The carbonate radical: its reactivity with oxygen, ammonia, amino acids, and melanins.

The carbonate radical (CO 3 (*-)) is of importance in biology and chemistry. We used pulse radiolysis to generate the CO 3 (*-) radical and show there is no reaction with oxygen. However, in the presence of ammonia the CO 3 (*-) radical is removed by NO (*), which itself arises from the scavenging of NH 2 (*) by oxygen, and the mechanism of this process is reported. The CO 3 (*-) radical shows complex decay patterns in the presence of ammonia, which can be understood as a balance between the radical-radical reaction CO 3 (*-) + CO 3 (*-) and CO 3 (*-) + NH 2 (*) (the amino radical). Also, we report reactivity with glycine and alanine and with melanin models. The CO 3 (*-) reacts with both dopa-melanin (DM, a model of black eumelanin) and with cysteinyl-dopa-melanin (CDM, a model of red/blond phaeomelanin). However, the reaction rate constant is much higher with CDM than with DM.

Site-specific interactions of copper(II) ions with heparin revealed with complementary (SRCD, NMR, FTIR and EPR) spectroscopic techniques.

The interactions between Cu(II) ions and heparin were investigated using several complementary spectroscopic techniques. NMR indicated an initial binding phase involving specific coordination to four points in the structure that recur in slightly different environments throughout the heparin chain; the carboxylic acid group and the ring oxygen of iduronate-2-O-sulfate, the glycosidic oxygen between this residue and the adjacent (towards the reducing end) glucosamine and the 6-O-sulfate group. In contrast, the later binding phase showed little structural specificity. One- and two-dimensional correlated FTIR revealed that complex out of phase (asynchronous) conformational changes also occurred during the titration of Cu(II) ions into heparin, involving the CO and N-H stretches. EPR demonstrated that the environments of the Cu(II) ions in the initial binding phase were tetragonal (with slightly varied geometry), while the later non-specific phases exhibited conventional coordination. Visible spectroscopy confirmed a shift of the absorbance maximum. Titration of Cu(II) ions into a solution of heparin indicated (both by analysis of FTIR and EPR spectra) that the initial binding phase was complete by 15-20 Cu(II) ions per chain; thereafter the ions bound in the non-specific mode. Hetero-correlation spectroscopy (FTIR-CD) improved resolution and assisted assignment of the broad CD features from the FTIR spectra and indicated both in-phase and more complex out of phase (synchronous and asynchronous, respectively) changes in interactions within the heparin molecule during the titration of Cu(II) ions.

Evaluation of an independent prestin mouse model derived from the 129S1 strain.

Studies using the prestin knockout mouse indicate that removal of the outer hair cell (OHC) motor protein is associated with loss of sensitivity, frequency selectivity and somatic electromotility. Here we provide data obtained from another prestin mouse model that was produced commercially. In vivo electrical recordings from the round window indicate that the phenotype is similar to that of the original knockout generated by the Zuo group at St. Jude Children's Research Hospital. Hence, compound action potential (CAP) thresholds are shifted in a frequency-dependent manner and CAP tuning curves at 12 kHz are flat for masker frequencies between 3 and 18 kHz. Although CAP input-output functions at 6 kHz show a shift in sensitivity at low levels, responses approach wild-type magnitudes at high levels where the cochlear amplifier has less influence. In order to confirm that the loss of sensitivity and frequency selectivity is due to loss of prestin, we performed immunohistochemistry using a prestin antibody. Cochlear segments from homozygous mutant mice showed no fluorescence, while wild-type mice displayed a fluorescent signal targeted to the OHC's lateral membrane. Absence of prestin protein was confirmed using LDS-PAGE/Western blot analysis. These results indicate that the loss of function phenotype is associated with loss of prestin protein. Lack of prestin protein also results in a shortening of OHC length to approximately 60% of wild-type, similar to that reported previously by Liberman's group. The linkage shown between the loss of prestin protein and abnormal cochlear function validates the original knockout and attests to the importance of OHC motor function in the auditory periphery.

