Cloning,Sequencing, and Expression of the Genes Encoding an Isocyclomaltooligosaccharide Glucanotransferase and an α-Amylase from a Bacillus circulans Strain

The gene for a novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), involved in the synthesis of a cyclomaltopentaose cyclized by an α-1,6-linkage [ICG5; cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}] from starch, was cloned from the genome of B. circulans AM7. The IgtY gene, designated igtY, consisted of 2,985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The deduced amino-acid sequence showed similarities to 6-α-maltosyltransferase, α-amylase, and cyclomaltodextrin glucanotransferase. The four conserved regions common in the α-amylase family enzymes were also found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8,325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme was purified. The enzyme acted on maltooligosaccharides that have a degree of polymerization (DP) of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme (IgtZ), which has a novel amino-acid sequence, should be assigned to α-amylase. It is notable that both IgtY and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to a family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.     

ubtree,TASS and an Analysis of the Genus Caragana

Subtree analysis and three area satements (TASS) procedure are used to deal with the area relationship in historical biogeography. On the basis of the taxon cladogram, the procedure could identify and eliminate the paralogy node and determine the informative subtree. The area relationships are generally illustrated in several subtrees and the consensus tree. The distribution pattern of the genus Caragana comprising about 72 species and occurring in 13 areas, was analyzed by using subtree method and TASS procedure in this study. The results showed seven subtrees representing the area relationships of section and series of the genus, and the consensus tree provided the 13 area relationships. These results are congruent with our former result using component analysis for Caragana.     

Hematoxylin and related compounds – an annotated bibliography concerning their origin,properties, chemistry,and certain applications

Abstract

This bibliography covers the historical aspects of hematoxylin and its close relative brazilin from their origin in dyewoods to their recently understood chemical reactions and properties. Until the end of the 19th century, dyewoods such as logwood were among the most commercially important natural dyes and investigations over a long period of time led to an extensive literature. The ready oxidation of hematoxylin to hematein, which subsequently can be complexed with metal ions or undergo reactions with acid to give isohematein, has provided a wealth of work for the organic chemist. The elucidation of the structure and subsequent synthesis of hematoxylin has proved to be a major incentive to chemists to discover novel chemical reagents for many years.     

Production,purification, and properties of an α-l-arabinofuranosidase from Cihomitus squalens

A white-rot fungus Dichomitus squalens, when grown on 1% wheat-straw glucuronoarabinoxylan under aerated submerged conditions, secreted an α-l-arabinofuranosidase (4.3 nkat/mL). The enzyme was purified 70-fold by ammonium sulfate precipitation, chromatofocusing on PBE 94, gel filtration on Ultrogel AcA 54, rechromatofocusing on PBE 94, and lectin affinity chromatography on Concanavalin A-Ultrogel. The enzyme is a glycoprotein having a molecular weight of 60,000 and a pI of 5.1. The enzyme exhibited maximal activity at pH 3.5 and at 60°, and was fully inactivated within 30 min at 70°. The K_m value for p-nitrophenyl α-l-arabinofuranoside was 1.64mm. The α-l-arabinofuranosidase liberated arabinose from sugar-beet arabinan, wheat-straw and oat-spelt arabinoxylans, and wheat-bran heteroxylan, and was inactive towards gum arabic.     

Fruit Fate, Seed Germination and Growth of an Invasive Vine – an Experimental Test of ‘sit and Wait’ Strategy

Oriental bittersweet (Celastrus orbiculatus Thunb.) is a non-indigenous, invasive woody vine in North America that proliferates in disturbed open sites. Unlike most invasive species, C. orbiculatus exhibits a sit and wait strategy by establishing and persisting indefinitely in undisturbed, closed canopy forest and responding to canopy disturbance with rapid growth, often overtopping trees. We compared fruit fates of C. orbiculatus and native American holly (Ilex opaca). We also explored mechanisms for this sit and wait invasion strategy by testing the effect of C. orbiculatus fruit crop density on removal rates and by examining the influence of seed treatment and light intensity on seed germination and seedling growth. More C. orbiculatus than I. opaca fruits became damaged, and damage occurred earlier. More fruit fell from C. orbiculatus than I. opaca, but removal rates by frugivores did not differ (76.0 ± 4.2% vs 87.5 ± 3.7%, respectively). Density (number of fruits in a patch) of C. orbiculatus did not influence removal rates. Scarification (bird-ingestion) of C. orbiculatus seed delayed germination but seeds germinated in similar proportion to manually defleshed seeds (sown either singly or all seeds from a fruit). Germination of seeds within intact fruits was inhibited and delayed compared to other treatments. Seed treatment did not affect seedling growth. The proportion of seeds germinating and time until germination was similar among five light intensity levels, ranging from full sun to closed-canopy. Seedlings in >70% photosynthetically active radiation (PAR) had more leaves, heavier shoots, and longer, heavier roots than seedlings at lower PAR levels. Results show that most (>75%) C. orbiculatus seeds are dispersed, seedlings can establish in dense shade, and plants grow rapidly when exposed to high light conditions. Control strategies for this highly invasive species should likely focus on minimizing seed dispersal by vertebrates.     

