Steady-state and femtosecond photoinduced processes of blepharismins bound to alpha-crystallin.

The interaction of blepharismin (BP) and oxyblepharismin (OxyBP) with bovine alpha-crystallin (BAC) has been studied both by steady-state and femtosecond spectroscopy, with the aim of assessing the possible phototoxicity of these compounds toward the eye tissues. We showed that these pigments form with BAC potentially harmful ground-state complexes, the dissociation constants of which have been estimated to be 6 +/- 2 micromol L(-1) for OxyBP and 9 +/- 4 micromol L(-1) for BP. Irradiation with steady-state visible light of solutions of blepharismins in the presence of BAC proved to induce a quenching of both the pigment and the intrinsic protein fluorescences. These effects were tentatively rationalized in terms of structural changes of alpha-crystallin. On the other hand, femtosecond transient absorption spectroscopy was used to check the occurrence of any type I photoactivity of oxyblepharismin bound to alpha-crystallin. The existence of a particular type of fast photoinduced reaction, not observed in former studies with human serum albumin but present in the natural oxyblepharismin-binding protein, could here be evidenced but no specific reaction was observed during the first few nanoseconds after excitation. Partial denaturation of alpha-crystallin was however found to alter the excited-state behaviour of its complex with oxyblepharismin, making it partly resemble that of free oxyblepharismin in solution.

Spectroscopic study of visible-light effects on hypericin-lens proteins systems.

Molecular interactions between hypericin and alpha-, beta- and gamma-crystallin proteins have been studied by means of absorption and steady-state fluorescence spectroscopy, aiming to clarify if and how the pigment binds to the proteins and to investigate the effects of visible-light irradiation on these molecular systems. Such a study is a prerequisite for assessing the possibility of using hypericin as a mild antidepressant and/or as a photodynamic agent for the treatment of eye tumors and eye viral and bacterial diseases without side injuries to the lens. We have shown that in dark-kept samples, with increasing alpha-crystallin concentration, both the fluorescence emission intensity and the ratio of the absorption maxima around 590 and 550 nm of hypericin increase. These effects have been attributed to the monomerization of nonfluorescent hypericin aggregates caused by the binding of the pigment to alpha-crystallin. The binding constant of hypericin has been evaluated to be of the order of 3.0 (mg/mL)-1, corresponding to a dissociation constant of the order of 0.3 mg/mL. Following irradiation with light of wavelengths over 400 nm, at an irradiance of 20 mW/cm2, both tryptophan and hypericin fluorescence emission intensities decrease. These effects are suggested to be the consequence of a spatial rearrangement of the protein framework which takes place following the alpha-crystallin photopolymerization sensitized by hypericin itself described in the literature. For the sake of comparison hypericin has been studied also in the presence of beta H-, beta L- and gamma-crystallins at the same concentration.

Photoresponsive polypeptides.

Polypeptides containing azobenzene or spiropyran units attached to the macromolecules respond to light or dark conditions giving reversible variations of their structure. In this Account we provide a short overview of current research in the field and describe the most significant experimental examples of photoresponse effects. They include photoinduced random coil/alpha-helix transitions, helix-sense reversal, photostimulated aggregation/disaggregation processes, and photomechanical effects. These fascinating properties suggest that photoresponsive polypeptides may become suitable materials for designing sensors and devices that can be photomodulated. Findings also demonstrate that it is possible to synthesize model systems which respond to light similarly to naturally occurring photoreceptors.

The uptake, location and fluorescence of hypericin in bovine intact lens.

