Mineral filters in sunscreen products--comparison of the efficacy of zinc oxide and titanium dioxide by in vitro method.

European legislation currently authorizes 26 sun filters among which, there is only one mineral filter: titanium dioxide. In the United States, two mineral filters are authorized: titanium dioxide in a maximum dose of 25% and zinc oxide. Zinc oxide is authorized in Europe, but its concentration level is not limited. A large number of commercial products are containing one of these mineral filters. The difference between these products lies in the percentage of the active substance, the way they are incorporated into the final product and the size of the primary particles. Depending on the ingredient used, there is a large variation in efficacy. The efficacy of the products tested was determined by an in vitro method using a spectrophotometer equipped with an integration sphere. Titanium dioxide was thus seen to be much more effective than zinc oxide; indeed no commercial form of zinc oxide tested can give a sun protection factor (SPF) higher than 10 at its maximum dose of use, unlike titanium dioxide which in its coated form (coated with alumina and with stearic acid, amongst others) gives a SPF of 38. This study has also allowed us to dispel the theory that talc--a raw material which has been used empirically for years in foundation in the belief that it has photoprotective effects--has an effect against sun rays. Talc proved to be particularly ineffective, as when it is used at a level of 25%, it only gives a totally negligible SPF of one unit.

Large-scale analysis of 73 329 physcomitrella plants transformed with different gene disruption libraries: production parameters and mutant phenotypes.

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73 329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11 068 polyploid transformants had morphological alterations. 9 % of the haploid transformants had deviations in the leaf shape, 7 % developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.

Spatial representation of corticofugal input in the inferior colliculus: a multicontact silicon probe approach.

The inferior colliculus (IC) is a well-established target of descending projections from the auditory cortex (AC). However, our understanding of these pathways has been limited by an incomplete picture of their functional influence within the three-dimensional space of the IC. Our goal was to study the properties and spatial representation of corticofugal input in the IC of guinea pigs with a high degree of spatial resolution. We systematically mapped neural activity in the IC using two types of silicon substrate probes that allow for simultaneous recording at multiple neural sites. One probe provided a high resolution in the dorsal-ventral plane and the other provided spatial resolution in the medial-lateral plane. Electrical stimulation of the ipsilateral AC produced excitatory responses in the IC with thresholds usually below 5-10 microA. First spike latencies were predominantly in the 6-20 ms range, although latencies from 3-5 ms were also observed. Broadly distributed unimodal spike patterns with modal latencies greater than 30 ms were occasionally seen. The excitatory responses to cortical stimulation were mostly unimodal and occasionally bimodal with a wide range of spike distribution patterns and response durations. Excitation was often followed by suppression of spontaneous activity. Suppression of acoustic responses was observed even when there was little or no response to electrical stimulation, suggesting spatial-temporal integration. A few of the responding neurons showed purely inhibitory responses to electrical stimulation, suggesting that there are disynaptic routes of corticocollicular inhibition. Detailed spatial mapping revealed that the response patterns and their durations had a characteristic spatial distribution in the IC.

Local application of sodium thiosulfate prevents cisplatin-induced hearing loss in the guinea pig.

Cisplatin (CDDP), an anticancer drug used extensively to treat a broad range of neoplasms, has strong ototoxic side effects. Sodium thiosulfate (STS) has been described as a protective agent against CDDP toxicity, but it also reduces CDDP's antitumoral cytotoxicity. To maintain the antitumoral effectiveness of systemic administration of CDDP, a strategy has been developed to apply STS directly into the cochlea. Perfusion of STS into the cochleae of guinea pigs completely prevented CDDP-induced hearing loss, with no change in either compound action potential (CAP) or distortion product otoacoustic emission (DPOAE) audiograms during the time course of the treatment. Histological analysis revealed a minimal loss of outer hair cells (OHCs) in the organ of Corti and no damage to the marginal cells of the stria vascularis as seen in animals exposed to CDDP. Cytocochleograms prepared 6 days after CDDP exposure showed that STS treatment protected more than 92.8% of OHCs and IHCs destined to die. Furthermore, it prevented CDDP-induced mitochondrial damage and subsequent translocation of cytochrome c, DNA fragmentation, and suppressed the apoptotic and necrotic hair cell degeneration. These results suggest that local application of STS may be an interesting strategy to prevent CDDP ototoxicity in patients undergoing CDDP chemotherapy.

