Reactivity of hypotaurine and cysteine sulfinic acid toward carbonate radical anion and nitrogen dioxide as explored by the peroxidase activity of Cu,Zn superoxide dismutase and by pulse radiolysis.

Hypotaurine and cysteine sulfinic acid are known to be readily oxidized to the respective sulfonates, taurine and cysteic acid, by several oxidative agents that may be present in biological systems. In this work, the relevance of both the carbonate anion and nitrogen dioxide radicals in the oxidation of hypotaurine and cysteine sulfinic acid has been explored by the peroxidase activity of Cu,Zn superoxide dismutase (SOD) and by pulse radiolysis. The extent of sulfinate oxidation induced by the system SOD/H2O2 in the presence of bicarbonate (CO3(•-) generation), or nitrite ((•)NO2 generation) has been evaluated. Hypotaurine is efficiently oxidized by the carbonate radical anion generated by the peroxidase activity of Cu,Zn SOD. Pulse radiolysis studies have shown that the carbonate radical anion reacts with hypotaurine more rapidly (k = 1.1 × 10(9) M(-1)s(-1)) than nitrogen dioxide (k = 1.6 × 10(7) M(-1)s(-1)). Regarding cysteine sulfinic acid, it is less reactive with the carbonate radical anion (k = 5.5 × 10(7) M(-1)s(-1)) than hypotaurine. It has also been observed that the one-electron transfer oxidation of both sulfinates by the radicals is accompanied by the generation of transient sulfonyl radicals (RSO2(•)). Considering that the carbonate radical anion could be formed in vivo at high level from bicarbonate, this radical can be included in the oxidants capable of performing the last metabolic step of taurine biosynthesis. Moreover, the protective effect exerted by hypotaurine and cysteine sulfinate on the carbonate radical anion-mediated tyrosine dimerization indicates that both sulfinates have scavenging activity towards the carbonate radical anion. However, the formation of transient reactive intermediates during sulfinate oxidation by carbonate anion and nitrogen dioxide radical may at the same time promote oxidative reactions.

Psodisk, a new visual method for assessing the burden of psoriasis on patients.

BACKGROUND: The last decades have witnessed an increasing interest for the psychosocial aspects of chronic skin diseases, such as psoriasis. Nonetheless, systematic assessments of the impact of psoriasis on patients' lives are rarely done in daily clinical practice. The existing instruments are mostly meant to be completed by patients alone, and rarely comprise a graphical representation of the results. OBJECTIVE: To develop a questionnaire allowing both a quick assessment of the impact of psoriasis on patients and, at the same time, an intuitive graphic visualization of the outcome of the test. METHODS: A preliminary version of an Italian questionnaire aimed to assess the global impact of psoriasis on patients, meant to be filled in together by the patient and the dermatologist and to produce visual, intuitive results, was developed through focus groups. The instrument was then the object of a Delphi survey addressed to a panel of experts, to assess both the need of possible improvements of the questionnaire (in terms of the formulations of the questions and of the domains to be explored) and the usefulness of the questionnaire. RESULTS: A 10-item questionnaire in Italian, taking into account different aspects of the burden of psoriasis on the patient, was developed. The answers are given on a 10-point visual analogue scale and graphically represented on a disc as a polygon. CONCLUSIONS: A formal validation of the questionnaire and a study to assess potential clinical and psychological benefits of a systematic implementation of the instrument in daily practice are planned.

Combined Raman and IR spectroscopic study on the radical-based modifications of methionine.

Among damages reported to occur on proteins, radical-based changes of methionine (Met) residues are one of the most important convalent post-translational modifications. The combined application of Raman and infrared (IR) spectroscopies for the characterisation of the radical-induced modifications of Met is described here. Gamma-irradiation was used to simulate the endogenous formation of reactive species such as hydrogen atoms (•H), hydroxyl radicals (•OH) and hydrogen peroxide (H(2)O(2)). These spectroscopic techniques coupled to mass experiments are suitable tools in detecting almost all the main radical-induced degradation products of Met that depend on the nature of the reactive species. In particular, Raman spectroscopy is useful in revealing the radical-induced modifications in the sulphur-containing moiety, whereas the IR spectra allow decarboxylation and deamination processes to be detected, as well as the formation of other degradation products. Thus, some band patterns useful for building a library of spectra-structure correlation for radical-based degradation of Met were identified. In particular, the bands due to the formation of methionine sulfoxide, the main oxidation product of Met, have been identified. All together, these results combine to produce a set of spectroscopic markers of the main processes occurring as a consequence of radical stress exposure, which can be used in a spectroscopic protocol for providing a first assessment of Met modifications in more complex systems such as peptides and proteins, and monitoring their impact on protein structure.