Acetate and hydrogen metabolism in intact and disintegrated granules from an acetate-fed, 55° C,UASB reactor

Summary To investigate mass-transfer resistance in granules, the effect of disintegration on the specific methanogenic activity (SMA) of acetate-grown anaerobic, thermophilic (55°C) granules was measured. Four different methods of disintegration were used; vortex mixing, ultra-sound, blending and repeated syrine aspiration. When H₂/CO₂ was used as the substrate, disintegrated granules showed a higher SMA than intact granules. However, with acetate as substrate, no effect was observed when granules disintegrated using a vortex mixer or ultra-sound, whereas both the blender- and syringe-treated granules had lower SMAs compared to intact granules. An effectiveness factor, , the ratio of the SMA of disintegrated granules to the SMA of intact granules, was presented and found useful for describing the effectiveness of disintegration to relieve mass-transfer limitation on the granules. Offprint requests to: B. K. Ahring     

Species–soil associations, disturbance, and nutrient cycling in an Australian tropical rainforest

Resource availability and disturbance are important factors that shape the composition, structure, and functioning of ecosystems. We investigated the effects of soil fertility and disturbance on plant–soil interactions and nutrient cycling in a diverse tropical rainforest. Our goal was to determine how common soil specialisation is among species and how plant–soil interactions affect ecosystem functioning in the presence of disturbance. Most species (59%) showed significant fidelity to either fertile (basalt) or infertile (schist) soils. Obligate schist specialists (six species) contributed 39 and 37% to total stand-level basal area and aboveground net primary productivity, respectively. High nutrient use efficiency of schist specialists reduced the rates of within-stand nutrient cycling through the production of nutrient-poor plant tissues and litter. Although forests on schist soils had higher basal area and similar rates of productivity to forests on basalt, uptake of Mg, K, P, and N were markedly less on schist than on basalt, particularly after a cyclone disturbance. Stands on schist soils were also less affected by the cyclone and, as a result, contributed less (ca. 50%) Mg, K, P, and N inputs to the forest floor (via litterfall) than stands on basalt soils. System “openness” (i.e. the risk of nutrient loss) from cyclone-affected basalt forests was minimised by high rates of uptake following disturbance and large effective cation exchange capacities of soils. Soil–plant-disturbance interactions are likely to engender different fitness-enhancing strategies on fertile and infertile soils, possibly leading to the development and/or maintenance of diversity in rainforests.     

Costly signaling,ritual and cooperation: evidence from Candomblé, an Afro-Brazilian religion

The apparent wastefulness of religious ritual represents a puzzle for rational choice theorists and evolutionary scholars. In recent years, it has been proposed that such rituals represent costly signals that promote intragroup cooperation precisely because of the effort and resources they require. This hypothesis was tested over the course of a 14-month long ethnographic study in the northeast of Brazil. The research focused on adherents of Candomblé, an African diasporic religion organized in autonomous congregations primarily located in low-income urban areas. Individuals who reported higher levels of religious commitment behaved more generously in a public goods economic game and revealed more instances of provided and received cooperation within their religious community. This suggests that ritual as a costly signaling may effectively predict willingness to cooperate with other group members and that the signaler may accrue benefits in the form of received cooperation. Socioeconomic variables are also shown to mediate religious signaling. This raises the possibility that signalers strategically alter their expressions of commitment as their needs and circumstances change.     

