Topography and spatial arrangement of reef-building corals on the fringing reefs of North Jamaica may influence their response to disturbance from bleaching.

Knowledge of factors that are important in reef resilience helps us understand how reefs react following major environmental disturbances such as hurricanes and bleaching. Here we test factors that might have influenced Jamaican reef resilience to, and subsequent recovery from, the 2005 bleaching event, and which might help inform management policy for reefs in the future: reef rugosity and contact of corals with macroalgae. In addition, we test in the field, on Dairy Bull reef, whether aggregated Porites astreoides colonies exhibit enhanced growth when exposed to superior competition from Acopora palmata, as has been found by experiment with the Indo-Pacific corals Porites lobata and the superior competitor Porites rus [Idjadi, J.A., Karlson, R.H., 2007. Spatial arrangement of competitors influences coexistence of reef-building corals. Ecology 88, 2449-2454]. There were significant linear relationships between rugosity and the increase in smallest size classes for Sidastrea siderea, Colpophyllia natans, P. astreoides and Agaricia species, and between rugosity and cover of the branching coral Acropora cervicornis. Linear extension rates of A. cervicornis and radial growth rates of P. astreoides were significantly lower (p<0.025; F>6) when in contact with macroalgae. Aggregated colonies of P. astreoides in contact with one another, one of which was in contact with the faster growing competitor A. palmata showed significantly greater growth rates than with just two aggregated P. astreoides colonies alone. These findings suggest that three dimensional topography and complexity is important for reef resilience and viability in the face of environmental stressors such as bleaching. Our findings also support the idea that aggregated spatial arrangements of corals can influence the outcome of interspecific competition and promote species coexistence, important in times of reef recovery after disturbance.

Modelling effects of geoengineering options in response to climate change and global warming: implications for coral reefs.

Climate change will have serious effects on the planet and on its ecosystems. Currently, mitigation efforts are proving ineffectual in reducing anthropogenic CO2 emissions. Coral reefs are the most sensitive ecosystems on the planet to climate change, and here we review modelling a number of geoengineering options, and their potential influence on coral reefs. There are two categories of geoengineering, shortwave solar radiation management and longwave carbon dioxide removal. The first set of techniques only reduce some, but not all, effects of climate change, while possibly creating other problems. They also do not affect CO2 levels and therefore fail to address the wider effects of rising CO2, including ocean acidification, important for coral reefs. Solar radiation is important to coral growth and survival, and solar radiation management is not in general appropriate for this ecosystem. Longwave carbon dioxide removal techniques address the root cause of climate change, rising CO2 concentrations, they have relatively low uncertainties and risks. They are worthy of further research and potential implementation, particularly carbon capture and storage, biochar, and afforestation methods, alongside increased mitigation of atmospheric CO2 concentrations.

Hurricanes and coral bleaching linked to changes in coral recruitment in Tobago.

Knowledge of coral recruitment patterns helps us understand how reefs react following major disturbances and provides us with an early warning system for predicting future reef health problems. We have reconstructed and interpreted historical and modern-day recruitment patterns, using a combination of growth modelling and in situ recruitment experiments, in order to understand how hurricanes, storms and bleaching events have influenced coral recruitment on the Caribbean coastline of Tobago. Whilst Tobago does not lie within the main hurricane belt results indicate that regional hurricane events negatively impact coral recruitment patterns in the Southern Caribbean. In years following hurricanes, tropical storms and bleaching events, coral recruitment was reduced when compared to normal years (p=0.016). Following Hurricane Ivan in 2004 and the 2005-2006 bleaching event, coral recruitment was markedly limited with only 2% (n=6) of colonies estimated to have recruited during 2006 and 2007. Our experimental results indicate that despite multiple large-scale disturbances corals are still recruiting on Tobago's marginal reef systems, albeit in low numbers.

Scleractinian coral population size structures and growth rates indicate coral resilience on the fringing reefs of North Jamaica.

