetonitrile. Both the mononuclear and the homobinuclear species ([Cu(1)](2+) and [Cu(2)(1)](4+)), previously detected through a UV-Vis investigation, have been studied by ESR, UV-Vis and cyclic voltammetry. ESR and UV-Vis data clearly indicate that in the [Cu(1)](2+) species the nitrogen atoms of dipyridyl pendants are tetrahedrally arranged around copper(II). Computer models, optimised through molecular mechanics methods, further support these spectroscopic findings. The tetrahedral arrangement, achieved thanks to the anchoring of the dipyridyl moieties onto the calixarene platform, accounts for the easy reversible reduction of this species. The aspects associated with the reduction-oxidation of the homobinuclear species are also discussed.
The electrochemically switchable Cu(2+) complex of a 1,3 alternate bis(dipyridyl)calix[4]arene derivative forms self-assembled monolayers on Au(111) surfaces. The receptor is patterned on the surface by using microcontact printing procedures and the resulting surface is imaged via SPR.
In order to examine the cyclic nucleotides (cGMP) role in carcinoma growth and invasivity. We analyzed two cell lines, LSHT29 and 17GT, and tissues in patients with carcinoma and malignant tissues with (N+) and without (N-) lymph node metastases. Higher cGMP levels in pathological samples suggest a strong correlation between intracellular cGMP concentration and carcinoma progression.
OBJECTIVES: During orthodontic treatment, changes in subgingival plaque colonization and tissue inflammation and remodelling have been described. This study uses a longitudinal design to examine subgingival colonization of Actinobacillus actinomycetemcomitans (Aa) and alkaline phosphatase (ALP) and aspartate aminotransferase (AST) activities in gingival crevicular fluid (GCF) in order to assess whether these parameters have potential as biomarkers of tissue responses to orthodontic tooth movement in humans. MATERIALS & METHODS: Twenty-one patients (ages: 11.2-22.5; mean 17.1 +/- 3.3 years) participated in the study. An upper canine from each patient undergoing treatment for distal movement served as the test tooth (DC), and its contralateral (CC) and antagonist (AC) canines were used as controls. The CC was included in the orthodontic appliance, but was not subjected to the orthodontic force; the AC was free from any orthodontic appliance. The subgingival plaque and GCF around the experimental teeth was harvested from both mesial and distal tooth sites immediately before appliance activation and on day 28. Clinical gingival condition was evaluated at the baseline and at the end of the experimental period. Aa colonization was determined by culture methods, while ALP and AST activities were evaluated spectrophotometrically. RESULTS: Throughout the study, the clinical conditions worsened in both the DCs and the CCs as compared with the baseline, whereas no significant differences were found between the DCs and the CCs, or between mesial and distal sites of each of these teeth on day 28. In the ACs, clinical parameters remained at baseline levels throughout the study. Similar results were found for Aa colonization, which increased significantly on day 28 in the DC and CC groups. On day 28, ALP and AST activities were significantly elevated in all sites from the DC and CC groups as compared with the ACs, where, conversely, enzymatic activities remained at the baseline levels. However, ALP activity in the DC group was significantly greater than in the CCs at mesial (tension) sites on day 28, while AST activity in the DCs was significantly elevated as compared with the CC group at the distal (compression) sites. Greater ALP activity in the DC group was observed at the tension sites compared with the compression sites on day 28. CONCLUSIONS: Our results suggest that Aa subgingival colonization, and ALP and AST activities in GCF reflect the tissue responses that occur in the periodontium during orthodontic treatment.
BACKGROUND: The phosphodiesterases (PDEs) are responsible for the hydrolysis of the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP), to their corresponding monophosphates with a fundamental role in the transduction of the intracellular signals. At least 11 different enzymatic isoforms have been identified, which are listed according to their specificity or affinity for the substratum, identity of the amino acid sequence, cofactor, and inhibitor sensitivity. Variations in PDE activity have been found in different pathologies, and they have also been correlated to different pathological e/o physiological mechanisms, such as cellular differentiation, apoptosis, and tumor invasivity. OBJECTIVES: In this study, we have evaluated cAMP PDE activity in patients with carcinoma of the gingiva, with the purpose of correlating differences in its development and progression. The same enzymatic activity has been used to evaluate differences between patients with lymph node involvement (group N(+)), and patients without lymph node involvement (N(-)). MATERIALS AND METHODS: The analysis of PDE activity and the cAMP assay was performed by reverse-phase HPLC on samples of fresh or frozen gingival tissues. Analysis of cAMP was confirmed with the enzyme-linked immunoabsorption assay (EIA). RESULTS AND CONCLUSIONS: The differences between control and N(-) groups (P = 0.0433), and between control and N(+) groups (P = 0.0156) were statistically significant. PDE3A was also evaluated immunohistochemically in lymph-node negative and lymph-node positive cases. The differences between the two groups were statistically significant (P = 0.0397).