Equilibrium,kinetic and structural properties of hemoglobin Cranston,an elongated β chain variant

Hemoglobin Cranston has an elongated β subunit owing to a frame shift mutation. Oxygen equilibrium measurements of stripped Hb Cranston³ at 20 °C in the absence of phosphate revealed a high affinity (P₅₀ = 0·2 mm Hg at pH 7), non-co-operative hemoglobin variant with markedly reduced Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). The addition of inositol hexaphosphate resulted in an overall decrease in oxygen affinity (P₅₀ = 0·7 mm Hg at pH 7), as well as an increase in co-operativity and Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). Rapid mixing and flash photolysis experiments reflected the equilibrium results. Over a pH range from 6 to 9 in the absence of phosphate, the rate of combination of carbon monoxide with Hb Cranston measured by a stopped-flow technique and following full or partial flash photolysis was extremely rapid (l′, l′₄, of ～ 6 × 10⁶m^?1s^?1). In rapid kinetic experiments the addition of inositol hexaphosphate lowered the value of l′ to ～ 0·5 × 10⁶m^?1s^?1 only after prior incubation with the deoxygenated protein. Inositol hexaphosphate had no effect on the rate of recombination of carbon monoxide following either full or partial flash photolysis. Overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement, were measured and found to be slow (k, k₄～ 11 s^?1), consistent with a high affinity hemoglobin. Sedimentation equilibrium experiments revealed that Hb Cranston, at concentrations used in the functional studies, is somewhat less tetrameric than Hb A but nonetheless does not exist solely as a non-co-operative dimer. These kinetic and centrifugational findings in conjunction with X-ray diffraction evidence suggested that a high affinity tetramer of Hb Cranston exists which may equilibrate slowly with inositol hexaphosphate. Oxygen equilibrium measurements, ligand binding kinetics and X-ray diffraction studies on equivalent mixtures of Hb Cranston and Hb A revealed an interaction between these two hemoglobins in vitro that most probably exists in vivo. The presence of asymmetric hybrid molecules, α₂β^Aβ^Cranston, in the difference Fourier maps indicated that the hydrophobic tail of Hb Cranston is accommodated in the central cavity of the hybrid molecule between the two β chains and is relatively protected from the water environment, thus aiding in the stability of Hb Cranston in the red cell.     

Escherichia coli Hmp, an “oxygen-binding flavohaemoprotein”, produces superoxide anion and self-destructs

Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with k_on 2.82×10⁷ M^–1 s^–1 and k_off 4.49×10³ s^–1. The dissociation constant of the primary O₂ compound was 160 M (25°C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a K_m <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.This paper is dedicated to Professor Dr Hans G. Schlegel, on the occasion of his 80th birthday.     

Design, synthesis, and evaluation of an α-tocopherol analogue as a mitochondrial antioxidant

An efficient synthesis has provided access to a novel α-tocopherol analogue (2), as well as its trifluoroacetate salt and acetate ester. An annulation reaction was used to establish the pyridinol core structure and a Stille coupling reaction was employed for conjugation with the tocopherol side chain. This analogue was shown to suppress the levels of reactive oxygen species in cultured cells, and to quench peroxidation of mitochondrial membranes.     

Production and Isolation of Dopastin,an Inhibitor of Dopamine β-Hydroxylase

A new inhibitor of dopamine β-hydroxylase, dopastin, has been isolated. The dopastin-producing strain was found in a mushroom culture, and after being separated, it was confirmed to be a member of Pseudomonas. Dopastin was obtained as colorless needles, mp 116~119°C, (c=0.5, С₂Н₅ОН), C₉H₁₇N₃O₃. The catalytic hydrogenation afforded dihydro-dopastin which also inhibits dopamine β-hydroxylase.     

Structure and Synthesis of Dopastin,an Inhibitor of Dopamine β-Hydroxylase

Dopastin, an inhibitor of dopamine β-hydroxylase of microbial origin, was shown to be N-[2(S)-nitrosohydroxylamino-3-methylbutyl] crotonamide based on chemical, spectroscopic and synthetic studies. The total synthesis of dopastin was completed in 8 steps starting from l-valinol. N-Nitrosohydroxylamino function was introduced through an oxaziran with retention of the absolute configuration in the final product. Thus, the 2S-configuration of dopastin was proved by the total synthesis. Racemic dopastin was also synthesized from isobutyraldehyde in 7 steps.     

