Saturating light and not increased carbon dioxide under ocean acidification drives photosynthesis and growth in Ulva rigida (Chlorophyta)

Carbon physiology of a genetically identified Ulva rigida was investigated under different CO2(aq) and light levels. The study was designed to answer whether (1) light or exogenous inorganic carbon (Ci) pool is driving growth; and (2) elevated CO2(aq) concentration under ocean acidification (OA) will downregulate CAext-mediated inline image dehydration and alter the stable carbon isotope (delta13C) signatures toward more CO2 use to support higher growth rate. At pHT 9.0 where CO2(aq) is <1 ?mol/L, inhibition of the known inline image use mechanisms, that is, direct inline image uptake through the AE port and CAext-mediated inline image dehydration decreased net photosynthesis (NPS) by only 56-83%, leaving the carbon uptake mechanism for the remaining 17-44% of the NPS unaccounted. An in silico search for carbon-concentrating mechanism elements in expressed sequence tag libraries of Ulva found putative light-dependent inline image transporters to which the remaining NPS can be attributed. The shift in delta13C signatures from -22 per mil toward -10 per mil under saturating light but not under elevated CO2(aq) suggest preference and substantial inline image use to support photosynthesis and growth. U. rigida is Ci saturated, and growth was primarily controlled by light. Therefore, increased levels of CO2(aq) predicted for the future will not, in isolation, stimulate Ulva blooms.

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO3-) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO3- by the surface-bound enzyme carbonic anhydrase (CAext). Here, we examined other putative HCO3- uptake mechanisms in M. pyrifera under pHT 9.00 (HCO3-: CO2 = 940:1) and pHT 7.65 (HCO3-: CO2 = 51:1). Rates of photosynthesis, and internal CA (CAint) and CAext activity were measured following the application of AZ which inhibits CAext, and DIDS which inhibits a different HCO3- uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO3- uptake by M. pyrifera is via an AE protein, regardless of the HCO3-: CO2 ratio, with CAext making little contribution. Inhibiting the AE protein led to a 55%-65% decrease in photosynthetic rates. Inhibiting both the AE protein and CAext at pHT 9.00 led to 80%-100% inhibition of photosynthesis, whereas at pHT 7.65, passive CO2 diffusion supported 33% of photosynthesis. CAint was active at pHT 7.65 and 9.00, and activity was always higher than CAext, because of its role in dehydrating HCO3- to supply CO2 to RuBisCO. Interestingly, the main mechanism of HCO3- uptake in M. pyrifera was different than that in other Laminariales studied (CAext-catalyzed reaction) and we suggest that species-specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO3-:CO2 due to ocean acidification.

Ocean acidification impacts mussel control on biomineralisation

Ocean acidification is altering the oceanic carbonate saturation state and threatening the survival of marine calcifying organisms. Production of their calcium carbonate exoskeletons is dependent not only on the environmental seawater carbonate chemistry but also the ability to produce biominerals through proteins. We present shell growth and structural responses by the economically important marine calcifier Mytilus edulis to ocean acidification scenarios (380, 550, 750, 1000 µatm pCO2). After six months of incubation at 750 µatm pCO2, reduced carbonic anhydrase protein activity and shell growth occurs in M. edulis. Beyond that, at 1000 µatm pCO2, biomineralisation continued but with compensated metabolism of proteins and increased calcite growth. Mussel growth occurs at a cost to the structural integrity of the shell due to structural disorientation of calcite crystals. This loss of structural integrity could impact mussel shell strength and reduce protection from predators and changing environments.

Protein fluctuations are sensed by stimulated infrared echoes of the vibrations of carbon monoxide and azide probes

The correlation functions of the fluctuations of vibrational frequencies of azide ions and carbon monoxide in proteins are determined directly from stimulated photon echoes generated with femtosecond infrared pulses. The asymmetric stretching vibration of azide bound to carbonic anhydrase II exhibits a pronounced evolution of its vibrational frequency distribution on the time scale of a few picoseconds, which is attributed to modifications of the ligand structure through interactions with the nearby Thr-199. When azide is bound in hemoglobin, a more complex evolution of the protein structure is required to interchange the different ligand configurations, as evidenced by the much slower relaxation of the frequency distribution in this case. The time evolution of the distribution of frequencies of carbon monoxide bound in hemoglobin occurs on the ≈10-ps time scale and is very nonexponential. The correlation functions of the frequency fluctuations determine the evolution of the protein structure local to the probe and the extent to which the probe can navigate those parts of the energy landscape where the structural configurations are able to modify the local potential energy function of the probe.

The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO2 permeability of the red blood cell membrane

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of 12C18O16O from a red cell suspension as the 18O exchanges between labeled CO2 + HCO3− and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO3− (Pm,HCO−3). To test this technique, we added sufficient 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) to inhibit all the HCO3−/Cl− transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced Pm,HCO−3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in Pm,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO2 and HCO3−, we found that DIDS inhibited both Pm,HCO−3 and Pm,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO2 exchange.