An Electron Paramagnetic Resonance (EPR) spectroscopy study on the γ-irradiation sterilization of the pharmaceutical excipient l-histidine: Regeneration of the radicals in solution.

The effects of γ-radiation sterilization on the parenteral excipient l-histidine were analysed by means of EPR spectroscopy. The irradiation process was found to induce the formation of a deamination radical which was persistent in the solid state. The nature and reactivity of the radicals following dissolution in water was evaluated using spin-trapping EPR experiments. The deamination radical was found to regenerate in solution in the presence of trace metals, potentially leading to radical induced degradation reactions occurring up to an hour after the dissolution process. Understanding this process is significant for the improved design of parental pharmaceutical formulations in which unwanted radical reactions after γ-radiation sterilization could lead to degradation of active ingredients.

Triplet-state and singlet oxygen formation in fluorene-based alternating copolymers.

Data are reported on the triplet states of a series of fluorene-based A-alt-B type alternating copolymers based on pulse radiolysis-energy transfer and flash photolysis experiments. From the pulse radiolysis experiments, spectra are given for eight copolymers involving phenylene, thiophene, benzothiadiazole, and oligothienylenevinylene groups. Quantum yields for triplet-state formation (PhiT) have been obtained by flash photolysis following laser excitation and in one case by photoacoustic calorimetry. In addition, yields of sensitized formation of singlet oxygen have been determined by time-resolved phosphorescence and are, in general, in excellent agreement with the PhiT values. In all cases, the presence of thiophene units is seen to increase intersystem-crossing quantum yields, probably because of the presence of the heavy sulfur atom. However, with the poly[2,7-(9,9-bis(2'-ethylhexyl)fluorene)-alt-1,4-phenylene] (PFP), thiophene S,S-dioxide (PFTSO2) and benzothiadiazole (F8BT) copolymers, low yields of triplet formation are observed. With three of the copolymers, the energies of the triplet states have been determined. With PFP, the triplet energy is virtually identical to that of poly[2,7-(9,9-bis(2'-ethylhexyl)fluorene)]. In contrast, with fluorene-thiophene copolymers PFaT and PF3T, the triplet energies are closer to those of thiophene oligomers, indicating that there is significant conjugation between fluorene and thiophene units but also that there is a more localized triplet state than with the homopolymers.

Beta-carotene with vitamins E and C offers synergistic cell protection against NOx.

The peroxynitrite anion and the nitrogen dioxide (radical) are important toxic species which can arise in vivo from nitric oxide. Both in vivo and in vitro cell protection is demonstrated for beta-carotene in the presence of vitamin E and vitamin C. A synergistic protection is observed compared to the individual anti-oxidants and this is explained in terms of an electron transfer reaction in which the beta-carotene radical is repaired by vitamin C.

One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments.

The one-electron reduction potentials of the radical cations of five dietary carotenoids (beta-carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980-1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.

The reduction potential of the beta-carotene.+ /beta-carotene couple in an aqueous micro-heterogeneous environment.

There is a resurgence of interest in the role of electron transfer reactions involving beta-carotene in photosynthesis. There is also current debate on the health benefits of dietary carotenoids and the possible deleterious effects on certain sub-populations such as smokers. The impact of dietary carotenoids on health may well be also related to radical reactions. A key parameter in biological systems is therefore the one-electron reduction potential of the carotenoid radical cation, now reported for the first time in a model biological aqueous environment. The value obtained is 1.06+/-0. 01 V and is sufficiently high to oxidise cell membrane proteins, but is low enough to repair P(680).+ in the photosynthetic reaction centre.

Development of the gerbil inner ear observed in the hemicochlea.