PURPOSE: To determine the uptake, location and fluorescence of hypericin, the active ingredient in St. John's Wort, in situ in the isolated intact calf lens. METHODS: The absorption and fluorescence spectra of hypericin 10(-5 ) M were measured in DMSO/phosphate buffer, pH 7.4) [PBS] (1/10 in volume) in the presence of alpha-crystallin (0.5 and 1.1 mg/ml). Bovine lenses were incubated in the dark for 24 hours in 10(-4) M hypericin in a DMSO/PBS (1/10 in volume) mixture. Diffused hypericin fluorescence emission was detected with a fluorescence stereomicroscope from the PBS washed lens surface. A lens-holder specially built for front-surface excitation-detection was used to measure fluorescence emission and excitation spectra of intact lenses incubated with hypericin solutions. RESULTS: As increasing concentrations of alpha-crystallin were added, the absorption and fluorescence spectra of hypericin in DMSO/PBS (1/10 in volume) changed, indicating a binding between the chromophore and the lens protein. Fluorescence emission spectra detected from the lens surface (lambda( em) = 601 and 651 nm; lambda(exc) = 550 nm) confirmed that hypericin does bind to the ocular tissues. CONCLUSIONS: The results we obtained in simplified model systems can provide clues to investigate the effects of hypericin on lens properties in physiological conditions. Hypericin could in fact bind to lens protein thus increasing the retention time of hypericin in the eye and possibly altering a-crystallin properties as a chaperone. Should therefore hypericin be taken up by the lens, this can be detected, non-invasively by its fluorescence. Therefore, ophthalmologists may use a slit-lamp or scanning fluorometry to monitor the uptake of hypericin in the eyes of patients using St. John's Wort or receiving high doses of hypericin while undergoing photodynamic therapy.

Photochromic polypeptides as synthetic models of biological photoreceptors: a spectroscopic study.

L-Glutamic acid polypeptides containing photochromic nitrospiropyran bound to the side chains at various percentages ("local" concentration) have been synthesized and investigated as possible artificial models of biological photoreceptors. Absorption and fluorescence spectroscopy have been utilized to investigate the photophysical and photochemical properties of nitrospiropyrans, both inserted in the polypeptide chain and in solution as "free" dye. Conformational variations produced by dark storage and light exposure of the photochromic polypeptides have been studied by means of circular dichroism. Dark-kept "free" dyes in hexafluoro-2-propanol solution in the merocyanine form ("open" form) give rise to molecular aggregates, which have been characterized as merocyanine dimers. The equilibrium constant between the monomer and the dimer, K, and their molar extinction coefficients, epsilon, at several wavelengths have been determined. Fluorescence measurements on "free" and polypeptide-bound nitrospiropyrans suggest that the dimerization process between merocyanines is favored when the photochromic units are inserted in the polypeptide chain and that under these conditions an efficient energy transfer from the monomer (donor) to the dimer (acceptor) occurs. By varying "local" as well as total nitrospiropyran concentration, it has been shown that the dimeric species result from intermolecular interactions between photochromic groups inserted in the same polypeptide chain. The alpha-helix --> random coil transition of the polypeptide structure after dark storage has eventually been shown to be the result of the dimerization process and not of the dark isomerization per se from the "closed" spiropyran form to the "open" merocyanine form of the dye.

Sensory perception and transduction of UV-B radiation by the ciliate Blepharisma japonicum.

A key question to answer studying the biological effects of ultraviolet radiation on planktonic micro-organisms is whether they can perceive UV-B radiation as a sensory signal, likewise they do with visible light. We have faced this problem performing an individual-cell analysis of Blepharisma japonicum photomotile responses to UV-B stimuli. Our results on spectral responsiveness and on the effects of a photoresponse inhibitor indicate that B. japonicum is capable to perceive and transduce UV-B radiation as an environmental sensory stimulus, which it escapes from gathering in shadowed areas. Similar UV-B avoidance motile reactions could serve as a behavioural defence mechanism contributing to avoid harmful overexposure to UV-B.

Spectroscopic and photoacoustic studies of hypericin embedded in liposomes as a photoreceptor model.

In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin-type chromophores packed in highly osmiophilic subpellicular granules. Lipopsomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light-induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady-state and time-resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L-alpha-phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.

Photosensory transduction in ciliates. Role of intracellular pH and comparison between Stentor coeruleus and Blepharisma japonicum.

To test the hypothesis that light signal transduction in the unicellular ciliates Stentor coeruleus and Blepharisma japonicum involves a change in intracellular pH as an initial signal following photoexcitation, we studied the dependence of the photophobic responses of the cells to changes in extracellular pH and to reagents that specifically affect intracellular pH. The extracellular pH can modify not only the intracellular pH, but can even reverse the sign of the pH gradient across the cell membrane. Thus, as predicted by the hypothesis, low extracellular pH reversibly inhibited the photophobic response of the ciliates. The intracellular pH-modulating reagents tested included ammonium chloride, a membrane-permeable weak acid that lowers the intracellular pH, and the protonophores carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and carbonylcyanide p-(trifluoromethoxy)-phenyl-hydrazone (FCCP), which collapse the pH gradient across the cell membrane. The low pH and protonophore treatments caused a gradual inhibition of the photophobic responses in both ciliates. The observed reduction of the responsiveness of the cells to visible light can be attributed to the alteration of the intracellular pH, which is suggested to play a specific role in the photosensory transduction in both Stentor coeruleus and Blepharisma japonicum.