Use of Raman spectroscopy for the identification of radical-mediated damages in human serum albumin.

Damages induced by free radicals on human serum albumin (HSA), the most prominent protein in plasma, were investigated by Raman spectroscopy. HSA underwent oxidative and reductive radical stress. Gamma-irradiation was used to simulate the endogenous formation of reactive radical species such as hydrogen atoms ((•)H), solvated electrons (e(aq)(-)) and hydroxyl radicals ((•)OH). Raman spectroscopy was shown to be a useful tool in identifying conformational changes of the protein structure and specific damages occurring at sensitive amino acid sites. In particular, the analysis of the S-S stretching region suggested the radical species caused modifications in the 17 disulphide bridges of HSA. The concomitant action of e(aq)(-) and (•)H atoms caused the formation of cyclic disulphide bridges, showing how cystine pairs act as efficient interceptors of reducing species, by direct scavenging and electron transfer reactions within the protein. This conclusion was further confirmed by the modifications visible in the Raman bands due to Phe and Tyr residues. As regards to protein folding, both oxidative and reductive radical stresses were able to cause a loss in α-helix content, although the latter remains the most abundant secondary structure component. ß-turns motifs significantly increased as a consequence of the synergic action of e(aq)(-) and (•)H atoms, whereas a larger increase in the ß-sheet content was found following the exposure to (•)OH and/or (•)H attack.

Raman and IR study on copper binding of histamine.

A comparative Raman and FTIR study of histamine (Hm), a small hormone present in a wide selection of living organisms, and its complexes with copper(II) at different pH values was carried out. Both the Raman and IR spectra present some marker bands useful for the identification of the structure of the species predominating in the Cu(II) aqueous and alcoholic systems. In particular, Raman spectroscopy appears to be a useful tool for analyzing the tautomeric equilibrium of the imidazole ring of Hm, because some bands (i.e., nuC(4)dbond;C(5)) appear at different wavenumbers, depending on whether the imidazole moiety is in the N(tau)-H (tautomer I) or N(pi)-H (tautomer II) protonated form. In aqueous solutions the manner in which Hm binds to Cu(II) depends on the pH. At basic pH the most relevant species formed are a dimer, [Cu(2)L(2)H(-2)](2+), and a monomeric complex, [CuL](2-) or [CuL(2)](+). On the contrary, by decreasing the pH, Hm acts as a mono- or bidentate ligand, giving rise to two types of monomeric complexes, [CuLH](2-) and [CuL](2-) or [CuL(2)](+). With respect to the Cu(II)-Hm alcoholic system, both the aminic group and the imidazole ring (tautomer I) take part in the Cu(II) coordination, leading to the formation of the [CuL](2-) or [CuL(2)](+) monomeric complex.

Frequency doubling perimetry in ocular hypertension and chronic open angle glaucoma.

The authors have studied the reliability of frequency doubling perimetry (FDP) in screening glaucomatous disease and in ocular hypertension. The study was made on 30 patients affected by chronic simple open-angle glaucoma (COAG) and 30 patients with ocular hypertension. MD and PSD variations were evaluated after a W/W perimetry and after FDP. In the COAG patients, FDP confirmed the perimetric alterations found by the W/W method. In the ocular hypertensive patients, with FDP we found a significant PSD increase over that found by the W/W technique: 4.051 +/- 0.822 against 2.501 +/- 0.928 (P < 0.001). FDP can therefore be put forward as a valid instrument for screening even precocious perimetric defects.

Characterization of dioxygenated cobalt(II)-carnosine complexes by Raman and IR spectroscopy.

