Testing the 'photoinhibition' model of coral bleaching using chemical inhibitors

Explants of the hard coral Seriatopora hystrix were exposed to sublethal concentrations of the herbicide diuron DCMU (N'-(3,4-dichlorophenyl,-N,N-dimethylurea)) and the heavy metal copper. Pulse amplitude modulated (PAM) chlorophyll fluorescence techniques were used to assess the effects on the photosynthetic efficiency of the algal symbionts in the tissue (in Symbio), and chlorophyll fluorescence and counts of symbiotic algae (normalised to surface area) were used to assess the extent of coral bleaching. At 30 mug DCMU l(-1), there was a reduction in both the maximum effective quantum yield (DeltaF/F-m') and maximum potential quantum yield (F-v/F-m) of the algal symbionts in symbio. Corals subsequently lost their algal symbionts and discoloured (bleached), especially on their upper sunlight-exposed surfaces. At the same DCMU concentration but under low light (5% of growth irradiance), there was a marked reduction in DeltaF/F-m' but only a slight reduction in F-v/F-m and slight loss of algae. Loss of algal symbionts was also noted after a 7 d exposure to concentrations as low as 10 mug DCMU l(-1) under normal growth irradiance, and after 14 d exposure to 10 mug DCMU l(-1) under reduced irradiance. Collectively the results indicate that DCMU-induced bleaching is caused by a light-dependent photoinactivation of algal symbionts, and that bleaching occurs when F-v/F-n, (measured 2 h after sunset) is reduced to a value of less than or equal to 0.6. Elevated copper concentrations (60 mug Cu l(-1) for 10 h) also induced a rapid bleaching in S. hystrix but without affecting the quantum yield of the algae in symbio. Tests with isolated algae indicated that substantially higher concentrations (300 mug Cu l(-1) for 8 h) were needed to significantly reduce the quantum yield. Thus, copper-induced bleaching occurs without affecting the algal photosynthesis and may be related to effects on the host (animal). It is argued that warm-water bleaching of corals resembles both types of chemically induced bleaching, suggesting the need for an integrated model of coral bleaching involving the effect of temperature on both host (coral) and algal symbionts.

The Equilibrium Flux Method for the calculation of flows with non-equilbrium chemical reactions

The Equilibrium Flux Method [1] is a kinetic theory based finite volume method for calculating the flow of a compressible ideal gas. It is shown here that, in effect, the method solves the Euler equations with added pseudo-dissipative terms and that it is a natural upwinding scheme. The method can be easily modified so that the flow of a chemically reacting gas mixture can be calculated. Results from the method for a one-dimensional non-equilibrium reacting flow are shown to agree well with a conventional continuum solution. Results are also presented for the calculation of a plane two-dimensional flow, at hypersonic speed, of a dissociating gas around a blunt-nosed body.

Triple objective team mentoring: achieving learning objectives with chemical engineering students

A sophisticated style of mentoring has been found to be essential to support engineering student teams undertaking technically demanding, real-world problems as part of a Project-Centred Curriculum (PCC) at The University of Queensland. The term ‘triple-objective’ mentoring was coined to define mentoring that addresses not only the student’s technical goal achievement but also their time and team management. This is achieved through a number of formal mentor meetings that are informed by a confidential instrument which requires students to individually reflect on team processes prior to the meeting, and a checklist of technical requirements against which the interim student team progress and achievements are assessed. Triple-objective mentoring requires significant time input and coordination by the academic but has been shown to ensure effective student team work and learning undiminished by team dysfunction. Student feedback shows they value the process and agree that the tools developed to support the process are effective in developing and assessing team work and skills with average scores mostly above 3 on a four point scale.

Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells

A method is presented for the direct extraction of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R-3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (

Prediction of the chemical composition of lamb carcasses from multi-frequency impedance data

