16Beta-hydroxylation of dehydroepiandrosterone sulphate by homogenates of human foetal liver.

The chemical syntheses of the 3-sulphates of 16 alpha-acetoxy-, 16 alpha-hydroxy- and 16 beta-hydroxy-dehydroepiandrosterone as their triethylammonium salts are described preparatory to studies of the metabolism of [7 alpha-3 H]dehydroepiandrosterone sulphate by homogenates of human foetal liver. Five main radioactive products of the incubation were separated by partition chromatography on a Celite column. Two were identified as 16 alpha-and 16 beta-hydroxydehydroepiandrosterone 3-sulphates by crystallization to constant specific radioactivity before and after solvolysis. The yields of the conversion to the two epimers were 24.4% (16 alpha) and 1.8% (16 beta).     

The sulphatase of ox liver. XXIV. The glycosulphatase activity of sulphatase a

The rhodizonic acid method for the determination of SO2-4 has been used to investigate the glycosulphatase activity of the sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver. Sulphatase A hydrolyses D-glucopyranose and D-galactopyranose 2-, 3-, 4- and 6-sulphates: glucose sulphates are hydrolysed more rapidly than galactose sulphates and the 3-sulphates more rapidly than the other isomers. 2-Acetamido-2-deoxyglucopyranose 6-sulphate is not hydrolysed, nor is 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose 1-sulphate. Sulphate is a competitive inhibitor of the glycosulphatase activity. Hydrolysis proceeds through fission of the O-S bond. Evidence is given that the hydrolysis of glucose 3-sulphate is accompanied by the formation of substrate-modified sulphatase A, although this has not been isolated. Sulphatase A has no detectable alkylsulphatase activity.     

Flavonoid sulphates in the Umbelliferae

A survey of over 250 representative taxa in the Umbelliferae has shown that sulphates only accumulate in three genera, Ammi, Daucus and Oenanthe. The presence of quercetin, rhamnocitrin, rhamnetin and isorhamnetin 3-sulphate in Ammi visnaga serves to distinguish it from the related A. majus which lacks sulphates. In Daucus carota leaf, the 7-and 4′-sulphates of luteolin both occur; the two characters are polymorphic and appear to be present more frequently in North temperate than in South temperate populations. Oenanthe is the only genus where sulphates are found abundantly; they occur in 7 of 9 species surveyed. In addition to the known isorhamnetin 3sulphate of O. stolonifera, quercetin 3-sulphate and luteolin 7-sulphate were identified for the first time in the genus. The synthesis of various kaempferol and quercetin sulphates is described.     

Synthesis of L-fucose 2-, 3-, and 4-Sulphates

Treatment of L-fucose with an excess of pyridine-sulphur trioxide gave an equilibrium mixture of mono-, di-,and tri-sulphates. L-Fucose was sulphated under optimal conditions for monosulphate formation, and the monoester fraction was isolated by chromatography on DEAE-cellulose. The isomeric L-fucose 2-, 3-, and 4-sulphates (1-3) were separated on a DEAE-cellulose column by elution with borate buffer. The structures of 1-3 were established by electrophoresis, colour tests, periodate oxidation, and, for the 2-isomer, by comparison with a specimen of 1 that had been definitively synthesised via methyl 3,4-O-isopropylidene-α-L-fucopyranoside (6) and methyl α-L-fucopyranoside 2-(barium sulphate) (5). The latter was rapidly hydrolysed in hot, dilute acetic acid to 1 and methyl α-L-fucopyranoside (4).     

Occurrence of sulphated flavones and caffeic acid esters in members of the fluviales

A survey of 50 species of the Fluviales showed that over 50% have either flavone or caffeic acid sulphates present. Flavone sulphates were detected in 16% of the sample and the 7-sulphates of luteolin, apigenin, diosmetin and chrysoeriol and the 7,3′-disulphate of luteolin were identified variously in Thalassia, Zannichellia and Zostera species. Anionic caffeic esters were found in 46% of the sample; preliminary studies indicate the presence of sulphated caffeylquinic acids in these plants. In confirmation of earlier studies, glycoflavones were found to be widespread and flavonols and proanthocyanidins to be rare. The taxonomic and ecological significance of these results are discussed.     