Raman and IR studies are carried out on carnosine (beta-alanyl-L-histidine, Carnos) and its complexes with cobalt(II) at different metal/ligand ratios and basic pH. Binuclear complexes that bind molecular oxygen are formed and information regarding the O-O bridge is obtained from the Raman spectra. When the Co(II)/Carnos ratio is

Dryness symptoms, diagnostic protocol and therapeutic management: a report on 1,200 patients.

PURPOSE: To report the diagnostic and therapeutic data obtained from 1,200 patients suffering from dry eye symptoms not due to Sjögren's syndrome or other auto-immune diseases. METHODS: Schirmer test I, ferning test, breakup time, vital dye staining, brush and imprint cytology were performed; data were grouped into diagnostic profiles, and the therapy was prescribed according to these. RESULTS: Eight diagnostic profiles were identified. Dry eye was diagnosed in 57.1% of patients; the remaining 42.9% were found to suffer from eye discomfort or conjunctivitis of different aetiologies. CONCLUSIONS: Subjective symptoms of dryness can hide diseases other than dry eye; combined clinical and laboratory tests are requested to make a diagnosis. Our experience indicates that a therapy prescribed on the basis of diagnostic profiles provides relief in 79.1% of cases.

The biocompatibility of silicone, fluorosilicone and perfluorocarbon liquids as vitreous tamponades. an ultrastructural and immunohistochemical study.

PURPOSE: To investigate the interactions occurring at the interface between some currently used vitreous tamponading substances and the ocular tissues, in an early follow-up after surgery. METHODS: Adult albino rabbit eyes underwent vitrectomy and were injected intravitreally with silicone oils at 1,000 or 3,000 cSt, fluorosilicone oil and perfluorodecalin. Different morphological techniques (light microscopy, scanning and electron microscopy, immunohistochemistry) were applied. RESULTS: All the tested materials, although non-toxic, penetrate the ocular tissues also at the anterior segment level, and in the long-term follow-up this can yield functional impairment. No massive inflammation has been detected in the zones in contact with the materials, but immunoglobulins and complement fractions are anyway present in the stroma of various tissues and around the droplets of emulsified materials, suggesting a local immune reaction. CONCLUSIONS: Data from this study confirmed that a permanent vitreous substitute, showing a perfect biocompatibility, still does not exist. The indication to remove the tamponading substance within 2 months after the surgery emerges from this investigation, confirming previous studies. Despite some negative features, silicone oil still appears the most biocompatible material for vitreous replacement.

Influence of environment on piroxicam polymorphism: vibrational spectroscopic study.

FTIR and FT-Raman spectroscopies were used to evaluate the mechanism of transformation of piroxicam into its different forms (alpha, beta, and monohydrate), depending on the environment. These vibrational techniques allowed us to identify the forms of piroxicam that crystallize from different solvents at different cooling rates and the conformation of the drug in some of its derivatives: piroxicam hydrochloride, piroxicam thallium and sodium salt hemihydrates, and piroxicam sodium salt. The usefulness of Raman spectroscopy in characterizing piroxicam:beta-cyclodextrin (PbetaCD) inclusion compounds was described. The Raman spectrum of 1:2 PbetaCD was discussed in comparison with that of the corresponding piroxicam sodium salt containing inclusion compound (1:2 PNabetaCD) in order to study the influence of the piroxicam derivative used on the structure of the inclusion compound. The Raman results showed that in both of the inclusion compounds the piroxicam mainly assumes the zwitterionic structure typical of a monohydrate; therefore, the kind of derivative used does not affect the conformation of the drug in its inclusion compound. The effect of the method of synthesis utilized (freeze-drying or freeze-thaw cycling) to obtain 1:2.5 PbetaCD was investigated. The inclusion compound obtained by freeze-thaw cycling proved to be more crystalline and to contain a higher amount of the beta form than the freeze-dried inclusion compound. Raman spectroscopy proved to be a useful technique for evaluating the effectiveness of the manufacturing process in relation to the pharmaceutical properties of the drug and to the nondestructive and noninvasive on-line quality control of the industrial products.

Frequent replacement and conventional daily wear soft contact lens symptomatic patients: tear film and ocular surface changes.