Fine alterations of distortion-product otoacoustic emissions after moderate acoustic overexposure in guinea pigs.

Guinea pigs were exposed to a pure tone at 6 kHz and 80 dB SPL for 30 minutes in order to induce mild reversible auditory fatigue over the 4 hours following exposure. Cochlear monitoring aimed to compare the shifts in round-window compound action potentials (CAP) thresholds to those of 2f1-f2 distortion-product otoacoustic emissions (DPOAE, frequency of stimuli f1 and f2). Both responses were evaluated every 1/10th octave between 6 and 12 kHz for CAP thresholds, and from 4 to about 14 kHz for DPOAEs in response to 50- and 70-dB SPL stimuli. The auditory fatigue turned out to be sufficiently mild that DPOAEs remained present, so that their microstructure could be followed up while the stimulus frequency ratio f2/f1 was swept from 1.06 to 1.30 (fixed f2) so as to derive DPOAE level profiles against f2/f1 and group latencies. CAP thresholds decreased by about 10 dB above 7.2 kHz, whereas DPOAE amplitudes decreased at most f2 frequencies from 6.6 kHz to 15.2 kHz, with the range of decrease being slightly narrower at higher stimulus intensities. While the mean DPOAE shift after 1 hour was around 5 dB irrespective of stimulus intensity, it tended to increase slightly after 2 hours despite stable CAP thresholds. DPOAE profiles against f2/f1 were slightly modified by the auditory fatigue, so that the maximum tended to be reached at lower ratio. No significant variation of DPOAE latencies was found after acoustic overstimulation. These experiments show that complex DPOAE changes were induced by auditory fatigue and their relationship to CAP threshold changes does not seem to be straight-forward. Nonetheless, fine DPOAE recordings might be useful to detect early changes in cochlear mechanics.

In vitro and in vivo efficacy of a novel fluoro-ketolide HMR 3562 against enterococci.

The in vitro activity of HMR 3562, a new 2-fluoro-ketolide drug, was investigated against 95 enterococci, including 36 vancomycin-resistant strains. HMR 3562 inhibited 90% of enterococci susceptible or resistant to erythromycin A at 0.005 and 0.6 mg/L, respectively. HMR 3562 was highly active in murine peritonitis induced by five enterococci, irrespective of resistance phenotype, displaying effective doses in the range 3.4-21.8 mg/kg. The results demonstrate the potential of HMR 3562 in the treatment of infections caused by multiresistant enterococci.

In vivo efficacy of the new ketolide telithromycin (HMR 3647) in murine infection models.

We compared the oral antibacterial activities of telithromycin (HMR 3647), a new ketolide drug, in different infections induced in mice by Staphylococcus aureus, Streptococcus pneumoniae, streptococci, enterococci, and Haemophilus influenzae with those of various macrolides and pristinamycin. Unlike all other comparators, telithromycin displayed a high therapeutic activity, particularly in septicemia induced by erythromycin A-resistant pathogens, where the ketolide was the only active compound, displaying effective doses between 3 and 26 mg/kg of body weight. Against H. influenzae, telithromycin was the most effective compound. Telithromycin displayed bacteriostatic behavior against S. pneumoniae and H. influenzae. The ketolide was also active against thigh muscle infection induced by S. aureus. The pharmacokinetic properties of telithromycin accounted for its outstanding well-balanced oral in vivo efficacy against both gram-positive cocci, whatever their phenotype of resistance, and H. influenzae.

On the spectral periodicity of transient-evoked otoacoustic emissions from normal and damaged cochleas.