Multi-frequency bioimpedance analysis (MFBIA) was used to determine the impedance, reactance and resistance of 103 lamb carcasses (17.1-34.2 kg) immediately after slaughter and evisceration. Carcasses were halved, frozen and one half subsequently homogenized and analysed for water, crude protein and fat content. Three measures of carcass length were obtained. Diagonal length between the electrodes (right side biceps femoris to left side of neck) explained a greater proportion of the variance in water mass than did estimates of spinal length and was selected for use in the index L-2/Z to predict the mass of chemical components in the carcass. Use of impedance (Z) measured at the characteristic frequency (Z(c)) instead of 50 kHz (Z(50)) did not improve the power of the model to predict the mass of water, protein or fat in the carcass. While L-2/Z(50) explained a significant proportion of variation in the masses of body water (r(2) 0.64), protein (r(2) 0.34) and fat (r(2) 0.35), its inclusion in multi-variate indices offered small or no increases in predictive capacity when hot carcass weight (HCW) and a measure of rib fat-depth (GR) were present in the model. Optimized equations were able to account for 65-90 % of the variance observed in the weight of chemical components in the carcass. It is concluded that single frequency impedance data do not provide better prediction of carcass composition than can be obtained from measures of HCW and GR. Indices of intracellular water mass derived from impedance at zero frequency and the characteristic frequency explained a similar proportion of the variance in carcass protein mass as did the index L-2/Z(50).

Modeling ex vivo hematopoiesis using chemical engineering metaphors

Ex vivo hematopoiesis is increasingly used for clinical applications. Models of ex vivo hematopoiesis are required to better understand the complex dynamics and to optimize hematopoietic culture processes. A general mathematical modeling framework is developed which uses traditional chemical engineering metaphors to describe the complex hematopoietic dynamics. Tanks and tubular reactors are used to describe the (pseudo-) stochastic and deterministic elements of hematopoiesis, respectively. Cells at any point in the differentiation process can belong to either an immobilized, inert phase (quiescent cells) or a mobile, active phase (cycling cells). The model describes five processes: (1) flow (differentiation), (2) autocatalytic formation (growth),(3) degradation (death), (4) phase transition from immobilized to mobile phase (quiescent to cycling transition), and (5) phase transition from mobile to immobilized phase (cycling to quiescent transition). The modeling framework is illustrated with an example concerning the effect of TGF-beta 1 on erythropoiesis. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.

Temperature-induced bleaching of corals begins with impairment of the CO2 fixation mechanism in zooxanthellae

The early effects of heat stress on the photosynthesis of symbiotic dinoflagellates (zooxanthellae) within the tissues of a reef-building coral were examined using pulse-amplitude-modulated (PAM) chlorophyll fluorescence and photorespirometry. Exposure of Stylophora pistillata to 33 and 34 degrees C for 4 h resulted in (1) the development of strong non-photochemical quenching (qN) of the chlorophyll fluorescence signal, (2) marked decreases in photosynthetic oxygen evolution, and (3) decreases in optimal quantum yield (F-v/F-m) of photosystern II (PSII), Quantum yield decreased to a greater extent on the illuminated surfaces of coral branches than on lower (shaded) surfaces, and also when high irradiance intensities were combined with elevated temperature (33 degrees C as opposed to 28 degrees C), qN collapsed in heat-stressed samples when quenching analysis was conducted in the absence of oxygen, Collectively, these observations are interpreted as the initiation of photoprotective dissipation of excess absorbed energy as heat (qN) and O-2-dependent electron flow through the Mehler-Ascorbate-Peroxidase cycle (MAP-cycle) following the point at which the rate of light-driven electron transport exceeds the capacity of the Calvin cycle. A model for coral bleaching is proposed whereby the primary site of heat damage in S, pistillata is carboxylation within the Calvin cycle, as has been observed during heat damage in higher plants, Damage to PSII and a reduction in F-v/F-m (i.e. photoinhibition) are secondary effects following the overwhelming of photoprotective mechanisms by light. This secondary factor increases the effect of the primary variable, temperature. Potential restrictions of electron flow in heat-stressed zooxanthellae are discussed with respect to Calvin cycle enzymes and the unusual status of the dinoflagellate Rubisco, Significant features of our model are that (1) damage to PSII is not the initial step in the sequence of heat stress in zooxanthellae, acid (2) light plays a key secondary role in the initiation of the bleaching phenomena.