We analyzed tear film changes, ocular surface tissue alterations and inflammatory conditions in Frequent Replacement Soft (FRS) Contact Lens (CL) or Conventional Soft (CS)-CL wearers suffering from eye discomfort, by applying complementary diagnostic tests. Our data suggest the following conclusions: i) eye discomfort symptoms begin after 1-3 years of successful wear, mainly in FRS-CL wearers; ii) tear film stability is affected by CL wear, mainly in FRS-CL wearers; iii) CL wear produces a subclinical moderate inflammation based on mononuclear cell recruitment in 3 out of 4 patients of both types of lenses; iv) CL wear induces dry eye conditions, more pronounced in FRS-CL users; v) discomfort symptoms, presence of subclinical inflammation, degree of dryness are not directly related to a clinical presentation of intolerance, mainly in FRS-CL wearers; vi) CL wear-related discomfort symptoms should never be underestimated, since a hidden inflammation or dryness can be present beyond an apparently good clinical picture.

Raman and IR spectroscopic investigation of zinc(II)-carnosine complexes.

The zinc(II)-L-carnosine system was investigated at different pH and metal/ligand ratios by Raman and IR spectroscopy. The Raman and IR spectra present some marker bands useful to identify the sites involved in metal chelation at a specific pH value. In particular, the neutral imidazole group gives rise to some Raman bands, such as the nu C(4)===C(5) band, that change in wave number, depending on whether the imidazole ring takes the tautomeric form I or II. Even if tautomer I is predominant in the free ligand, metal coordination can upset tautomeric preference and N(tau)- and N(pi)-ligated complexes can be identified. Although weak compared to those of aromatic residues, these Raman marker bands may be useful in analyzing metal-histidine interaction in peptides and proteins. On the basis of the vibrational results, conclusions can be drawn on the species existing in the system. Depending on the available nitrogen atoms, various complexes can be formed and the prevalent form of the species depends mainly on the pH. At basic pH carnosine gives rise to two different neutral complexes: a water-insoluble polymeric species, [ZnH(-1)L](0)(n), and a dimer, [Zn(2)H(-2)L(2)](0). The first is predominant and involves the tautomeric I form of the imidazole ring in metal chelation; the second contains tautomer II and increases its percentage by going from a 2 to 0.25 metal/ligand ratio. Conversely, the dimeric species dominates at pH 7, whereas two charged species, [ZnHL](2+) and [ZnL](+), are formed under slightly acidic conditions. In the [ZnHL](2+) complex the imidazole ring takes part in the Zn(II) coordination in the tautomeric I form, whereas in [ZnL](+) the ring is protonated and not bound to the Zn(II) ion. In addition, the curve fitting analysis of the 1700-1530 cm(-1) Raman region was helpful in indicating the predominant species at each pH.

Binding of copper(II) to carnosine: Raman and IR spectroscopic study.

A comparative Raman and FTIR study of carnosine, a dipeptide present in several mammalian tissues, and its complexes with copper(II) at different pH values was carried out. The neutral imidazole ring gives rise to some bands that appear at different wavenumbers, depending on whether the imidazole ring is in the tautomeric form II or I. At pH 7 and 9 the molecule exists in equilibrium between the two tautomeric forms; tautomer I is predominant. Metal coordination is a factor that affects the tautomeric equilibrium, and the copper(II) coordination site can be monitored by using some Raman marker bands such as the vC(4)=C(5) band. On the basis of the vibrational results, conclusions can be drawn on the functional groups involved in the Cu(II) chelation and on the species existing in the Cu(II)-carnosine system. At neutral and basic pH the most relevant species formed when the Cu(II)/carnosine molar ratio is not very different from unity is a dimer, [Cu(2)L(2)H(-2)](0). In this complex the ligand coordinates the metal via the N (amino), O (carboxylate), and N (amide) donor atoms while the N(tau) nitrogen atoms of the imidazole rings (tautomer II) bridge the copper(II) ions. At a slightly acidic pH the two monomeric complexes [CuLH](2+) and [CuL](+) were present. In the former the imidazole ring takes part in the Cu(II) coordination in the tautomeric I form whereas in the latter it is protonated and not bound to Cu(II).

Free radical scavenging and copper chelation: a potentially beneficial action of captopril.

