The production of azurin and similar proteins

Summary It has been shown that, in the organisms tested, the production of azurin or similar blue, copper-protein complexes is confined to bacterial species of the three genera Bordetella, Alcaligenes and Pseudomonas. In the strain of Pseudomonas aeruginosa used, there appeared to be no difference in the amount of azurin occurring when the strain was grown aerobically and anaerobically. The amount of azurin produced by representative strains of Ps. aeruginosa, B. bronchiseptica and A. denitrificans varied with the copper content of the medium. Above a level of 5 g copper/ml of medium, the azurin content was constant for the three species tested; below a copper level of 0.5 g/ml there was an almost total absence of azurin although good growth occurred. Under similar growth conditions, the azurin content of the three bacterial species studied was not significantly different.The possible role of azurin is discussed.     

Copper and cobalt uptake by metallophytes from Zaïre

Summary Pot trials were carried out on plants ofHaumaniastrum katangense, Haumaniastrum robertii andAeolanthus biformifolius. These metallophytes from Zaïre were grown in substrates containing from 0–10,000 g/g (0–1%) copper or cobalt. The tolerance of each species was determined and for cobalt was highest inH. robertii (4000 g/g) and for copper was highest inAeolanthus biformifolius (9000 g/g). Discontinuities in the plant-soil curves for each element indicate an exclusion mechanism operating for all three species at lower concentrations of the element in the soil. All species would grow in soils containing only traces of cobalt or copper and this indicated that uptake of heavy metals was not linked to a physiological requirement for either element. The plant-soil relationship for cobalt (P<0.001) was sufficiently good for all three species for them to be useful in biogeochemical prospecting for this element.     

Copper and cobalt uptake byHaumaniastrum species

Summary Herbarium specimens of the genus Haumaniastrum (mainly from Shaba Province Zaïre) were analysed for copper and cobalt.H. homblei showed elevated copper levels and indicated its tolerance to mineralized ground.H. robertii (previously-known as a copper flower) showed the expected high copper concentrations, but contained abnormally high concentrations of cobalt (av. 4304 g/g dry weight) far exceeding those of copper. This species has the highest cobalt content of any phanerogam and its distribution may be controlled by cobalt rather than by copper. re]19761220     

Copper requirement and its influence on the growth of the obligate methylotrophic bacterial strain 4025

Summary Requirement of copper for growth of the obligate methanol-utilizing bacterium, strain 4025 was investigated. Strong demand for copper compounds was determined in the surface growth on methanol and methylamine. A pronounced requirement for copper and iron was evident during submerged growth of the strain on methanol; both elements affected growth and methanol utilization of the cultures. The optimal concentration of several copper compounds was 1.2 moles per liter. The copper effect was more pronounced with ammonium than with nitrate as the nitrogen source.     

Heavy metals in Lake George, Uganda, with relation to metal concentrations in tissues of common fish species

The northern end of Lake George, Uganda, and its associated wetlands receive localized metal pollution from a former copper mine and tailings left after metal extraction. The aim of this study was to determine (i) whether the heavy metals are a threat to the biology of the major commercial fish species and (ii) whether consumption of the fish threatens human health. Concentrations of copper, zinc, cobalt and nickel in detrital sediments, plankton, and five fish species from sites in Lake George, the Kazinga Channel and Lake Edward (which are inter-connected) were determined using atomic absorption spectroscopy. The detrital sediments of Hamukungu Bay, Lake George, had average concentrations (g/g dry weight) of 96.3 zinc, 270.4 copper, 57.4 cobalt and 42.8 nickel. There were no significant differences between the Hamukungu Bay and the North Lake George site of Bushatu: both receive inflows from the mining activities. Concentrations of copper and zinc were significantly higher than background values from unpolluted freshwater ecosystems. Plankton samples showed a metal concentration gradient consistent with a gradient from the source of pollution in northern Lake George, along the Kazinga Channel to Lake Edward. The liver tissues of fish had markedly higher concentrations of copper and zinc than flesh. Concentrations of cobalt and nickel were relatively low. The highest mean concentrations of metals in liver tissue occurred in Oreochromis leucostictus (189.0 g/g Cu) and Bagrus docmac (187.5 g/g Zn) whilst the lowest occurred in Oreochromis niloticus (15.3 g/g and 78.2 g/g dry weight copper and zinc, respectively). However, O. niloticus contained the highest concentrations of cobalt (11.2 g/g) and nickel (3.8 g/g). Liver Somatic Indices (LSI) of the fish species from the different sites indicated a reduction of LSI in those fish from the most contaminated zones of northern Lake George compared with all other sites. This suggests there could be anatomical and physiological abnormalities linked to the heavy metal pollution. The flesh had only low concentrations of metals; well within international guidelines for consumption. A person would have to consume 9 kg of fresh flesh of Clarias sp. and 65 kg of O. leucostictus daily to exceed the WHO recommended intake for copper, and even more for other metals. This implies that currently metal pollution in Lake George presents an ecological rather than a human health concern.     