A frequency-dependent change in hearing sensitivity occurs during maturation in the basal gerbil cochlea. This change takes place during the first week after the onset of hearing. It has been argued that the mass of a given cochlear segment decreases during development and thus increases the best frequency. Changes in mass during cochlear maturation have been estimated previously by measuring the changes in cochlear dimensions. Fixed, dehydrated, embedded, or sputter-coated tissues were used in such work. However, dehydration of the tissue, a part of most histological techniques, results in severe distortion of some aspects of cochlear morphology. The present experiments, using a novel preparation, the hemicochlea, show that hydrated structures, such as the tectorial membrane and the basilar membrane hyaline matrix, are up to 100% larger than estimated previous studies. Therefore, the hemicochlea was used to study the development of cochlear morphology in the gerbil between the day of birth and postnatal day 19. We used no protocols that would have resulted in severe distortion of cochlear elements. Consequently, a detailed study of cochlear morphology yields several measures that differ from previously published data. Our experiments confirm growth patterns of the cochlea that include a period of remarkably rapid change between postnatal day 6 and 8. The accelerated growth starts in the middle of the cochlea and progresses toward the base and the apex. In particular, the increase in height of Deiters' cells dominated the change, "pushing" the tectorial membrane toward scala vestibuli. This resulted in a shape change of the tectorial membrane and the organ of Corti. The tectorial membrane was properly extended above the outer hair cells by postnatal day 12. This time coincides with the onset of hearing. The basilar membrane hyaline matrix increased in thickness, whereas the multilayered tympanic cover layer cells decreased to a single band of cells by postnatal day 19. Before and after the period of rapid growth, the observed gross morphological changes are rather small. It is unlikely that dimensional changes of cochlear structures between postnatal days 12 and 19 contribute significantly in the remapping of the frequency-place code in the base of the cochlea. Instead, structural changes affecting the stiffness of the cochlear partition might be responsible for the shift in best frequency.

Scleral perforation during retrobulbar and peribulbar anesthesia: risk factors and outcome in 50,000 consecutive injections.

PURPOSE: To measure the frequency of scleral perforation and identify related risk factors during local anesthetic injection for intraocular surgery. SETTING: Multispecialty eye hospital. METHODS: All patients (n = 50,000) having retrobulbar (26,857) or peribulbar (23,143) injections at the King Khaled Eye Specialist Hospital were reviewed. Cases of scleral perforation were analyzed for potential technical and ophthalmic risk factors, management of injuries, and visual and anatomic outcomes. Mean follow-up was 14.4 months (range 8 to 24 months). RESULTS: Seven (0.014%) needlestick injuries were identified, all of which had posterior staphyloma as the only identifiable risk factor. Applying a previously measured prevalence of 10.7% for posterior staphyloma in our surgery patients gave a scleral perforation rate of 0.13% (7 of 5350) for staphylomatous eyes. All perforated globes had originally planned cataract extraction within 8 weeks of injury. Additional management consisted of observation (2 cases), cryotherapy (2 cases), and vitreoretinal procedures for retinal detachment (3 cases) and subretinal hemorrhage (1 case). At last follow-up, all retinas were attached and 3 cases (42.8%) had a visual acuity of worse than 20/160. Both cases requiring multiple retinal detachment surgeries developed proliferative vitreoretinopathy and poor visual acuity. CONCLUSIONS: Eyes with posterior staphyloma sustained needlestick injuries at a rate of 1 in 760 compared with 0 injection perforations in more than 44,000 nonstaphylomatous eyes.

Axial length and posterior staphyloma in Saudi Arabian cataract patients.

PURPOSE: To determine the axial length distribution and prevalence of posterior staphyloma in a group of Saudi Arabian patients having cataract extraction. SETTING: Tertiary care referral center. METHODS: Approximately 75% of all cataract patients at the eye hospital have preoperative B-scan ultrasonography. The last 1000 B-scans performed for any reason were screened for patients having uncomplicated cataract surgery; 629 cases were included. The relationship between the presence of posterior staphyloma and patient age, sex, and axial length was studied. RESULTS: There were 371 (59.0%) men and 258 (41.0%) women with a mean age of 62.4 years +/- 15.7 (SD). Posterior staphyloma was identified in 67 patients (10.7%). The presence of staphyloma was not significantly related to patient sex or age. Although the mean axial length was significantly longer in eyes with staphyloma (27.43 +/- 2.36 mm) than in those without (23.18 +/- 1.64 mm), 9.3% of eyes without staphyloma had an axial length longer than 25.0 mm. No staphylomas were present in eyes with an axial length shorter than 23.3 mm. The longest axial length in an eye without a staphyloma was 32.5 mm. CONCLUSIONS: Posterior staphyloma was present in a high percentage of Saudi Arabian patients having cataract extraction.