Coral reefs throughout the world are under severe challenges from many environmental factors. This paper quantifies the size structure of populations and the growth rates of corals from 2000 to 2008 to test whether the Discovery Bay coral colonies showed resilience in the face of multiple acute stressors of hurricanes and bleaching. There was a reduction in numbers of colonies in the smallest size class for all the species at all the sites in 2006, after the mass bleaching of 2005, with subsequent increases for all species at all sites in 2007 and 2008. Radial growth rates (mm yr(-1)) of non-branching corals and linear extension rates (mm yr(-1)) of branching corals calculated on an annual basis from 2000-2008 showed few significant differences either spatially or temporally. At Dairy Bull reef, live coral cover increased from 13+/-5% in 2006 to 20+/-9% in 2007 and 31+/-7% in 2008, while live Acropora species increased from 2+/-2% in 2006 to 10+/-4% in 2007 and 22+/-7% in 2008. These studies indicate good levels of coral resilience on the fringing reefs around Discovery Bay in Jamaica.

Dietary glycated protein modulates the colonic microbiota towards a more detrimental composition in ulcerative colitis patients and non-ulcerative colitis subjects.

AIM: To investigate the effect of native, heated and glycated bovine serum albumin (BSA) on the ulcerative colitis (UC) and non-UC colonic microbiota in vitro. METHODS AND RESULTS: Continuous flow culture (CFC) models of the human colonic microbiota inoculated with faeces from UC and non-UC volunteers were maintained on BSA as growth substrate. Changes in bacterial populations and short-chain fatty acids were determined. UC and non-UC microbiota differed significantly in microbial populations, with elevated numbers of sulfate-reducing bacteria (SRB) and clostridia in the microbiota from UC patients. Compared with native BSA, glycated BSA modulated the gut microbiota of UC patients in vitro towards a more detrimental community structure with significant increases in putatively harmful bacteria (clostridia, bacteroides and SRB; P < 0.009) and decreases in dominant and putatively beneficial bacterial groups (eubacteria and bifidobacteria; P < 0.0004). The levels of beneficial short-chain fatty acids were significantly decreased by heated or glycated BSA, but were increased significantly by native BSA. CONCLUSION: The UC colonic microbiota maintained in CFC was significantly modified by glycated BSA. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that dietary glycated protein may impact upon the composition and activity of the colonic microbiota, an important environmental variable in UC.

Aldose reductase and the importance of experimental design.

Kinetic studies on the AR (aldose reductase) protein have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. As with non-enzymatic glycation reactions, there is probably a free-radical element involved derived from monosaccharide autoxidation. In the case of AR, there is free radical oxidation of NADPH by autoxidizing monosaccharides, which is enhanced in the presence of the NADPH-binding protein. Thus any assay for AR based on the oxidation of NADPH in the presence of autoxidizing monosaccharides is invalid, and tissue AR measurements based on this method are also invalid, and should be reassessed. AR exhibits broad specificity for both hydrophilic and hydrophobic aldehydes that suggests that the protein may be involved in detoxification. The last thing we would want to do is to inhibit it. ARIs (AR inhibitors) have a number of actions in the cell which are not specific, and which do not involve them binding to AR. These include peroxy-radical scavenging and effects of metal ion chelation. The AR/ARI story emphasizes the importance of correct experimental design in all biocatalytic experiments. Developing the use of Bayesian utility functions, we have used a systematic method to identify the optimum experimental designs for a number of kinetic model data sets. This has led to the identification of trends between kinetic model types, sets of design rules and the key conclusion that such designs should be based on some prior knowledge of K (m) and/or the kinetic model. We suggest an optimal and iterative method for selecting features of the design such as the substrate range, number of measurements and choice of intermediate points. The final design collects data suitable for accurate modelling and analysis and minimizes the error in the parameters estimated, and is suitable for simple or complex steady-state models.

Chaperone function of mutant versions of alpha A- and alpha B-crystallin prepared to pinpoint chaperone binding sites.