Copper but not iron limitation increases astaxanthin production by Phaffia rhodozyma in a chemically defined medium

The influence of copper (0–32 M) and iron (0–108 M) on growth and astaxanthin production by Phaffia rhodozyma was studied. Copper below 3.2 M increased the astaxanthin content of the cells (from 220 to 287 g g^–1) but at the expense of a slightly decreased growth (from 11.3 to 10.2 mg ml^–1). In contrast, iron below 1 M decreased both the growth and astaxanthin content of the cells. Using copper limitation instead of toxic respiratory inhibitors to improve astaxanthin production has obvious advantages from the product quality, environmental and process operation points of view.     

Distinct morphological and mito-inhibitory effects induced by TGF-β1, HGF and EGF on mouse,rat and human hepatocytes

TGF-1 is known as a potent inhibitor of proliferation of rat and human hepatocytes. In this study we show that the effects of TGF-1 are quite different on mouse hepatocytes. In rat and human hepatocytes, TGF-1 inhibited DNA synthesis and also inhibited the morphological changes induced by growth factors in rat and human hepatocytes. In contrast, addition of TGF-1 to mouse hepatocytes resulted in pronounced alterations in morphology of these cells. These changes were similar to those induced by HGF and EGF. The induction of structural changes by TGF-1 was noted only in mouse hepatocytes. Mouse hepatocytes were also much more resistant to the mito-inhibitory effect of TGF-1. These findings suggest profound differences in hepatocyte growth regulation between these species and may relate to observed differences in susceptibility to carcinogenesis.Abbreviations EGF epidermal growth factor - HGF hepatocyte growth factor - SF scatter factor - TGF-1 transforming growth factor beta type one     

Tolerance and bioaccumulation of copper in Phragmites australis and Zea mays

The effects of copper on the growth, tolerance indices, mineral composition (N, P, K, Fe, Zn and Mn) and metal uptake of reed (Phragmites australis [Cav. Trin. ex Steudel]) and maize (Zea mays L.) were investigated in hydroponic experiments at copper concentrations ranging from 0.5 to 157 M Cu. A reduction in root length was shown to be a good indicator of copper toxicity, concentrations of 15.7 and 78.7 M Cu inhibiting root growth in maize and reed, respectively. The reed was significantly more tolerant of copper than maize and at 7.85 M Cu (external concentration), reed can be described as a Cu tolerant plant, and maize as a Cu non-tolerant species. As a result of Cu toxicity, the concentrations of macronutrients N, P and K decreased in both shoot and root of maize, while the concentrations were hardly affected in reed tissues. Fe concentration increased in shoots and roots of maize and in roots of reed with increasing Cu treatments, leading to highly significant (p<0.01) linear relationships between tissue Fe and Cu concentrations. The bioconcentration factor (BCF) of Cu was higher in roots than in shoots of both plant species, ranging from 612 to 1592 in reed for the Cu treatments tested. In the roots of maize, BCF of Cu increased from 349 to 1931 when increasing Cu in nutrient solution from 7.85 M to 78.5 M. Therefore, reed could be useful in wastewater treatments for the removal of Cu. However, the use of reed in phytoextraction of Cu from contaminated soils is limited by the low accumulation rate in shoots and although reed can be more efficient than maize for Cu phytoextraction, harvesting the full biomass, including roots, may be required.     

Somatic embryogenesis and plant regeneration inMedicago suffruticosa

A successful procedure has been designed for the regeneration of plantlets from leaf sections of the self-pollinating species,Medicago suffruticosa. Callus growth was promoted by a 4-week culture period on liquid Kao's medium containing 4.9 M benzyladenine and 4.5 M 2,4-dichlorophenoxyacetic acid (2,4-d), followed by a 4-day treatment in which the benzyladenine was elevated to 44.4 M. Shoots/plantlets were observed after 3–4 weeks culture on growth regulator-free agar-solidified medium. Under these conditions, the regeneration frequency from callus was 18% and a histological study showed that this regeneration was through somatic embryogenesis. The growth regulator treatment, with a relatively high concentration of growth regulators (44.4 M benzyladenine) for a short time period (4 days), is important for inhibiting polyphenol compounds and for stimulating callus growth and plant regeneration.Abbreviations 2,4-d 2,4-dichiorophenoxyacetic acid - BA benzyladenine - NAA -napthaleneacetic acid     