Captopril (CpSH), an angiotensin converting enzyme (ACE) inhibitor, is reported to provide protection against free-radical mediated damage. The purpose of this study was to investigate, by means of pulse radiolysis technique, the behaviour of CpSH towards radiation-induced radicals in the absence and in the presence of copper(II) ions, which can play a relevant role in the metal catalysed generation of reactive oxygen species. The results indicate that the -SH group is crucial in determining the radical scavenging action of CpSH and the nature of the resulting CpSH transient products in the absence or in the presence of oxygen. In the presence of Cu(II), the -SH group is still involved in the biological action of the molecule participating both in the one-electron reduction of Cu(II) with formation of CpSSCp, and in Cu(I) chelation. This conclusion is supported by the Raman spectroscopic data which allow to identify the CpSH sites involved in the copper complex at different pH. These results suggest that CpSH may potentially inhibit oxidative damage both through free radical scavenging and metal chelation. Considering the low CpSH concentration in vivo, the metal chelation mechanism, more than the direct radical scavenging, could play the major role in moderating the toxicological effects of free radicals.

Hydroxyl radical scavenging by carnosine and Cu(II)-carnosine complexes: a pulse-radiolysis and spectroscopic study.

PURPOSE: To obtain a wider insight into the general properties of carnosine and to provide support to its anti-oxidative role. This property is hypothesized to be linked to various mechanisms including free-radical scavenging and metal chelation (i.e. Cu(II)). METHODS: Pulse-radiolysis experiments were performed by a 12 MeV electron linear accelerator (LINAC) on carnosine/copper(II) (2:1) and carnosine aqueous solutions at different pH. Raman spectra of solid samples were obtained by a Bruker IFS 66 spectrometer. RESULTS: As well as for free carnosine, in the presence of copper ions the interaction of carnosine with *OH radicals involves the imidazole group of the molecule. The oxidation of copper (II)-carnosine system by *OH radicals is related to the pH-dependent structure of the copper(II)-carnosine complex. Raman spectra indicate that at alkaline pH the formation of a dimeric species containing two carnosine molecules complexed to two Cu2+ ions takes place. This structure can address the *OH attack more selectively than carnosine itself to different sites of the imidazole ring. The formation of at least two different *OH-radical adducts occurs and positions C(2) and C(5) of the imidazole ring are the preferential sites for the *OH attack, as the heterocyclic ring is mainly present as its N(1)-protonated tautomeric form. CONCLUSION: These studies provide further evidence about the formation of carnosine-copper complexes and the predominance of a dimeric structure at slightly basic pH. The chelation of Cu(II) is not detrimental to the scavenging ability of carnosine. Raman spectra are helpful in identifying the structure of the copper(II)-carnosine complexes and in predicting the preferential sites for the *OH attack to the carnosine-copper system.

Adhesion mechanisms of human lens epithelial cells on 4 intraocular lens materials.

PURPOSE: To evaluate lens epithelial cell (LEC) adhesion on different intraocular lens (IOL) materials with particular attention to the distribution of proteins located in the focal contacts. SETTING: Center of Biotechnological and Clinical Research in Ophthalmology, University of Bologna, Italy. METHODS: The IOL materials tested were poly(methyl methacrylate) (PMMA), heparin-surface-modified PMMA (HSM PMMA), polyHEMA, and silicone. Primary cultures of human LECs were established from human anterior capsules obtained during cataract surgery. The mean number of cells attached per square millimeter was calculated for each material after 24 and 72 hours. Transmission electron microscopy and immunocytochemical analysis were performed to detect the proteins actin, vinculin, and talin. RESULTS: Mean adhesiveness of human LECs increased over time with PMMA and decreased with the other materials. At 72 hours, mean LECs ranged from 54.8 cells/mm2 +/- 12.8 (SD) on PMMA to 2.1 +/- 0.7 cells/mm2 on silicone. The means for HSM PMMA and polyHEMA fell in between. The cytoskeletal proteins were arranged to produce focal contacts in only the LECs cultured on PMMA. The LECs cultured on polyHEMA, HSM PMMA, and silicone attached but failed to develop focal contacts or stress fibers. CONCLUSION: This study confirms the multifactorial pathogenesis of posterior capsule opacification and suggests its incidence will be reduced by improving surgical techniques and using IOL surfaces that discourage cell adhesion.