Two gossypetin methyl ethers as ultraviolet patterning guides in the flowers of Coronilla valentina

The 3′-monomethyl and 8,3′-dimethyl ethers of gossypetin have been identified in the flowers of Coronilla valentine where they occur as the 3-rutinosides. These two yellow flavonols occur specifically in the wings and thus provide both visible yellow colour and UV absorption to bees, which land on the wings and trigger the self-fertilization mechanism. These yellow pigments are absent from the flowers of the related C. emerus, where their role in UV patterning is taken over by colourless kaempferol and quercetin glycosides.     

Low-Pressure UV Inactivation and DNA Repair Potential of Cryptosporidium parvum Oocysts

Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25°C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm² (=30 J/m²), the reduction reached the cell culture assay detection limit of ~3 log₁₀. At UV doses of 1.2 and 3 mJ/cm², the log₁₀ reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.     

Cyanobacteria toxins in the Salton Sea

Background

Sequenced archaeal genomes contain a variety of bacterial and eukaryotic DNA repair gene homologs, but relatively little is known about how these microorganisms actually perform DNA repair. At least some archaea, including the extreme halophile Halobacterium sp. NRC-1, are able to repair ultraviolet light (UV) induced DNA damage in the absence of light-dependent photoreactivation but this 'dark' repair capacity remains largely uncharacterized. Halobacterium sp. NRC-1 possesses homologs of the bacterial uvrA, uvrB, and uvrC nucleotide excision repair genes as well as several eukaryotic repair genes and it has been thought that multiple DNA repair pathways may account for the high UV resistance and dark repair capacity of this model halophilic archaeon. We have carried out a functional analysis, measuring repair capability in uvrA, uvrB and uvrC deletion mutants.

Results

Deletion mutants lacking functional uvrA, uvrB or uvrC genes, including a uvrA uvrC double mutant, are hypersensitive to UV and are unable to remove cyclobutane pyrimidine dimers or 6–4 photoproducts from their DNA after irradiation with 150 J/m² of 254 nm UV-C. The UV sensitivity of the uvr mutants is greatly attenuated following incubation under visible light, emphasizing that photoreactivation is highly efficient in this organism. Phylogenetic analysis of the Halobacterium uvr genes indicates a complex ancestry.

Conclusion

Our results demonstrate that homologs of the bacterial nucleotide excision repair genes uvrA, uvrB, and uvrC are required for the removal of UV damage in the absence of photoreactivating light in Halobacterium sp. NRC-1. Deletion of these genes renders cells hypersensitive to UV and abolishes their ability to remove cyclobutane pyrimidine dimers and 6–4 photoproducts in the absence of photoreactivating light. In spite of this inability to repair UV damaged DNA, uvrA, uvrB and uvrC deletion mutants are substantially less UV sensitive than excision repair mutants of E. coli or yeast. This may be due to efficient damage tolerance mechanisms such as recombinational lesion bypass, bypass DNA polymerase(s) and the existence of multiple genomes in Halobacterium. Phylogenetic analysis provides no clear evidence for lateral transfer of these genes from bacteria to archaea.     

Molecular Mechanisms of Pyrimidine Dimer Excision in Saccharomyces cerevisiae: Incision of Ultraviolet-Irradiated Deoxyribonucleic Acid In Vivo