Effects of copper on algal communities at different current velocities

This study examines the influence of current velocity in the toxiceffect of copper in diatom-dominated biofilms grown in artificial channels.Effects on community structure, algal biomass and photosynthesis (carbonincorporation) caused by 15 g L^–1 of copperwere tested at contrasting (1 and 15 cm s^–1)velocities. Moreover, a possible threshold on the effect of copper on algalbiomass and photosynthesis related to current velocity was examined by usingprogressively increasing current velocity (1 to 50 cms^–1) at 15 g L^–1 Cu.Chlorophyll-a decreased ca. 50% as a result of addition of15 g L^–1 Cu. Chlorophyll decrease occurredearlier at 15 cm s^–1 than at 1 cms^–1 when adding 15 g L^–1Cu. Copper also caused a remarkable decrease in carbon incorporation(from 30 to ca. 50%), which was produced earlier at 15 cms^–1 (three days) than at 1 cms^–1 (seven days). Some taxa were affected by thecombination of copper and current velocity. Both Achnanthesminutissima and Stigeoclonium tenue becomedominant at 15 cm s^–1 in the presence of copper.Significant inhibition of algal growth in 15 g L^–1Cu occurred at low (1 cm s^–1) and highvelocities (50 cm s^–1), but not at intermediatevelocity (20 cm s^–1). The experiments indicatethat current velocity triggers the effect that copper has on diatom-dominatedbiofilms, and that the effect is more remarkable at low and high than atintermediate current velocities.     

Electron paramagnetic resonance studies and effects of vanadium in Saccharomyces cerevisiae

Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO₄ ^3- concentration and the growth was completely arrested at a concentration of 20 mM of VO₄ ^3- in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A mobile and an immobile species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rotational correlation time _r indicated the relative motional freedom at the macromolecular site. A strongly immobilized vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO²⁺ ions with isolated cell walls.     

Copper exposure and potential biomarkers of copper metabolism

Relevant biological effects associated with mild to moderate copper deficiency and copper excess are unknown. It is difficult to identify markers of these early changes because limits of the homeostatic range are still undefined and early changes may represent adaptive responses that do not imply necessarily risk of damage. We report here a series of studies carried out to shed light on the responses within the homeostatic range, by assessing classic parameters of copper status in humans at different copper exposure. In adult healthy volunteers that had an estimated daily intake of 0.9 mg Cu/day (approximately 15 g/kg/d), exposure to additional 50–60 g of copper/kg/day for three months or up to 150 g/kg/d for two months resulted in no significant changes of SOD activity in erythrocytes, of copper concentration (in serum, erythrocytes and mononuclear cells) and of serum ceruloplasmin (ANOVA). Neither were found differences by gender or age. As in previous studies in infants, the non-ceruloplasmin copper fraction was positively correlated to serum copper (r=0.58). Assessing variations on copper absorption, infants supplemented/not supplemented with oral copper (80 ug/kg/14 days), at age 1 and 3 months, showed copper absorption close to 80% at both ages; no effect was observed for age or supplementation, suggesting that either these concentrations do not elicit regulatory mechanisms or that at this age down regulation for copper absorption is not efficient. These studies indicate that in the range of the copper homeostasis area the markers tested are not suitable to detect mild changes (within the homeostatic range) of copper metabolism.     

Plant uptake and leaching of copper during EDTA-enhanced phytoremediation of repacked and undisturbed soil

EDTA-enhanced phytoremediation of copper contaminated soil was evaluated. Up to 740 g g^–1 of Na₂H₂ EDTA in solution was added to repacked soil columns, and intact cores of a sandy loam of volcanic origin, that was growing Agrostis tenuis. The soil contained up to 400 g g^–1 of copper due to a history of fungicide spraying. EDTA application increased the herbage copper concentration of the grass growing in repacked soil from 30 to 300 g g^–1, but the same application to an intact core only brought about an increase from 10 to 60 g g^–1. More copper accumulated in the herbage when the EDTA was applied in numerous small doses than in just one or two larger amounts. Calculation of the concentration of copper in the water taken up by the grass revealed this to be two orders of magnitude lower than that in the soil solution. As a result of the EDTA applications, about 100 times more copper was leached than was taken up by the herbage. This means that a strategy for managing leaching losses needs to be part of any plan for EDTA-enhanced phytoremediation.     