The spectral quasi-periodicity of transient-evoked otoacoustic emissions (TEOAE) is well acknowledged since Zwicker described a preferred spacing of 0.4 bark between consecutive peaks in the spectrum of otoacoustic emissions from normal ears. While there is scarce evidence of any anatomical reason for this regularity, several functional models of the cochlea have predicted that the structure of emission spectra reflects important characteristics of cochlear filters. In an attempt to check such predictions, the average regularity of TEOAE spectra was studied in three groups of human subjects, normally hearing adults, healthy neonates, and adults suffering from noise-induced hearing loss. Significant differences in emission periodicities were found. Around 1 kHz, the preferred spacing was close to 130 Hz in normally hearing adult ears and neonates. In contrast, no clear periodicity was found in the group of damaged ears, even though they had clinically normal pure-tone audiometry below 2 kHz. Around 4 kHz, the preferred spacing was close to 240 Hz in normal adults and neonates, whereas TEOAEs were absent in many impaired ears. A phenomenological model assuming that TEOAEs stem from the responses of a slightly disarrayed bank of highly tuned filters predicts that the filter width would be the same in healthy young adults and neonates. In contrast, ears suffering from high-frequency hearing loss could exhibit early damaged filters. The proposed method might provide an objective assessment of parameters otherwise difficult to evaluate, especially in neonatal cochleas.

Detection and tissue distribution of potato spindle tuber viroid in infected tomato plants by tissue print hybridization.

Potato spindle tuber viroid (PSTVd) was detected in two cultivars of tomato (Lycopersicon esculentum Mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35S-labeled PSTVd RNA probe. PSTVd was detectable in the viroid-sensitive and symptom-developing cv "Rutgers" 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv "Goldkugel" 3 weeks p.i. In both tomato cultivars, PSTVd accumulated in the upper parts of the plants newly grown after inoculation. It was predominantly found in association with the ring formed by the vascular tissue. The final accumulation of PSTVd as well as its spatial distribution were similar in the sensitive and in the tolerant tomato cultivar, as estimated from the tissue print autoradiographs. Thus, tissue print hybridization provides a rapid and sensitive means for viroid diagnosis and for the assessment of tissue-specific localization of the viroid RNA.

Detection and tissue distribution of potato spindle tuberviroid in infected tomato plants by tissue print hybridization.

Potato spindle tuber viroid (PSTVd) was detected in two cultivars of tomato (Lycopersicon esculentum Mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35S-labeled PSTVd RNA probe. PSTVd was detectable in the viroid-sensitive and symptom-developping cv "Rutgers" 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv "Goldkugel" 3 weeks p.i. In both tomato cultivars, PSTVd accumulated in the upper parts of the plants newly grown after inoculation. It was predominantly found in association with the ring formed by the vascular tissue. The final accumulation of PSTVd as well as its spatial distribution were similar in the sensitive and in the tolerant tomato cultivar, as estimated from the tissue print autoradiographs. Thus, tissue print hybridization provides a rapid and sensitive means for viroid diagnosis and for the assessment of tissue-specific localization of the viroid RNA.

Isolation of viroid-RNA-binding proteins from an expression library with nonradioactive-labeled RNA probes.

The detection and isolation of cDNAs of tomato proteins that are able to bind to viroid RNA molecules are described. They were found by screening of a lambda gt11 cDNA expression library using a modification of the previously established ligand-blotting procedure to detect DNA- and RNA-binding proteins. The essentials of our modifications are the use of (i) digoxigenin-labeled viroid RNA, (ii) low concentration of the labeled probes and (iii) an expression library that allows the direct isolation of cDNA clones. The analysis of various isolated clones showed that this method is reliable for RNA-ligand screening and North-Western blotting. Applied to viroid RNA, these experimental tools provide the precondition for further studies on the specificity of the isolated proteins.

Photosynthetically active suspension cultures of potato spindle tuber viroid infected tomato cells as tools for studying viroid - host cell interaction.

Photosynthetically active callus and cell suspension cultures were established from uninfected Lycopersicon peruvianum plants and from uninfected and potato spindle tuber viroid (PSTVd) infected plants of Lycopersicon esculentum cv. Rutgers. Viroid infection was maintained in photoheterotrophic culture on media containing 3% sucrose, but during continuous photo-mixotrophic culture in low sucrose media (1% sucrose), the level of PSTVd accumulation decreased. Photoautotrophic cell suspensions could be established with uninfected, but not with viroid infected tomato cells. As compared to uninfected cells, PSTVd infected cells grew slowly, were morphologically different in size and shape, and formed tight cell aggregates. Electronmicroscopy showed that starch accumulation in chloroplasts, deformation of the chloroplast envelope and irregular plasmalemmasomes at the cell membrane were associated with PSTVd infection.

Cloning of a cDNA for the S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum.