A major stress protein, alpha-crystallin, functions as a chaperone. Site-directed mutagenesis has been used to identify regions of the protein necessary for chaperone function. In this work we have taken some of the previously described mutants produced and assessed their chaperone function by both a traditional heat-induced aggregation method at elevated temperature and using enzyme methods at 37 degrees C. In general the different assays gave parallel results indicating that the same property is being measured. Discrepancies were explicable by the heat lability of some mutants. Most mutants had full chaperone function showing the robust nature of alpha-crystallin. A mutant corresponding to a minor component of rodent alpha A-crystallin, alpha Ains-crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139--> Arg, Thr144-->Arg, Ser59-->Ala mutants of alpha B-crystallin and double mutants Ser45-->Ala/Ser59-->Ala, Lys103--> Leu/His104-->Ile, and Glu110-->His/His111-->Glu. A mutant Phe27-->Arg that was the subject of previous controversy was shown to be fully active at physiological temperatures.

A heterologous expression system for bovine lens transmembrane main intrinsic protein (MIP) in Nicotiana tabacum plants.

We have developed a heterologous expression system for transmembrane lens main intrinsic protein (MIP) in Nicotiana tabacum plant tissue. A native bovine MIP26 amplicon was subcloned into an expression cassette under the control of a constitutive Cauliflower Mosaic Virus promoter, also containing a neomycin phosphotransferase operon. This cassette was transformed into Agrobacterium tumefaciens by triparental mating and used to infect plant tissue grown in culture. Recombinant plants were selected by their ability to grow and root on kanamycin-containing media. The presence of MIP in the plant tissues was confirmed by PCR, RT-PCR and immunohistochemistry. A number of benefits of this system for the study of MIP will be discussed, and also its application as a tool for the study of heterologously expressed proteins in general.

The study of renin-angiotensin-aldosterone in experimental diabetes mellitus.

It is generally accepted that hypertension and other vascular pathologies increase in diabetes mellitus (DM) patients as a result of the renin-angiotensin-aldosterone (RAA) system. In this study, changes in the renin-angiotensin-aldosterone (RAA) system level was determined in Streptozotocin (STZ)-injected rats. A total of 46 female Wistar albino rats (180-220 g body weight) was utilized in these experiments. STZ was given intraperitoneally to induce diabetes in rats. Streptozotocin (60 mg kg(-1) body weight) was dissolved in 0.1 m citrate--phosphate buffer (pH 4-5). The non-diabetic rats were injected with sterilized buffer alone to act as a control group. Blood glucose levels were 398+/-8.2 mg dl(-1), 488+/-11.75 mg dl(-1) and 658+/-29.6 mg dl(-1) at days 3, 12 and 30 respectively. The level of plasma renin activity (PRA) was measured as 7.69+/-1.07 ng ml(-1) h(-1); 1.82+/-0.22 ng ml(-1) h(-1) and 0. 67+/-0.12 ng ml(-1) h(-1) at days 3, 12 and 30, respectively. These values showed that the PRA levels are decreased with increased time period. Serum angiotensin converting enzyme (ACE, E.C. 3.4.15.1) levels were increased at days 12 and 30 (p<0.05 and p<0.005), whereas serum aldosterone levels were increased at days 3 and 12 (p<0.05). The level of urea and creatinine increased at days 12 and 30 (p<0.05 and p<0.005, respectively) when compared to the control group. The data from these experiments indicate that the PRA level decreased whereas ACE activity level increased in diabetic rats compared with the control. Aldosterone levels increased at the first stage of the experiment, but then decreased by the end of the experiment as a result of changes in renin and ACE levels.

Maintenance of chaperone-like activity despite mutations in a conserved region of murine lens alphaB crystallin.

PURPOSE: To understand the relationship between certain conserved residues in alphaB crystallin and the chaperone-like function of the protein. METHODS: In alphaB crystallin, residues H101 to R120 are highly conserved between alphaB crystallin and alphaA crystallin (85% identity), and between alphaB crystallin and the small heat shock protein hsp 27 (80% identity). We made three substitution mutants of alphaB crystallin: the single mutant F118A, and the double mutants K103L/H104I, and E110H/H111E. RESULTS: Polyacrylamide gel electrophoresis revealed no decrease in aggregate size or measureable structural changes between them and the native structure. Using the insulin aggregation assay, all three mutants had identical chaperone-like activity to the wild-type recombinant alphaB crystallin. CONCLUSIONS: Despite the high conservation in this area of sequence between alphaB crystallin, alphaA crystallin, and the small heat shock protein hsp 27, mutations F118A, K103L/H104I, and E110H/H111E did not significantly alter chaperone-like activity.