Effects of cyanide on coral photosynthesis: implications for identifying the cause of coral bleaching and for assessing the environmental effects of cyanide fishing

Modulated chlorophyll fluorescence techniques were used to examine the effects of cyanide (NaCN) from cyanide fishing on photosynthesis of the symbiotic algae (zooxanthellae) located within the tissues of the zooxanthellate hard coral Plesiastrea versipora. Incubating corals for 3 h in a cyanide concentration of >10(-5) M NaCN under a saturating light intensity (photosynthetically active radiation [PAR] intensity of 250 mu mol quanta m(-2) s(-1)) caused a long-term decrease in the ratio of variable to maximal fluorescence (dark-adapted F-v/F-m). The effect of cyanide on dark-adapted F-v/F-m was Light dependent; thus F-v/F-m only decreased in corals exposed to 10(-4) M NaCN for 3 h under PAR of 250 mu mol quanta m(-2) s(-1). In corals where dark-adapted F-v/F-m was significantly lowered by cyanide exposure, we observed significant loss of zooxanthellae from the tissues. causing the corals to discolour (bleach). To further examine the light-dependent effect of cyanide and its relation to loss of zooxanthellae, corals were exposed to 10-4 M NaCN or seawater only (control), either in darkness or under 250 mu mol quanta m(-2) s(-1). ill significant decrease in dark-adapted F-v/F-m and loss of zooxanthellae only occurred in corals exposed to cyanide in the light. These results suggest cyanide causes the dissociation of the symbiosis (bleaching) by affecting photosynthesis of the zooxanthellae. Quenching analysis using the saturation-pulse technique revealed the development of high levels of non-photochemical quenching in cyanide-exposed coral. This result is consistent with the known property of cyanide as an inhibitor of the dark reactions of the Calvin cycle, specifically as an inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Therefore, chronic photoinhibition and an impairment of photosynthesis of zooxanthellae provides an important 'signal' to examine the environmental effects of cyanide fishing during controlled releases in situ.

Determination of chemical shielding tensor of an indole carbon and application of tryptophan orientation of a membrane peptide

Using tryptophan C-13-enriched at the C-4 (C epsilon(3)) of the indole, the orientation of the C epsilon(3) chemical shift tensor relative to the C epsilon(3)-H dipolar axis was determined from the C-13 chemical shift/C-13-H-1 dipolar 2D NMR powder pattern. The principal values obtained were 208, 137 and 15 ppm with sigma(33) perpendicular to the indole plane, and sigma(11) (least shielded direction) 5 degrees off the C epsilon(3)-H bond toward C xi(3). The side off the C epsilon(3)-H bond was determined by comparing the reduced chemical shift anisotropies obtained by solid-state NMR and from molecular dynamics calculations of [4-C-13] tryptophans in gramicidin A aligned in phospholipid membranes. (C) 1999 Elsevier Science B.V. All rights reserved.

Chemical synthesis and folding pathways of large cyclic polypeptide: Studies of the cystine knot polypeptide kalata B1

Kalata B1 is a member of a new family of polypeptides, isolated from. plants, which have a cystine knot structure embedded within an amide-cyclized backbone. This family of molecules are the largest known cyclic peptides, and thus, the mechanism of synthesis and folding is of great interest. To provide information about both these phenomena, we have synthesized kalata B1 using two distinct strategies. In the first, oxidation of the cysteine residues of a linear precursor peptide to form the correct disulfide bonds results in folding of the three-dimensional structure and preorganization of the termini in close proximity for subsequent cyclization. The second approach involved cyclization prior to oxidation. In the first method, the correctly folded peptide was produced only in the presence of partially hydrophobic solvent conditions. These conditions are presumably required to stabilize the surface-exposed hydrophobic residues. However,; in the synthesis,involving cyclization prior to oxidation, the cyclic reduced peptide folded to a significant degree in the absence of hydrophobic solvents and even more efficiently in the presence of hydrophobic solvents. Cyclization clearly has a major effect on the folding pathway and facilitates formation of the correctly disulfide-bonded form in aqueous solution; In addition to facilitating folding to a compact stable structure cyclization has an important effect on biological activity as assessed by hemolytic activity.

Submarine groundwater discharge and associated chemical input to a coastal sea

This paper presents a theoretical model of flow and chemical transport processes in subterranean estuaries (unconfined brackish groundwater aquifers at the ocean-land interface). The model shows that groundwater circulation and oscillating flow, caused by wave setup and tide, may constitute up to 96% of submarine groundwater discharge (SGWD) compared with 4% due to the net groundwater discharge. While these local flow processes do not change the total amount of land-derived chemical input to the ocean over a long period (e.g., yearly), they induce fluctuations of the chemical transfer rate as the aquifer undergoes saltwater intrusion. This may result in a substantial increase in chemical fluxes to the ocean over a short period (e.g., monthly and by a factor of 20 above the averaged level), imposing a possible threat to the marine environment. These results are essentially consistent with the experimental findings of Moore [1996] and have important implications for coastal resources management.