On the structure of flavin-oxygen intermediates involved in enzymatic reactions.

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421--505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C(4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the subsituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavoproteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides.     

Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.     

Covalent adducts of lactate oxidase. Photochemical formation and structure identification.

Lactate oxidase forms tight complexes with a variety of mono- and dicarboxylic acids. Most of these undergo facile photoreactions involving decarboxylation of the carboxylic acid and formation of covalent adducts at position N(5) of the flavin, characterized by absorption maxima from 325 to 365 nm and fluorescence emission in the range 440 to 490 nm. The properties of the adducts are strongly dependent on the nature of the substituent. Enzyme-bound N(5)-acyl adducts and N(5)-CH2-R derivatives are stable in the dark. Glycollyl- and alpha-lactyl adducts, however, decay to oxidized enzyme with half-lives in the order of minutes. Upon denaturation of the enzyme, the N(5)-alkyl adducts decay rapidly or are oxidized by oxygen. Reduced lactate oxidase is also photoalkylated in the presence of halogenated carboxylic acids. Bromoacetate yields an N(5)-carboxymethyl adduct; with beta-bromopropionate, a C(4a)-beta-propionyl derivate is formed. The N(5) adduct is identical with that from the photochemical reaction of oxidized enzyme and malonic acid. When the native coenzyme FMN is substituted by 2-S-FMN, qualitatively the same photoproducts are formed. The adducts obtained with the 2-S-FMN enzyme show the expected bathochromic shifts in absorption spectra. The results indicate that the photoreactivity of the enzyme is restricted to the positions C(4a) and N(5) of the flavin.     

Photoreduction of flavoproteins and other biological compounds catalyzed by deazaflavins

Deazaflavins have been found to act as potent catalysts in the photoreduction of flavoproteins in the presence of EDTA and other "photosubstrates". In distinction to the catalysis brought about by normal flavins which involves dark reaction of the photoreduced flavin catalyst, the mechanism of the catalysis by deazaflavins has been shown to involve unstable, strongly reducing radicals which are generated by photolysis of a preformed covalent dimer. By this new method it is possible to reduce not only flavoproteins but a variety of other redox proteins, including heme proteins and iron-sulfur proteins. By virtue of its great catalytic efficiency, it is possible to employ concentrations of deazaflavin sufficiently low as not to interfere with the spectral evaluation of the reduced proteins obtained.     

Common and rare species respond to similar niche processes in macroinvertebrate metacommunities

Ecologists have long investigated why communities are composed of a few common species and many rare species. Most studies relate rarity to either niche differentiation among species or spatial processes. There is a parallel between these processes and the processes proposed to explain the structure of metacommunities. Based on a metacommunity perspective and on data on stream macroinvertebrates from different regions of Brazil, we answer two questions. 1) Are sets of common and rare species affected by similar niche and spatial processes? 2) How does the community composition of common and of rare species differ? The main hypothesis we test is that common species are mainly affected by environmental factors, whereas rare species are mostly influenced by dispersal limitation. We used variation partitioning to determine the proportion of variation explained by the environment and space in common and rare species matrices. Contrary to our expectations, evidence supported the idea that both common and rare species are affected mainly by environmental factors, even after controlling for the differing information content between common and rare species matrices. Moreover, the abundance of some common species is also a good predictor of variation in rare species matrices. Niche differences are unlikely to be the sole cause of patterns of rarity in these metacommunities. We suggest that sets of common and rare species react to similar major environmental gradients and that rare species also respond to processes that operate at a more fine‐grained spatial scale, particularly biotic interactions. We extend the view that species sorting is the dominant process structuring metacommunities and argue that future studies focusing on rarity would benefit from a metacommunity perspective.     