Cataract as a conformational disease--the Maillard reaction, alpha-crystallin and chemotherapy.

Cataract, the major cause of blindness world-wide, is associated with conformational changes and unfolding of proteins in the lens, which can arise directly as a result of post-translational modifications, induced by the Maillard reaction. In the lens, the stress protein alpha-crystallin, which is related to small heat-shock proteins and forms GroEL-like functional aggregates, can act as a chaperone-like protein to maintain transparency, sequestering unfolded protein, and inhibiting subsequent aggregation and insolubilisation. There are a number of criteria which enable the classification of cataract as a conformational disease, including not only the protein conformational change itself, resulting in aggregation and tissue deposition, but also the mechanisms for preventing such unfolding and aggregation. Post-translational modification of alphabeta-crystallin results in loss of chaperone-like activity, and aspirin, ibuprofen and paracetamol can inhibit in vitro cross-linking events responsible for the loss of this activity. Of the many avenues available to block protein aggregation, common analgesics--and vitamin C--may provide a cost-effective route to explore further in the treatment of a range of conformational diseases.

Effect of mutations of murine lens alphaB crystallin on transfected neural cell viability and cellular translocation in response to stress.

We examined the influence of over-expressed native and mutant murine lens alphaB crystallin on the response of a murine neural cell line to heat and ionic strength shock. Native and mutant (F27R and KK174/175LL) murine alphaB crystallin amplicons were subcloned into a Lac-Switch IPTG-inducible RSV promoter eukaryotic vector, and transfected into N1E-115 cells using lipofectin. Expression was induced maximally 8 h after addition of IPTG (optimal final concentration 1 mM) to the medium. Cells grew normally after transfection with native and mutant murine alphaB crystallin. We demonstrated expression of the protein using specific anti-alpha crystallin antibodies. Viability under severe heat and ionic strength stress increased when cells had been transfected with native alphaB crystallin, but not with mutants F27R or KK174/175LL. Heat shock caused translocation of both native and mutant alphaB crystallins into the central region of the cells. These results show that mutations in alphaB crystallin that effect its chaperone-like activity may also influence viability of N1E-115 neural cells under stress, while not influencing the distribution of the protein within the cell.

Aldose reductase: a window to the treatment of diabetic complications?

Kinetic studies on the aldose reductase protein (AR2) have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. These results have been confirmed by X-ray crystallography studies, which have pinpointed binding sites for pharmacological "aklose reductase inhibitors" (ARIs). As with non-enzymic glycation reactions, there is probably a free-radical element involved derived from monosaccharide autoxidation. In the case of AR2, there is free radical oxidation of NADPH by autoxidising monosaccharides, enhanced in the presence of the NADPH-binding protein. Whatever the behaviour of AR2, many studies have showed that sorbitol production is not an initiating aetiological factor in the development of diabetic complications in humans. Vitamin E (alpha-tocopherol), other antioxidants and high fat diets can delay or prevent cataract in diabetic animals even though sorbitol and fructose levels are not modified; vitamin C acts as an AR1 in humans. Protein post-translational modification by glyc-oxidation or other events is probably the key factor in the aetiology of diabetic complications. There is now no need to invoke AR2 in xylitol biosynthesis. Xylitol can be produced in the lens from glucose, via a pathway involving the enzymes myo-inositol-oxygen oxidoreductase, D-glucuronate reductase. L-gulonate NAD(+)-3-oxidoreductase and L-iditol-NAD(+)-5-oxidoreductase, all of which have recently been found in bovine and rat lens. This chapter investigates the molecular events underlying AR2 and its binding and kinetics. Induction of the protein by osmotic response elements is discussed, with detailed analysis of recent in vitro and in vivo experiments on numerous ARIs. These have a number of actions in the cell which are not specific, and which do not involve them binding to AR2. These include peroxy-radical scavenging and recently discovered effects of metal ion chelation. In controlled experiments, it has been found that incubation of rat lens homogenate with glucose and the copper chelator o-phenanthroline abolishes production of sorbitol. Taken together, these results suggest AR2 is a vestigial NADPH-binding protein, perhaps similar in function to a number of non-mammalian crystallins which have been recruited into the lens. There is mounting evidence for the binding of reactive aldehyde moieties to the protein, and the involvement of AR2 either as a 'housekeeping' protein, or in a free-radial-mediated 'catalytic' role. Interfering with the NADPH binding and flux levels--possibly involving free radicals and metal ions--has a deleterious effect. We have yet to determine whether aldose reductase is the black sheep of the aldehyde reductase family, or whether it is a skeleton in the cupboard, waiting to be clothed in the flesh of new revelations in the interactions between proteins, metal ions and redox metabolites.