Copper toxicity in young maize (Zea mays L.) plants: effects on growth, mineral and chlorophyll contents, and enzyme activities

Changes in the growth parameters and in enzyme activities were studied in roots and leaves of 14-days old maize grown in a nutrient solution containing various copper concentrations (i.e. 0.01 to 10 M). A significant decrease in root and leaf biomass was only found at 10 M Cu. In contrast, changes in several enzyme activities occured at lower copper concentrations in the solution, corresponding to different threshold values which are lower than those observed for growth parameters. Peroxidase (POD) activity significantly increased in all investigated plant organs (i.e. 3rd-leaf, 4th-leaf and roots) in relation to their copper content. Additionally, glucose-6-phosphate dehydrogenase (G-6-PDH), and isocitrate dehydrogenase (ICDH) activities decreased in the leaves, especially in the 4th-leaf. However, the activity of malic enzyme (ME), G-6-PDH, glutamate dehydrogenase (GDH) and ICDH increased with the copper content in roots. According to the relationship between POD activity and copper content, the toxic critical value was set at 26 mg Cu per kg dry matter (DM) in roots and 21 mg Cu per kg DM in the 3rd-leaf. In roots, a new isoenzyme of peroxidase appeared for copper content above 12.6 mg Cu kg DM^–1. Measurement of enzyme activity, especially that of POD and Cu-specific changes in the (iso)peroxidase pattern, might be used as biomarkers to assess the phytotoxicity for maize grown on copper-contaminated substrata.     

Determination of copper, manganese and zinc in human liver

Autopsied liver tissue samples collected from 42 males and 31 females were analyzed for copper, manganese and zinc using atomic absorption spectrometry (AAS). With the exception of two liver samples for which the copper levels were determined to be 74.8 and 104.0 g/g (dry weight), hepatic copper concentrations were found to range from 1.7 to 32.4 g/g with a mean concentration of 14.2 g/g and standard deviation of 7.0 g/g. Manganese concentrations (with the exception of one sample having 12.9 g/g) ranged from 0.22 to 4.6 g/g with a mean of 2.26 ± 1.00 g/g. Hepatic zinc levels averaged 118.3 ± 44.4 g/g and ranged from 38.5 to 231.3 g/g. There were no apparent trends for the levels of any metals versus age nor were there any differences in average hepatic metal concentrations for males and females. © Rapid Science 1998.     

Reusability of immobilised Saccharomyces cerevisiae with successive copper adsorption-desorption cycles

Summary Immobilised Saccharomyces cerevisiae in batch reactors effectively removed copper from solution with a binding equilibrium of 70 % being attained within 20 minutes of contact. Maximum uptake was between pH 3 to 5 (V_max = 24.1 mol/g) and was substantially reduced at pH 2. Bound copper was readily recovered by addition of 1.0 M HCl ( % v/v). In adsorption-desorption studies metal removal and recovery was high, and uptake was increased with repeated use. Electron microscopy confirmed that no morphological changes occur to the cells during repeated adsorption-desorption.     

Superoxide dismutase: An enzyme system for the study of micronutrient interactions in plants

The effect of different Mn levels on the isozyme pattern of superoxide dismutase was investigated. Pisum sativum L. plants were grown in nutrient solutions containing three Mn concentrations: 0.005 g/ml (deficient), 0.05 g/ml (low), and 0.5 g/ml (optimum). Leaf extracts contained three electrophoretically distinct superoxide dismutases (SOD), two of which were inhibited by cyanide and were probably Cu-Zn-SODs, while the third one was CN-insensitive and could be either an Mn- or an Fe-SOD. At 0.005 g/ml Mn supply the CN-insensitive SOD was significantly depressed at 15, 30, and 45 days of growth, whereas at 0.05 g/ml Mn this isozyme was significantly decreased only at 45 days growth. The two CN-sensitive SODs were inversely related to the CN-resistant enzyme, the activities of the former enzymes being significantly increased at Mn-deficient levels throughout plant growth. Metal determinations of the plants showed that at low concentrations of Mn in the nutrient media, copper and zinc content of leaves increased: the lower the Mn level, the higher the increase produced. The CN-resistant SOD activity, as judged by its dependency on Mn, appears to be an Mn-SOD rather than an Fe-SOD. In the light of the results obtained, the use of the enzyme system superoxide dismutase for the study of the role and interactions between Mn, Cu, and Zn in the plant cell is proposed.Abbreviations EDTA ethylenediaminotetraacetic acid - NBT nitro blue terazolium - SOD superoxide dismutase (EC 1.15.1.1)     