Five New Guaiane Sesquiterpenes from the Endophytic Fungus Xylaria sp. YM 311647 of Azadirachta indica

Five new guaiane sesquiterpenes, 1 – 5 , were isolated from the culture broth of the endophytic fungus Xylaria sp. YM 311647, isolated from Azadirachta indica A. Juss . The structures of these compounds were elucidated on the basis of spectroscopic analyses, and their inhibitory activities against five pathogenic fungi were evaluated. All guaiane sesquiterpenes showed moderate or weak antifungal activities in a broth microdilution assay.     

Growth hormone affects behaviour of wild brown trout Salmo trutta in territorial owner–intruder conflicts

The effects of growth hormone (GH) implants on aggression, and ability to win dyadic territorial conflicts were studied in brown trout Salmo trutta parr. Bovine GH or vehicle (C) was given to either the territory owner or the intruder in four treatment combinations: C and C, C and GH, GH and C, GH and GH (owner and intruder). GH‐treated intruders initiated significantly more conflicts compared to control intruders. Furthermore, GH treatment of either the owner or intruder tended to increase aggression of the intruder. This indicates that intruders have more scope for motivational increase, while the motivation of owners is already at a maximum. The GH treatment, however, did not affect the outcome of the conflict. It thus appears that growth enhancement increases intruder aggression without increasing the chance of winning the conflict, which may have implications for the effect of growth‐selected or growth‐enhanced farmed Atlantic salmon Salmo salar on wild populations.     

What shapes the altitudinal range of a high mountain Mediterranean plant? Recruitment probabilities from ovule to seedling stage

Recruitment is a complex process consisting of sequential stages affected by biotic interactions and abiotic factors. Assessment of these sequential stages and corresponding subprocesses may be useful in identifying the most critical stages. Accordingly, to assess the factors that may determine the altitudinal range limits of the high mountain Mediterranean plant Silene ciliata, a set of demographic stages, from flower production to establishment of 2‐yr‐old plants, and their influence on recruitment probability were examined using a step‐by‐step approach. We integrated florivory, pollination and pre‐dispersal seed predation as pre‐dispersal factors, and seedling emergence and survival as post‐dispersal determinants of recruitment. Three populations were monitored at the southernmost margin of the species along its local altitudinal range. Previous studies suggest that seediness is strongly limited by summer drought especially at the lower boundary of the species, a situation that may worsen under current global warming. Our results showed that recruitment was mainly limited by low seed production in the pre‐dispersal stage and low seedling emergence and survival in the post‐dispersal stage, probably due to environmental harshness in summer. By contrast, biotic factors responsible for propagule loss, such as flower and fruit predation, had a minor effect on the probability of plant recruitment. Although the relative importance of transition probabilities was similar among populations along the altitudinal range, comparatively lower flower production significantly reduced the number of recruited plants at the lowest altitude population. This demographic bottleneck, together with increased competition with other species favoured by climate warming, might collapse population growth and limit persistence at the lower altitudinal range of the species, raising its low local altitudinal edge.     

Landscape simplification increases vineyard pest outbreaks and insecticide use

Diversifying agricultural landscapes may mitigate biodiversity declines and improve pest management. Yet landscapes are rarely managed to suppress pests, in part because researchers seldom measure key variables related to pest outbreaks and insecticides that drive management decisions. We used a 13‐year government database to analyse landscape effects on European grapevine moth (Lobesia botrana) outbreaks and insecticides across c. 400 Spanish vineyards. At harvest, we found pest outbreaks increased four‐fold in simplified, vineyard‐dominated landscapes compared to complex landscapes in which vineyards are surrounded by semi‐natural habitats. Similarly, insecticide applications doubled in vineyard‐dominated landscapes but declined in vineyards surrounded by shrubland. Importantly, pest population stochasticity would have masked these large effects if numbers of study sites and years were reduced to typical levels in landscape pest‐control studies. Our results suggest increasing landscape complexity may mitigate pest populations and insecticide applications. Habitat conservation represents an economically and environmentally sound approach for achieving sustainable grape production.