Translocation of beta crystallin in neural cells in response to stress.

While extralenticular expression of proteins in the alpha crystallin (small heat shock protein) family is well documented, that for proteins in the beta/gamma-superfamily is less well established. Here we show, using SDS-PAGE, Western blotting and confocal microscopy, that there is a constitutive level of beta crystallin expression in mouse N1E-115 neural cells. Furthermore, upon heat shock at 43 degrees C or 55 degrees C, or cold shock at 30 degrees C, beta crystallin immunoreactivity translocated predominantly from the nuclear region into the cytoplasmic region of the cells. In conditions of stress, it may be important for beta crystallin to be recruited into the cytoplasm to stabilise other proteins via its high beta-sheet content, and/or to ensure that storage levels of cytoplasmic Ca2+ are maintained.

Studies on the binding of alpha-crystallin to recombinant prochymosins and chymosin.

PURPOSE: To further investigate the binding of alpha-crystallin to other proteins as part of its chaperone-like activity, we studied interactions of alpha-crystallin with recombinant calf prochymosins and chymosin. METHODS: Recombinant calf prochymosin B and one C-terminal mutant (PC+2, with two additional residues, Histidine-Glycine) were expressed as inclusion bodies in E. coli. Native and mutant proteins were denatured in 8 M urea before being refolded by dilution slowly in phosphate buffer, pH 10.7, in the presence and absence of alpha-crystallin at different concentration ratios. After dialysis, the folded proteins were converted to the active chymosin by acidification. The resulting enzyme activities at standard protein concentrations were determined by a microtitre milk-clotting assay. RESULTS: Refolding of 1.0 mg/ml of protein inclusion bodies diluted in phosphate buffer at 0.32 M urea in the presence of alpha-crystallin resulted in enhanced chymosin activity relative to the control without alpha-crystallin. When lower inclusion body concentrations were used, enzyme activity was not enhanced relative to the control. The mutant enzyme (PC+2) showed no conversion to the active form in the presence of alpha-crystallin. alpha-Crystallin formed a complex with refolded prochymosin, as well as with prochymosin during refolding, but not with active chymosin. Removal of the 43-residue propeptide resulted in loss of alpha-crystallin binding. The addition of two residues (Histidine-Glycine) to the prochymosin C-terminus resulted in precipitation of the mutant prochymosin-alpha-crystallin complex and loss of enzyme activity. CONCLUSIONS: Our experiments show that even under stringent refolding conditions, alpha-crystallin, which retains its gross oligomeric integrity, can bind to unfolded proteins in inclusion bodies and enhance the apparent yield of chymosin activity from high concentrations of inclusion bodies. alpha-Crystallin shows some specificity for binding to its target protein; this specificity may be based on steric considerations as well as residue-specific interactions.

Effects of controlled mutations on the N- and C-terminal extensions of chick lens beta B1 crystallin.