Habitat use and preference by guanacos,vicuñas,and livestock in an altitudinal gradient in northwest Argentina

Distribution of ungulates is regulated by natural factors, such as presence of other species, climate, and habitat variability, as well as human activities, including livestock grazing and mining. Understanding the spatial dynamics of landscape use can help solve problems of access to key resources by native herbivores. Laguna Brava Reserve is a protected area of Argentina where the two wild South American camelids, guanacos and vicuñas, coexist with cattle, mules, horses, and goats. Information about the effects of livestock and mining activities, which are widespread throughout the region, on wild camelids is scarce. We used variable-width transects to determine the distribution of guanacos, vicuñas, equids, and cattle in relation to five habitat types defined according to vegetation and topography in the reserve. We carried out a correspondence analysis between the proportion of groups observed in and the proportion of area occupied by each habitat, and a χ ² goodness of fit test to establish if camelids and livestock selected a particular habitat type. Vicuñas were associated with grasslands at high altitudes while guanacos and livestock were associated with shrublands at lower elevations. This coevolutionary segregation between guanacos and vicuñas possibly reduced competition between the two species. Competition between camelids and livestock is probably low because of the low density of livestock. Vegas were preferred by all species year round but used more intensively in summer. In order to conserve the wild camelids of the region, governments must implement measures to conserve the critical habitat provided by the vegas.     

Nitrogen,phosphorus and Daphnia grazing in controlling phytoplankton biomass and composition – an experimental study

The role of nitrogen as a factor controllingphytoplankton biomass was studied in nutrientenrichment incubations in the laboratory using waterfrom pelagic region of two mesotrophic lakes ineastern Finland, Lake Kallavesi (in year 1994) andLake Juurusvesi (in year 1995). We used differentcombinations of phosphorus and nitrogen additions ina total of eight experiments. Furthermore, we includedDaphnia grazing treatment to the experimentaldesign in Lake Juurusvesi experiments. The nitrogentreatments did not increase chlorophyll aconcentration in any of the experiments compared withthe controls. Chlorophyll a content was highestin those nutrient treatments where phosphorus wasadded with or without nitrogen. Daphnia grazingdecreased chlorophyll a concentration comparedwith non-grazed treatments. In some cases grazing alsocaused higher ammonium concentrations. Theseexperiments, as well as the nutrient ratio of the lakewater used, suggest that phosphorus is likely tocontrol the amount of phytoplankton biomass. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tropical forest fragmentation and nest predation – an experimental study in an Eastern Arc montane forest, Tanzania

When a habitat becomes fragmented and surrounded by another habitat this generally causes an increase in predation pressure at habitat transitions, often referred to as an edge effect. Edge effect in the form of enhanced nest predation intensities is one of the most cited explanations for bird population declines in fragmented landscapes. Here, we report results from a nest predation experiment conducted in a tropical montane forest landscape in the Uzungwa Mts., Tanzania. Using artificial nests with chicken eggs, we determined predation rates across a fragmentation gradient. The proportion of indigenous forest in four landscapes used in the study were 0.29, 0.58, 0.75 to 1.0. Nest predation intensities on artificial nests were about 19% higher inside intact forest than at edges in fragmented forest landscapes. Furthermore, predation intensities were relatively constant across a forest fragmentation gradient. Our results thus challenge the applicability and generality of the edge effect, derived from studies almost exclusively conducted in temperate regions rather than tropical forest ecosystems. Nest predation levels differences between tropical montane forest and that reported in other forest ecosystems are discussed.     

2��-Deoxyriboguanylurea, the primary breakdown product of 5-aza-2��-deoxyribocytidine, is a mutagen, an epimutagen, an inhibitor of DNA methyltransferases and an inducer of 5-azacytidine-type fragile sites

5-Aza-2′-deoxycytidine (5azaC-dR) has been employed as an inhibitor of DNA methylation, a chemotherapeutic agent, a clastogen, a mutagen, an inducer of fragile sites and a carcinogen. However, its effects are difficult to quantify because it rapidly breaks down in aqueous solution to the stable compound 2′-deoxyriboguanylurea (GuaUre-dR). Here, we used a phosphoramidite that permits the introduction of GuaUre-dR at defined positions in synthetic oligodeoxynucleotides to demonstrate that it is a potent inhibitor of human DNA methyltransferase 1 (hDNMT1) and the bacterial DNA methyltransferase (M.EcoRII) and that it is a mutagen that can form productive base pairs with either Guanine or Cytosine. Pure GuaUre-dR was found to be an effective demethylating agent and was able to induce 5azaC-dR type fragile sites FRA1J and FRA9E in human cells. Moreover, we report that demethylation associated with C:G → G:C transversion and C:G → T:A transition mutations was observed in human cells exposed to pure GuaUre-dR. The data suggest that most of the effects attributed to 5azaC-dR are exhibited by its stable primary breakdown product.