Eye discomfort and air pollution.

Discomfort eye syndrome (DES) comprises a series of 'minor' subjective symptoms in patients where no relevant clinical signs are observed suggesting ocular disease. Our study includes 100 DES patients, excluding video terminal users, selected from the First Aid Service of our Department over two peak periods in both winter and summer time. The Schirmer test I, ferning test, breakup time and conjunctival cytology (scraping and imprint) were performed and data were related to sex, age and air pollution indexes, recorded in the patients' living zones. Our results demonstrate that: (i) the ocular surface cytology and the analysis of tear film changes provide significant information in those patients where no other clinical signs are evident; (ii) DES symptoms are more frequent in women than in men (ratio about 2:1), both with ages over 51 years; (iii) DES is significantly associated with ocular surface inflammation, as detected by cytological methods, and (iv) ocular surface subclinical inflammation and ocular dryness are related to high concentrations of atmospheric polluters, in both sexes.

A pulse radiolysis study of carnosine in aqueous solution.

PURPOSE: To investigate the potential antioxidative role of carnosine (beta-alanyl-L-histidine) through its interaction with radiation induced radicals. MATERIALS AND METHODS: Pulse radiolysis experiments were performed by a 12 MeV electron linear accelerator (LINAC) on carnosine aqueous solutions at different pHs. The Raman spectra of solid samples were obtained by a Bruker IFS 66 spectrometer. RESULTS: The protonation state of the imidazole ring was observed to affect the site(s) of .OH attack on carnosine as well as the decay rates of the resulting adducts. Reasonably, a base catalysed water elimination from the adducts leading to a resonance-stabilized radical, which absorbs at lambda = 270 nm, takes place. Raman spectra in slightly alkaline medium have indicated that carnosine is mainly present as tautomer I and, of consequence, positions C(2) and C(4) of the imidazole ring are the preferential sites for .OH attack. CONCLUSIONS: These studies have shown that carnosine is a good scavenger of .OH radicals giving rise to a quite stable intermediate which should be less reactive towards other biological components than .OH itself. Raman data have been helpful in predicting the preferential sites for the .OH attack.

The binding of biotin analogues by streptavidin: a Raman spectroscopic study.

Raman spectra of anhydrous complexes of streptavidin (Strep) with biotin (Bio) and some Bio analogues [Biotin methyl ester (MEBio), desthiobiotin (DEBio), 2'-iminobiotin (IMBio), and diaminobiotin (DABio)] were recorded. The vibrational results indicate that the interaction with some of these ligands is able to modify the overall structure of the protein and this binding results in a decrease in the beta-sheet content and an increase in the alpha-helix content. To further confirm the conformational changes of the protein structure due to Bio analogue binding, the curve-fitting analysis of the amide I Raman band of neat Strep and of the complexes were performed. The intensity ratio of the components due to the beta-sheet and alpha-helix conformations decreased in the Strep-MEBio, Strep-IMBio (pH 11), and Strep-Bio systems, whereas in all the other systems the changes were not significant. This behavior differs from that of Avi bound to the same ligands and suggests that Strep and Avi differ in their binding selectivity. A good correlation was found between the secondary structure percentages of the Avi and of the Strep complexes and deltaG(o). On the basis of this linear relationship, the vibrational results allow for an acceptable evaluation of the dissociation constants of the Strep complexes, not previously reported in the literature. The present results indicate a correlation between the type of interaction and the effects of the protein-substrate bonding on the overall structure of the proteins. The amino acid residues in the binding site appear to be positioned in a such a way as to provide a precise fit of Bio. Even slight change in the substrate structure causes a weakness in the strength of the binding. The vibrational results confirm that both the imidazolidinone and the thiophan rings are important in the Strep-Bio interactions, but the former is more responsible for the high affinity of the binding. One of the Tyr residues is hydrogen bound with the ureido ring and another Tyr could be involved in the binding pocket. Trp residues do not directly bind the ligand and probably stabilize other binding site residues which in turn interact directly with Bio.