A group of genetically related ultraviolet (UV)-sensitive mutants of Saccharomyces cerevisiae has been examined in terms of their survival after exposure to UV radiation, their ability to carry out excision repair of pyrimidine dimers as measured by the loss of sites (pyrimidine dimers) sensitive to a dimer-specific enzyme probe, and in terms of their ability to effect incision of their deoxyribonucleic acid (DNA) during post-UV incubation in vivo (as measured by the detection of single-strand breaks in nuclear DNA). In addition to a haploid RAD⁺ strain (S288C), 11 different mutants representing six RAD loci (RAD1, RAD2, RAD3, RAD4, RAD14, and RAD18) were examined. Quantitative analysis of excision repair capacity, as determined by the loss of sites in DNA sensitive to an enzyme preparation from M. luteus which is specific for pyrimidine dimers, revealed a profound defect in this parameter in all but three of the strains examined. The rad14-1 mutant showed reduced but significant residual capacity to remove enzyme-sensitive sites as did the rad2-4 mutant. The latter was the only one of three different rad2 alleles examined which was leaky in this respect. The UV-sensitive strain carrying the mutant allele rad18-1 exhibited normal loss of enzyme-sensitive sites consistent with its assignment to the RAD6 rather than the RAD3 epistatic group. All strains having mutant alleles of the RAD1, RAD2, RAD3, RAD4, and RAD14 loci showed no detectable incubation-dependent strand breaks in nuclear DNA after exposure to UV radiation. These experiments suggest that the RAD1, RAD2, RAD3, RAD4 (and probably RAD14) genes are all required for the incision of UV-irradiated DNA during pyrimidine dimer excision in vivo.     

Sodium borohydride reduction of anthocyanidins

The reduction of anthocyanidins with NaBH₄ in EtOH or MeOH produces inter alia racemates of epicatechins. Thus, the (±) racemates of 3′,5′-di-O-methylepigallocatechin, 3′-O-methylepigallocatechin, and 3′-O-methylepicatechin have been identified as the reduction products of malvidin, petunidin, and peonidin, respectively, by their UV, MS and NMR spectra.     

Development-dependent effects of UV radiation exposure on broccoli plants and interactions with herbivorous insects

The responses of plants to stress can highly depend on their developmental stage and furthermore influence biotic interactions. Effects of outdoor exposure to different ambient radiation conditions including (+UV) or excluding (?UV) solar ultraviolet radiation were investigated in broccoli plants (Brassica oleracea L. convar. botrytis) at two developmental stages. Plants either germinated directly under these different outdoor UV conditions, or were first kept for three weeks in a climate chamber under low radiation before outside exposure at +UV and ?UV. Access of herbivores to the plants was possible under the outdoor conditions. Plants of both groups protected their tissue against destructive UV by increasing concentrations of phenolic compounds (flavonoids and hydroxycinnamic acids) after +UV exposure. But only plants that germinated under +UV conditions kept smaller than plants grown under ?UV conditions, indicating certain costs for production of phenolics or for other potential metabolic processes specifically in young, growing plants. In contrast, growth of plants transferred at a later stage did not differ under both UV conditions. Thus, plants responded much more sensitive to the environment they experienced at first growth. Glucosinolates, the characteristic secondary compounds of Brassicaceae, as well as proteinase inhibitors, remained unaffected by UV in all plants, demonstrating independent regulation pathways for different metabolites. Plant infestation by phloem-feeding insects, Aleyrodidae and Aphididae, was more pronounced on +UV exposed plants, whereas cell content feeders, like Thripidae were more abundant on plants under the ?UV condition. Choice experiments with the cabbage whitefly Aleyrodes proletella L. (Aleyrodidae), commonly found on Brassica spp., revealed that the key environmental cue navigating their behaviour seems to be the radiation composition, rather than plant quality itself. In conclusion, stress mediated changes of plant chemistry and morphology depend on the plant life cycle stage and are not necessarily mirrored in the behavioural responses of herbivorous insects.     

Effects of newly synthesised platinum(ii) complexes on mitochondrial functions

Protease deficient recA431 mutants of Escherichia coli are defective in their capacity for induction of SOS responses and were intermediate in their sensitivities to ultraviolet light (UV) and cis-platinum(II)diamminodichloride (cis-PDD). Survival after treatment determined as colony forming ability was greater in rec⁺ strains and decreased in recA13 mutants which are defective in both recA proteolytic and recombination capabilites. In contrast, recA431 mutants were as sensitive to N-methyl-N′-nitro-N-nitrosoguanidine (NTG) as the recA13 cells. When cells carried either the pKM101 or N3 plasmid, survival after treatment with the three mutagens was increased. Presence of these plasmids in cells also resulted in hypermutagenicity as indicated by reversion of the argE3 mutation using a modified Ames test. Mutagenesis by NTG and cis-PDD was increased, as was survival of cells treated with UV light, cis-PDD and NTG in both recA⁺ and recA431 (protease deficient) strains. No plasmid mediated enhancement of mutagenesis or cell survival was observed in recA13 mutants. Thus, the ability of the plasmids to enhance cell survival and mutagenesis was dependent on recombination proficiency of the recA gene product and not its regulatory proteolytic activity. Unlike UV or NTG, presence of one of these plasmids was needed to detect reversion of the argE3 mutation by cis-PDD.     