Toxigenic fungi and the deterioration of Nigerian poultry feeds

Growth of toxigenic strains of Aspergillus clavatus Des. and Aspergillus flavus Link at 30°C on milled poultry feeds led to a considerable decrease in the protein, oil and crude fibre contents of the feed substrate. A corresponding increase in the free fatty acid fractions of the feeds due to the activities of these microbes was also recorded. Rapid degradation of the feedstuff by both species was recorded at a temperature of 25°C and 30°C and a pH range of 4.8–6.4. When grown on feed infusion broth at 30°C, the highest amounts of mycelial production with sporulation of both fungal species occurred within the 8-day incubation period. A determination of their extra-cellular enzyme profile showed the production of amylases, pectate lyase, cellulases, proteases, lipases, xyalanases, DNase and RNase.All the carbon and nitrogen sources used (except L-sorbose and DL-tryptophan), supported good mycelial growth with sporulation. An optimal CN ratio of 5.04.5 and 7.53.0 was recorded for growth and sporulation of A. clavatus. For A. flavus, a CN ratio of 7.54.5 was found best for growth and 5.03.0 for sporulation.     

Growth and competition between seedlings of an invasive plantation tree, <Emphasis Type="Italic">Acacia mangium,</Emphasis> and those of a native Borneo heath-forest species, <Emphasis Type="Italic">Melastoma beccarianum</Emphasis>

An introduced plantation tree species, Acacia mangium Willd., is becoming invasive in the Brunei region of Borneo. To examine its invasive potential, a greenhouse, additive series experiment (target–neighbour) involving seedlings of A. mangium and those of a common native heath-forest (kerangas), Melastoma beccarianum Cogn. was carried out under low and high light regimes in intra- and interspecific combinations over a 6-month period. Significant variations in growth parameters (other than biomass allocation patterns) existed amongst seedlings from different treatments. A major part of this variation in growth could be attributed to the main factors of target species, neighbour species, and competition (seedling density). For the growth variables examined, the target–species response was not consistent across light regimes. Under high light conditions, Acacia was the better competitor; the Lotka-Volterra competition coefficient effect of Melastoma on Acacia was lower (=0.30) than the effect of Acacia on Melastoma (=0.54). However, the reverse occurred under low light conditions with Melastoma gaining the upper hand (=1.45 and =0.44). These results show that light (and hence disturbance) can strongly influence the pattern and intensity of both intra- and interspecific competition between invasive and local flora species. Relatively intact forest is unlikely to be invaded by Acacia trees (as they are poor competitors under this scenario). On the other hand, the Acacia trees can easily invade disturbed forests, especially those prone to recurring drought and fire, and over time convert the habitats to nearly monospecific stands, as is presently being observed in Brunei.     

Water relations and growth of shrubs before and after fire in a semi-arid woodland

Summary Plant water relations and shoot growth rate of shrubs resprouting after fire or unburnt were measured in a semi-arid poplar box (Eucalyptus populnea) shrub woodland of eastern Australia. In vegetation unburnt for about 60 years, the dawn xylem water potential (_x) of the dominant shrub species was about-1.0 MPa when the soil was wet and-8.0 MPa when the soil was very dry. At any one time, the dominant shrub species,Eremophila mitchellii, E. sturtii, Geijera parviflora andCassia nemophila, were similar in _x butAcacia aneura andDodonaea viscosa were consistently higher in _x than this group when the soil was moist and lower when the soil was dry. The dominant tree species,Eucalyptus populnea andE. intertexta, appeared to have access to additional water beneath the hardpan which is located 60–80 cm below the surface. When shrubs were under extreme water stress (_x of-8 MPa), the trees had a _x of-3 to-3.6 MPa. Following a fire, both _x and leaf stomatal conductance (g _s) of resprouting shrubs were higher for about 5 years than comparable-aged unburnt vegetation, with relative differences in _x increasing with drought stress. Elongation rate of resprouts was positively linked to prefire shrub height in 3 of 4 species. However, shrubs resprouting after high intensity fires had substantially higher rates of shoot elongation than after low intensity fires which were in turn higher than for foliar expansion of unburnt shrubs. It is concluded that the growth rate of resprouting shrubs is primarily determined by physiological/ morphological factors associated with plant size but is also assisted by greater availability of water and possibly nutrients for a period after fire.