BACKGROUND: While gamma-crystallin exists as a monomer, beta-crystallin, which unlike gamma-crystallin contains N- and C-terminal arms, associates to form homodimers. METHODS: In order to answer the question of whether the extensions are involved in dimerisation of chick lens beta B1 crystallin, we have developed a heterologous expression system for chicken beta B1 crystallin in Escherichia coli, and produced three mutations by site-directed mutagenesis. We have substituted residues in the PAPA segment of the N-terminal extension, curtailed the N-terminal extension by five residues, and deleted 16 residues from the C-terminal extension. RESULTS: High-resolution gel filtration chromatography and non-denaturing gel electrophoresis show that the mutations did not influence dimerisation of the beta B1 crystallin, while circular dichroism and tryptophan fluorescence indicated that the mutations did not have a major influence on beta B1 crystallin structure or its heat stability. CONCLUSIONS: Our experiments show that as with rat lens beta B2 crystallin, dimerisation of beta B1 crystallin is not affected by alterations to the conserved PAPA region and that the peptide linker region rather than the N- and C-terminal extensions must be important in dimerisation.

Ibuprofen protects alpha-crystallin against posttranslational modification by preventing protein cross-linking.

Posttranslational modification of bovine alpha-crystallin by D-erythrose-4-phosphate, fructose-6-phosphate, D-ribose-5-phosphate and carbamylation using potassium cyanate induced the loss of chaperone-like activity, as assessed by gamma-crystallin aggregation. The presence of high-molecular-weight aggregates indicated that erythrosylated, fructosylated and carbamylated alpha-crystallins were modified by non-reducible cross-linking. In contrast, ribosylation of alpha-crystallin induced the formation of reducible cross-links. Analysis of ribosylated, erythrosylated and carbamylated alpha-crystallin using non-denaturing acrylamide gels showed that the cross-linking did not sterically inhibit the normal aggregate formation or alter the oligomerisation of the aggregate. Co-incubation of ibuprofen in the presence of alpha-crystallin and the modifying agents protected the chaperone-like activity of alpha-crystallin, enabling the inhibition of gamma-crystallin aggregation. In addition, ibuprofen inhibited the formation of both reducible and non-reducible cross-linked high-molecular-weight alpha-crystallin aggregates. We show in this paper that ibuprofen can inhibit in vitro cross-linking events responsible for the loss of chaperone-like activity of alpha-crystallin and suggest that the protective effect of ibuprofen may be exerted by the binding of ibuprofen breakdown products to alpha-crystallin lysine groups, preventing posttranslational modification responsible for the loss of chaperone-like activity.

Effects of site-directed mutations on the chaperone-like activity of alphaB-crystallin.

Recombinant alphaB-crystallin has been shown to exhibit chaperone-like activity, suppressing the thermal aggregation of gamma-crystallin and aggregation of the reduced insulin B chain conferring thermotolerance to Escherichia coli BL21(DE3) cells. Mutations were made in three specific areas of the alphaB-crystallin, the N terminus D2G, the conserved phenylalanine-rich region, F24R, F27R, F27A, and the two C-terminal lysines K174L/K175L, K174G/K175G. Biophysical characterization of the mutant alphaB-crystallins using far-UV CD revealed no change in secondary structural elements. Tryptophan fluorescence demonstrated global structural changes. Heat stability of the mutant alphaB-crystallins was not significantly affected as indicated by tryptophan fluorescence of heat-treated proteins. Mutations within the phenylalanine-rich region abolish the chaperone-like activity as measured by both in vivo and in vitro assays. Proteins with mutations at the C terminus demonstrated no significant chaperone-like activity, failing to confer thermotolerance on E. coli and demonstrating no significant inhibition of protein aggregation in either gamma-crystallin or reduced insulin B chain assays. The N-terminal mutation D2G demonstrated a significant reduction in efficiency of the chaperone-like activity although some thermotolerance was conferred in the E. coli assay. In vitro assays showed that complete inhibition of aggregation was only achieved at 10-fold higher concentrations of D2G than that required by the native alphaB-crystallin. Consistent changes in the chaperone-like activity of the site-directed mutants were demonstrated by the three assays. The results suggested that both charge-charge and hydrophobic interactions are important in protein binding by alphaB-crystallin and that the conserved RLFDQFF region is vital for chaperone-like activity.