Studies on the ultraviolet light sensitivity of Bloom's syndrome fibroblasts

The sensitivity of Bloom's syndrome (bl/bl) fibroblasts to ultraviolet light (254 nm) has been estimated by 4 criteria: sister-chromatid exchange (SCE) formation, micronucleus production, cell survival, and host-cell reactivation of UV-irradiated adenovirus 2. In general, bl/bl strains did not differ significantly from the normal (+/+) strains in their response to UV treatment by any of the 4 criteria. One bl/bl strain, GM1492, was exceptional: It was abnormally sensitive to UV light in the SCE, micronucleus, and host-cell reactivation assays, but was not sensitive to UV as estimated by colony-forming ability. Thus, although one of the bl/bl strains studied in the experiments was sensitive to UV light as judged by some criteria, UV sensitivity is not a universal characteristic of Bloom's syndrome cells. It is nuclear whether the UV sensitivity of the GM1492 strain reflects genetic diversity within the syndrome or some unrelated property of this strain.     

Phosphoinositide-specific phospholipase C forms a complex with 14-3-3 proteins and is involved in expression of UV resistance in fission yeast

The fission yeast plc1 ⁺ gene encodes phosphoinositide-specific phospholipase C. The two- hybrid interaction assay with plexA-plc1 ⁺ as a bait revealed that Plc1p interacted with the 14-3-3 proteins Rad24p and Rad25p. Formation of a complex containing Plc1p and Rad24p in vivo was confirmed by an immunological method. As predicted from the fact that rad24 null mutant cells are hypersensitive to UV irradiation, plc1 null mutant cells were almost as sensitive to UV irradiation as rad24 null mutant cells. In addition, deletion of rad24 in the plc1 null mutant cells did not enhance the UV sensitivity, indicating that plc1 ⁺ and rad24 ⁺ belong to the same epistasis group with respect to UV sensitivity. Whereas Rad24p has been reported to be involved in the DNA damage checkpoint pathway, the delay to mitosis after UV irradiation was not defective either in rad24 null mutant cells or in plc1 null mutant cells in our analysis. Thus, Plc1p is responsible for resistance to UV irradiation, but not for the DNA damage checkpoint pathway, in cooperation with 14-3-3 proteins.     

Yeast Deubiquitinase Ubp3 Interacts with the 26 S Proteasome to Facilitate Rad4 Degradation

Deubiquitinating enzymes (DUBs) function in a variety of cellular processes by removing ubiquitin moieties from substrates, but their role in DNA repair has not been elucidated. Yeast Rad4-Rad23 heterodimer is responsible for recognizing DNA damage in nucleotide excision repair (NER). Rad4 binds to UV damage directly while Rad23 stabilizes Rad4 from proteasomal degradation. Here, we show that disruption of yeast deubiquitinase UBP3 leads to enhanced UV resistance, increased repair of UV damage and Rad4 levels in rad23Δ cells, and elevated Rad4 stability. A catalytically inactive Ubp3 (Ubp3-C469A), however, is unable to affect NER or Rad4. Consistent with its role in down-regulating Rad4, Ubp3 physically interacts with Rad4 and the proteasome, both in vivo and in vitro, suggesting that Ubp3 associates with the proteasome to facilitate Rad4 degradation and thus suppresses NER.     

Lichens as survivors in space and on Mars

Many lichens are able to live and photosynthesize under harsh conditions, characterized by low temperatures, aridity and high UV radiation fluxes. Some lichen species are even able to survive simulated and real space conditions. Many tests after space exposure on the satellite FOTON M3 and on the International Space Station have shown their capacity to maintain physiological and photosynthetic activity, and their capacity to germinate and grow after being exposed to space parameters. Further tests using simulated Martian atmospheres, temperatures, humidity profiles and UV radiation spectra and fluxes have shown maintenance of photosynthetic activity of Xanthoria elegans. Results from space and Mars simulation experiments on lichens such as X. elegans are valuable for determining the habitability of a planet and for the search for possible life-supporting habitats on planets like Mars.     