During orthodontic treatment, the early response of periodontal tissues to mechanical stress involves several metabolic changes that allow tooth movement. Many studies have evaluated such modifications by analysis of various host metabolites released into the gingival crevicular fluid (GCF). This study used a cross-sectional design to examine the lactate dehydrogenase (LDH) activity in GCF to assess whether GCF LDH can be proposed as a sensitive marker for periodontal tissue modifications during orthodontic tooth movement. Thirty-seven subjects, 16 males and 21 females (mean age 18.7 years, range 14.0 to 26.7 years), participated in this study. Each subject underwent a session of professional oral hygiene and received oral hygiene instructions; 2 weeks later, a fixed orthodontic appliance was placed on the maxillary arch. A randomly selected maxillary canine was considered as the test tooth, and its antagonist, which had no appliance, was used as the control tooth. From 2 to 12 weeks after orthodontic appliance placement, GCF was harvested from both experimental teeth at the mesiobuccal angle, for GCF volume and LDH activity determinations. Clinical monitoring consisted of recording supragingival plaque presence, bleeding on probing, and probing depth at the same collection sites. The results showed that no differences in clinical conditions and GCF volume occurred between the experimental teeth. On the contrary, GCF LDH activity in the test teeth was significantly greater than that of the control teeth (P <.01). Moreover, no differences were found in the enzymatic activity between the sexes by experimental tooth, and no significant correlation was present between GCF LDH activity and patients' ages within experimental teeth. Our enzymatic results initially indicated a possible role of GCF LDH during the early phases of orthodontic treatment and therefore warrant further study as a possible diagnostic tool for tissue response during orthodontic treatment.
BACKGROUND: Cyclic guanosine monophosphate (cGMP) is an essential second messenger metabolized by phosphodiesterases (PDEs). OBJECTIVES: We looked for a possible correlation of PDE activities in human oral squamous cell carcinoma (OSCC) with and without lymph node metastases. MATERIALS AND METHODS: The analysis of phosphodiesterase activity and the cGMP assay were done by reverse-phase HPLC on samples of fresh or frozen gingival tissues. Analysis of cGMP was confirmed with the enzyme-linked immunoabsorption assay. RESULTS AND CONCLUSIONS: cGMP PDE activity was 34.92 +/- 7.17 SD, 12.89 +/- 4.43 SD, and 35.88 +/- 8.76 SD (nmols/mg of protein), respectively, in controls, samples without lymph node involvement (N-), and specimens with lymph node metastases (N+). cGMP values were 1.97 +/- 0.63 SD, 3.30 +/- 1.47 SD, and 3.49 +/- 1.47 SD (nmols/mg of protein). Our data support the hypothesis of a role for cGMP and PDE in the progression of OSCC.
In this study, we analyzed the intracellular mechanisms leading to basic fibroblast growth factor (bFGF)-dependent production of NO in Chinese hamster ovary (CHO)-K1 cells and a possible physiological role for such an effect. bFGF induces NO production through the activation of the endothelial form of NO synthase (eNOS), causing a subsequent increase in the cGMP levels. In these cells, the activation of eNOS by bFGF is Ca(2+)- and mitogen-activated protein kinase-independent. The translocation of the enzyme from the plasma membrane, where it is located in caveolae bound to caveolin 1, to the cytosol is the crucial step for the synthesis of NO through the eNOS isoform. We demonstrate that bFGF activates a sphingomyelinase to synthesize ceramide, which, in turn, allows the dissociation of eNOS from caveolin 1 and its translocation to the cytosol in the active form, where it catalyzes the synthesis of NO. In fact, drugs interfering with sphingomyelinase activity blocked bFGF activation of eNOS, and an increase in ceramide content was detected after bFGF treatment. Moreover, in fibroblasts derived from patients with Niemann-Pick disease, in which the enzyme is genetically inactive, bFGF is unable to elicit eNOS activation. The NO produced after bFGF treatment, through the activation of guanylyl cyclase and protein kinase G, mediates a mitogen-activated protein kinase-independent cell proliferation. In conclusion, our data show that, in CHO-K1 cells, bFGF regulates the activity of eNOS through a novel intracellular pathway, involving the induction of ceramide synthesis and that the NO released participates in bFGF proliferative activity.