Compositional analysis of endogenous porphyrins from Helicobacter pylori.

Bacteria able to accumulate porphyrins can be inactivated by visible light irradiation thanks to the photosensitizing properties of this class of aromatic pigments (photodynamic therapy, PDT). Since the bacterial resistance to antibiotic is growing, PDT is becoming a valid alternative. In this context, the pathogen Helicobacter pylori (Hp) is a suitable target for PDT since it spontaneously produces and accumulates porphyrins. It is then important to understand the spectroscopic behavior of these endogenous species to exploit them as photosensitizers, thus improving the results given by the application of PDT in the treatment of Hp infections. In this work we extracted porphyrins from both a laboratory-adapted and a virulent strain of Hp, and we performed spectroscopic and chromatographic experiments to collect information about the composition and the spectrophotometric features of the extracts. The main components of the porphyrin mixtures were identified and their relative contribution to the global red fluorescence was examined.

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.

[Set-up, portals and normal exploration in wrist arthroscopy.] / Installation, voies d'abord et exploration normale en arthroscopie du poignet.

Arthroscopy is an accepted technique for evaluation of intra-articular pathology and treatment of a variety of disorders even in the wrist joint. Dedicated miniaturized instrumentation is needed along with a specific traction system. The external distraction alone (dry technique) allows for complete joint exploration and several type of arthroscopic surgery, avoiding annoying leaking in the subcutaneous tissues, though further distension of the articular pouches can be achieved by saline infusion (fluid distension or wet technique). Knowledge of surface anatomic landmarks and careful surgical technique are required for proper portal placement and in order to avoid injury to the numerous noble structures crossing nearby. Description of radio- and medio-carpal portals is provided along with the different bony, condral, synovial and ligamentous structures that can be visualised or treated through each portal. Surgeon can choose the most suitable portal for scope or instruments, according to specific needs for diagnostic or therapeutic purposes.

[Set-up, portals and normal exploration in wrist arthroscopy]. / Installation, voies d'abord et exploration normale en arthroscopie du poignet.

Arthroscopy is an accepted technique for evaluation of intra-articular pathology and treatment of a variety of disorders even in the wrist joint. Dedicated miniaturized instrumentation is needed along with a specific traction system. The external distraction alone (dry technique) allows for complete joint exploration and several type of arthroscopic surgery, avoiding annoying leaking in the subcutaneous tissues, though further distension of the articular pouches can be achieved by saline infusion (fluid distension or wet technique). Knowledge of surface anatomic landmarks and careful surgical technique are required for proper portal placement and in order to avoid injury to the numerous noble structures crossing nearby. Description of radio- and medio-carpal portals is provided along with the different bony, condral, synovial and ligamentous structures that can be visualised or treated through each portal. Surgeon can choose the most suitable portal for scope or instruments, according to specific needs for diagnostic or therapeutic purposes.

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.

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.

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.