Outcome of cataract surgery associated with posterior staphyloma.

PURPOSE: To assess the outcome of cataract surgery in eyes with posterior staphyloma and identify predictors of surgical success. SETTING: Tertiary care referral center. METHODS: A retrospective analysis was conducted of 107 consecutive patients with posterior staphyloma and 107 control patients who had cataract surgery. Potential associations of preoperative variables with surgical success were analyzed using the chi-square test, multivariate linear regression, and Student t test. RESULTS: The staphyloma group was younger, had more women, and had a lower mean visual acuity preoperatively than the control group. All patients in the control group and all but 1 in the staphyloma group had intraocular lens (IOL) implantation at the time of cataract extraction. The rates of posterior capsule tear and vitreous loss were similar in the staphyloma and control groups. There was 1 case each of retinal detachment and IOL dislocation in the staphyloma group. The staphyloma group had significantly lower postoperative visual acuity than the control group; however, the percentage in the staphyloma group with a visual acuity of finger counting or worse decreased from 80.4% preoperatively to 16.8% postoperatively and the percentage with an uncorrected visual acuity of 20/160 or better increased from 5.6% to 67.3%. Myopic degeneration was judged to be the cause of postoperative acuity worse than 20/100 in 22.4% in the staphyloma and 0% in the control group; other causes for poor postoperative acuity were similar in the 2 groups. In the staphyloma but not the control group, decreased postoperative acuity was independently associated with age greater than 65 years and axial length greater than 29.0 mm. CONCLUSIONS: Myopic degeneration may limit the results of cataract extraction in up to one fourth of patients with posterior staphyloma. Nevertheless, a substantial improvement in mean visual acuity resulted that was not associated with a significant increase in surgical complications.

Isolation of cochlear inner hair cells.

In order to identify hair cell specific genes, it is essential to obtain isolated hair cells in quantity. While whole-cell recordings have been made from isolated inner hair cells (IHCs) from guinea pigs, detailed methods for obtaining a fairly large amount of isolated inner hair cells have not been published. Here we describe a protocol that can yield a fairly large amount of isolated gerbil IHCs. This technique can provide sufficient numbers of solitary IHCs for either electrophysiological studies of the cell's membrane properties or identifying genes related to IHC functions using techniques of molecular biology.

Morphology of the unfixed cochlea.

Our knowledge of cochlear geometry is based largely upon anatomical observations derived from fixed, dehydrated, embedded and/or sputter-coated material. We have now developed a novel preparation, the hemicochlea, where for the first time living cochlear structures can be observed in situ and from a radial perspective. The experiments were performed on the Mongolian gerbil. Ion substitution experiments suggest that no significant swelling or shrinkage occurs when the preparation is bathed in normal culture medium, so long as calcium concentration is kept at endolymph-like (20 microM) levels. The tectorial membrane-reticular lamina relationship appears to remain well preserved. Hensen's stripe maintains a close relationship with the inner hair cell stereociliary bundle, unless the mechanical coupling becomes disturbed. In addition, standard fixation and/or dehydration procedures are used to quantify changes due to shrinkage artifacts. Various morphometric gradients are examined in unfixed specimens from apical, middle, and basal turns.

The carotenoids as anti-oxidants--a review.

Carotenoids are abundant in many fruits and vegetables and they play diverse roles in photobiology, photochemistry and medicine. This review concerns the reactivity of carotenoids with singlet oxygen and the interaction of carotenoids with a range of free radicals. Mechanisms associated with the anti- and pro-oxidant behaviour of carotenoids are discussed including carotenoid interactions with other anti-oxidants.

Dietary uptake of lycopene protects human cells from singlet oxygen and nitrogen dioxide - ROS components from cigarette smoke.