Enantiometric enrichment of R(−)-alkan-2-ols using a stereospecific alkylsulphatase

Summary When grown on nutrient broth, Pseudomonas C12B produces two secondary alkyl 2-sulphatases, designated S1 and S2 on the basis of electrophoretic mobility. Although both enzymes are synthesised at the end of the exponential phase in batch culture, the appearance of S1 preceded S2 by a few hours in either fermentor or shake-flask cultures. Thus extracts prepared from cells harvested in the intervening period contained only the S1 enzyme. Because S1 and S2 are stereospecific in hydrolysing S(+)- and R(–)-alkyl 2-sulphates to R(–)- and S(+)-alkan-2-ols respectively, the exclusive presence of S1 was exploited in the enantiomeric enrichment of R(–)-alkan-2-ols. Extracts containing only S1 according to gel zymography, hydrolysed only about one-half of added racemic octyl or undecyl 2-sulphates, as expected. Extraction and analysis of liberated octan-2-ol by spectral polarimetry or high resolution gas chromatography with chiral derivatisation, indicated an enantiomeric excess around 60%. Offprint requests to: G. F. White     

Sensitive assay for measuring amoxicillin in human plasma and middle ear fluid using solid-phase extraction and reversed-phase high-performance liquid chromatography

We developed a sensitive assay to measure amoxicillin in human plasma and midle ear fluid (MEF) using solid-phase extraction and reversed-phase HPLC. Amoxicillin and cefadroxil, the internal standard, were extracted from 50–200 μl of sample with Bond Elut C₁₈ cartridges. The exact was analyzed on a 15 cm × 2 mm, 5μm Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer (pH = 6.5) and 5 mM tetrabutylammonium. The average absolute recovery of amoxicillin and cefadroxil were 91.2 ± 16.6% and 91.0 ± 6.8%, respectively. The limit of quantitation was 0.125 μg/ml with 200 μl sample size. The linear range was from 0.125 to 35.0 μg/ml with correlation coefficients greater than 0.999. These analytic conditions produced a highly sensitive amoxicillin assay in human body fluids without derivatization.     

Optimizing the colour and fabric of targets for the control of the tsetse fly Glossina fuscipes fuscipes

Background

Most cases of human African trypanosomiasis (HAT) start with a bite from one of the subspecies of Glossina fuscipes. Tsetse use a range of olfactory and visual stimuli to locate their hosts and this response can be exploited to lure tsetse to insecticide-treated targets thereby reducing transmission. To provide a rational basis for cost-effective designs of target, we undertook studies to identify the optimal target colour.

Methodology/Principal Findings

On the Chamaunga islands of Lake Victoria , Kenya, studies were made of the numbers of G. fuscipes fuscipes attracted to targets consisting of a panel (25 cm square) of various coloured fabrics flanked by a panel (also 25 cm square) of fine black netting. Both panels were covered with an electrocuting grid to catch tsetse as they contacted the target. The reflectances of the 37 different-coloured cloth panels utilised in the study were measured spectrophotometrically. Catch was positively correlated with percentage reflectance at the blue (460 nm) wavelength and negatively correlated with reflectance at UV (360 nm) and green (520 nm) wavelengths. The best target was subjectively blue, with percentage reflectances of 3%, 29%, and 20% at 360 nm, 460 nm and 520 nm respectively. The worst target was also, subjectively, blue, but with high reflectances at UV (35% reflectance at 360 nm) wavelengths as well as blue (36% reflectance at 460 nm); the best low UV-reflecting blue caught 3× more tsetse than the high UV-reflecting blue.

Conclusions/Significance

Insecticide-treated targets to control G. f. fuscipes should be blue with low reflectance in both the UV and green bands of the spectrum. Targets that are subjectively blue will perform poorly if they also reflect UV strongly. The selection of fabrics for targets should be guided by spectral analysis of the cloth across both the spectrum visible to humans and the UV region.