[Guided regeneration of the periodontal tissues with resorbable and nonresorbale membranes. I. Clinical evaluation]. / Rigenerazione guidata dei tessuti parodontali con membrane riassorbibili e non. Nota I. Valutazioni cliniche.

The present experimental study has been undertaken in order to analyse clinically, in humans, the regeneration of periodontal tissue following two different surgical techniques of guided tissue regeneration therapy using a resorbable and a nonresorbable membrane. Clinical results have demonstrated no statistically significant difference between the techniques employed, both of the showing a favourable outcome if perfect plaque control in the postoperative period is performed.

[Guided regeneration of the periodontal tissues with resorbable and nonresorbable membranes. II. Microbiological evaluation]. / Rigenerazione guidata dei tessuti parodontali con membrane riassorbibili e non. Nota II: Valutazioni microbiologiche.

The present experimental study has been undertaken in order to analyze the influence of bacterial recolonization on periodontal wound healing following guided tissue regeneration therapy in humans. Two different surgical techniques has been carried out: one using a non absorbable membrane and the other a absorbable one. Microbiological data indicate no statistically significant difference between the techniques employed. Furthermore our data have confirmed the necessity of maintaining the healing sites free from the main periodontal pathogens.

[Bacterial recolonization in the guided regeneration of the inter-radicular periodontal tissues]. / La ricolonizzazione batterica nella rigenerazione guidata dei tessuti parodontali interradicolari.

The present experimental study has been undertaken in order to analyse clinically and microbiologically the formation of new periodontal attachment in class II furcation lesions in mandibular molars, comparing healing after surgical conventional therapy and after guided periodontal tissue regeneration technique using non-resorbable membranes and evaluating at the same time, with cultural anaerobic technique, the influence on healing of the bacterial recolonization of some periodontally pathogenic species. Clinical results have demonstrated that, after therapy, may occur a certain discrepancy between soft and hard tissue regeneration and that the bone tissue shows a different pattern of apposition in the horizontal and vertical direction. Microbiological data have underlined the significance of surgical instrumentation in the guided inter-radicular periodontal tissue regeneration and have confirmed the necessity of maintaining the healing sites free from the main periodontal pathogens.

[Current trends in therapy of periodontal disease]. / Attuali orientamenti della terapia della malattia parodontale.

A comprehensive literature review on biological principles guiding present research lines of Periodontal Therapy has been carried out, focusing mainly on the repair potential of the periodontal tissues.

Photoreceptor pigments for photomovement of microorganisms: some spectroscopic and related studies.

Optical spectroscopy of photoreceptor pigments can substantially contribute to our understanding of the molecular processes which are the basis of photoreception and sensory transduction in photomotile microorganisms. The main spectroscopic techniques are briefly illustrated, together with the most significant types of progress that can be achieved. A few "case examples" are discussed in some detail: Halobacterium, with particular attention to the contribution of flash photolysis studies to the identification and characterization of sensory rhodopsins; Euglena, and the role of in vivo microspectrofluorometry in confirming the flavin nature of its photoreceptor pigment; the first suggestions on the rhodopsin-like nature of the Chlamydomonas photosensing system; Stentor and Blepharisma and the contribution of static and time-resolved fluorescence studies to a molecular model of the primary events in their photoreceptor pigments (stentorin and blepharismin) and systems.

[Periodontal osseous surgery: osteoplasty and osteotomy]. / Chirurgia ossea parodontale: osteoplastica e osteotomia.

Elimination of the periodontal pocket is a prime objective of periodontal therapy. Normally the gingiva is consistent with the underlying bone, and aberration in gingival form can be corrected by gingivectomy or gingivoplasty if the gingiva is supported by good bony architecture. If periodontitis has caused marginal irregularities or septal deformities the complete elimination of the periodontal pocket is done by osteoplasty or osteoectomy. The Authors through a review of the literature have examined these surgical techniques.