We screened a cDNA library of the primitive eukaryote Dictyostelium discoideum constructed in the expression vector lambda gt11 with a specific antiserum directed against S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase) and isolated cDNA clones coding for fusion proteins with beta-galactosidase. The identity of the largest of these clones was further assessed by analysis of hybrid-selected in vitro translation products. Using the cDNA as a probe, we showed that only one gene coding for AdoHcy hydrolase is present per genome and that a single transcript of 1.6 kb could be detected at various stages of differentiation. The nucleotide sequence of the cDNA was determined. It corresponds to the carboxyterminal half of the protein whose primary structure is highly homologous to the AdoHcy hydrolase from rat liver. The significance of this strong conservation of AdoHcy hydrolase in the course of evolution is discussed.

S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase variations during differentiation of Dictyostelium discoideum.

We have analyzed the level of substrate (AdoMet) and products (AdoHcy) of transmethylations throughout the developmental cycle of the primitive eukaryote Dictyostelium discoideum. The ratio AdoMet/AdoHcy varied dramatically during differentiation. The intracellular level of AdoHcy decreased sharply after the beginning of starvation reaching a value of 18% of that in vegative cells within 4 h. In contrast, there was a two-fold transient increase in AdoMet at the time of aggregation. However, these changes were not related to changes in AdoHcy hydrolase since constant levels of both the protein and the activity were found until 16 h of differentiation. In particular, there was no indication of an in vivo inactivation of the enzyme by cAMP at the time of aggregation. These results are discussed with respect to the previously postulated role of AdoHcy hydrolase in the regulation of the AdoMet/AdoHcy ratio in eukaryotic cells.

Inactivation of S-adenosyl-L-homocysteine hydrolase by cAMP results from dissociation of enzyme-bound NAD+.

S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) is inactivated by cAMP and also by 2'-deoxyadenosine, and in both cases, activity is restored by incubating the inactivated enzyme with NAD+. We have previously presented evidence that, despite these similarities, inactivation by these two ligands proceeds by different mechanisms. We have now used a fluorescence technique to quantitate enzyme-bound NAD+ and NADH on S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum, and we have confirmed that cAMP and 2'-deoxyadenosine inactivate by different mechanisms. Whereas inactivation by 2'-deoxyadenosine is due to reduction of the enzyme-bound NAD+ to NADH, incubation of S-adenosyl-L-homocysteine hydrolase with cAMP results in dissociation of the enzyme-bound NAD+. The dissociation is reversible, and reactivation likely occurs by restoration of the initial NAD+ content. This reversible inactivation by cAMP may be a mechanism of controlling biological methylation reactions by adjusting intracellular concentrations of S-adenosyl-L-homocysteine through action of S-adenosyl-L-homocysteine hydrolase.

Purification of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum: reversible inactivation by cAMP and 2'-deoxyadenosine.

S-Adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum has been purified to homogeneity. It is composed of four subunits, each with a molecular mass of 47,000. In the hydrolysis direction, the enzyme has a pH optimum of 7.5, a Km for S-adenosyl-L-homocysteine (SAH) of 6 microM, and a Vmax of 0.22 mumol min-1 mg-1. In the synthesis direction, the pH optimum is 8.0, the Km for adenosine is 0.4 microM, and the Vmax is 0.30 mumol min-1 mg-1. Although the enzyme binds beta-nicotinamide adenine dinucleotide, as well as adenosine 3',5'-cyclic monophosphate and 2'-deoxyadenosine, these ligands have no effect on enzymatic activity when added to the assay mixture. However, preincubation of SAH hydrolase with NAD+ results in a 25% activation of the enzyme. In addition, this ligand has a striking effect on subunit-subunit interactions, as shown by stabilization of quaternary structure during polyacrylamide gel electrophoresis. Preincubation with cAMP or 2'-deoxyadenosine inactivates the enzyme. Although in both cases the activity is restored upon further incubation with NAD+, we show that inactivation by these two ligands proceeds by different mechanisms. NAD+-reversible inactivation by cAMP and 2'-deoxyadenosine was also observed with the SAH hydrolase from rabbit erythrocytes. Thus, these previously unreported properties of SAH hydrolase also occur with mammalian enzymes and are not restricted to D. discoideum.