There is current interest in the health benefits of dietary carotenoids and the possible deleterious effects on certain sub-populations such as smokers. Here we report in vivo protection of human lymphocytes, conferred by dietary supplementation of lycopene rich foods against the reactive oxygen species, NO(2)(*) radical (by electron transfer) and 1(O)(2) (by energy transfer). It was found that a lycopene rich diet, maintained for 14 days, increased the serum lycopene level 10 fold compared to serum obtained after the same period, where a typical western European diet had been consumed. Relative lymphocyte protection factors of 17.6 and 6.3 against NO(2)(*) radical and 1(O)(2), respectively, were obtained, which re-enforce epidemiological data, showing protection against several chronic diseases by tomato lycopene.

Antioxidant inhibition of porphyrin-induced cellular phototoxicity.

Porphyrins such as protoporphyrin IX (PP IX) and uroporphyrin I (UP I) can be phototoxic to human cells. To study the protective ability of antioxidants (beta-carotene, lycopene, ascorbic acid and alpha-tocopherol), against such porphyrin phototoxicity, membrane destruction experiments (Jurkat cells) and human cell cultures (fibroblasts) were performed. Both beta-carotene and lycopene and also the combination of beta-carotene, ascorbic acid and alpha-tocopherol offered cell protection against PP IX phototoxicity. Investigations of both cell membrane protection and of cell growth showed differences in terms of the protection afforded by the anti-oxidants. Thus, for PP IX, carotenoids alone, and in combination with ascorbic acid and alpha-tocopherol, showed higher protection factors in general than UP I. However, for membrane protection there was significant protection against UP I by the combination of beta-carotene, ascorbic acid and alpha-tocopherol but not by any of these anti-oxidants alone. The membrane protection against PP IX by beta-carotene, and especially lycopene, is significant presumably because of the high lipophilicity of all these molecules. However, the hydrophilic UP I will cause phototoxicity mainly via H(2)O(2), radical or singlet oxygen production in the aqueous phase, and these reactive species may be generated some distance from the cell membrane. This may lead to the little or no protection observed for UP I by the individual antioxidants. Nevertheless, a combination of beta-carotene, ascorbic acid and alpha-tocopherol offers membrane protection against the phototoxicity of both porphyrins. This is believed to occur as a result of synergistic processes. Our results suggest that the treatment of porphyria cutanea tarda and erythropoietic protoporphyria may be improved by the use of a combination of the antioxidants studied.

Characterisation of carotenoid radical cations in liposomal environments: interaction with vitamin C.

Pulse radiolysis was used to generate the radical cations of beta-carotene and two xanthophylls, zeaxanthin and lutein, in unilamellar vesicles of dipalmitoylphosphatidyl choline. The rate constants for the reaction (repair) of these carotenoid radical cations with the water-soluble vitamin C were found to be similar (approximately 1x10(7) M(-1) s(-1)) for beta-carotene and zeaxanthin and somewhat lower (approximately 0.5x10(7) M(-1) s(-1)) for lutein. The results are discussed in terms of the microenvironment of the carotenoids and suggest that for beta-carotene, a hydrocarbon carotenoid, the radical cation is able to interact with a water-soluble species even though the parent hydrocarbon carotenoid is probably entirely in the non-polar region of the liposome.

Enhanced protection of human cells against ultraviolet light by antioxidant combinations involving dietary carotenoids.

Antioxidants like beta-carotene, alpha-tocopherol and ascorbic acid should be able to protect human cells against damage due to ultraviolet light. Cultured human fibroblasts have been irradiated with UVA or UVB light after incubation with the antioxidants or combinations of them. The efficiency of the protection by the antioxidants in dietary concentrations is estimated by cell counting following cell culture. In the case of UVA irradiation we find synergistic effects of combinations with beta-carotene as the main protector. On the other hand, only additive effects of the tested combinations are observed in the experiments with UVB light. Our experiments show a protective effect of dietary antioxidants against human tissue cell damage by ultraviolet light.