Energy Sources for HCO3− and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 6251

Light-dependent inorganic C (Ci) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO2 or HCO3− uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular Ci pool of 20 mm, even though CO2 fixation was completely inhibited, indicating that cyclic electron flow was involved in the Ci-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar Ci accumulation was observed, suggesting that linear electron flow could support the transport of Ci. When carbonic anhydrase was not present, initial CO2 uptake was greatly reduced and the extracellular [CO2] eventually increased to a level higher than equilibrium, strongly suggesting that CO2 transport was inhibited and that Ci accumulation was the result of active HCO3− transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, Ci transport and accumulation were inhibited by inhibitors of CO2 transport, such as COS and Na2S, whereas Li+, an HCO3−-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, Ci transport and accumulation were not inhibited by COS and Na2S but were inhibited by Li+. These results suggest that CO2 transport is supported by cyclic electron transport and that HCO3− transport is supported by linear electron transport.

Fast Induction of High-Affinity HCO3− Transport in Cyanobacteria1

The induction of a high-affinity state of the CO2-concentration mechanism was investigated in two cyanobacterial species, Synechococcus sp. strain PCC7002 and Synechococcus sp. strain PCC7942. Cells grown at high CO2 concentrations were resuspended in low-CO2 buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was reached. Thereafter, more than 95% of a high-affinity CO2-concentration mechanism was induced in both species. Mass-spectrometric analysis of CO2 and HCO3− fluxes indicated that only the affinity of HCO3− transport increased during the fast-induction period, whereas maximum transport activities were not affected. The kinetic characteristics of CO2 uptake remained unchanged. Fast induction of high-affinity HCO3− transport was not inhibited by chloramphenicol, cantharidin, or okadaic acid. In contrast, fast induction of high-affinity HCO3− transport did not occur in the presence of K252a, staurosporine, or genistein, which are known inhibitors of protein kinases. These results show that induction of high-affinity HCO3− transport can occur within minutes of exposure to low-inorganic-C conditions and that fast induction may involve posttranslational phosphorylation of existing proteins rather than de novo synthesis of new protein components.

Scorpion toxins as natural scaffolds for protein engineering.

A compact, well-organized, and natural motif, stabilized by three disulfide bonds, is proposed as a basic scaffold for protein engineering. This motif contains 37 amino acids only and is formed by a short helix on one face and an antiparallel triple-stranded beta-sheet on the opposite face. It has been adopted by scorpions as a unique scaffold to express a wide variety of powerful toxic ligands with tuned specificity for different ion channels. We further tested the potential of this fold by engineering a metal binding site on it, taking the carbonic anhydrase site as a model. By chemical synthesis we introduced nine residues, including three histidines, as compared to the original amino acid sequence of the natural charybdotoxin and found that the new protein maintains the original fold, as revealed by CD and 1H NMR analysis. Cu2+ ions are bound with Kd = 4.2 x 10(-8) M and other metals are bound with affinities in an order mirroring that observed in carbonic anhydrase. The alpha/beta scorpion motif, small in size, easily amenable to chemical synthesis, highly stable, and tolerant for sequence mutations represents, therefore, an appropriate scaffold onto which polypeptide sequences may be introduced in a predetermined conformation, providing an additional means for design and engineering of small proteins.

A paradigm for drug discovery employing encoded combinatorial libraries.

Very large combinatorial libraries of small molecules on solid supports can now be synthesized and each library element can be identified after synthesis by using chemical tags. These tag-encoded libraries are potentially useful in drug discovery, and, to test this utility directly, we have targeted carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) as a model. Two libraries consisting of a total of 7870 members were synthesized, and structure-activity relationships based on the structures predicted by the tags were derived. Subsequently, an active representative of each library was resynthesized (2-[N-(4-sulfamoylbenzoyl)-4'-aminocyclohexanespiro]-4-oxo-7 -hydroxy- 2,3-dihydrobenzopyran and [N-(4-sulfamoylbenzoyl)-L-leucyl]piperidine-3-carboxylic acid) and these compounds were shown to have nanomolar dissociation constants (15 and 4 nM, respectively). In addition, a focused sublibrary of 217 sulfamoylbenzamides was synthesized and revealed a clear, testable structure-activity relationship describing isozyme-selective carbonic anhydrase inhibitors.

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.

Orbscan Topography in Primary Open-Angle Glaucoma

Purpose: To compare anterior and posterior corneal curvatures between eyes with primary open-angle glaucoma (POAG) and healthy eyes. Methods: This is a prospective, cross-sectional, observer-masked study. A total of 138 white subjects (one eye per patient) were consecutively recruited; 69 eyes had POAG (study group), and the other 69 comprised a group of healthy control eyes matched for age and central corneal pachymetry with the study ones. Exclusion criteria included any corneal or ocular inflammatory disease, previous ocular surgery, or treatment with carbonic anhydrase inhibitors. The same masked observer performed Goldmann applanation tonometry, ultrasound pachymetry, and Orbscan II topography in all cases. Central corneal thickness, intraocular pressure, and anterior and posterior topographic elevation maps were analyzed and compared between both groups. Results: Patients with POAG had greater forward shifting of the posterior corneal surface than that in healthy control eyes (p < 0.01). Significant differences in anterior corneal elevation between controls and POAG eyes were also found (p < 0.01). Conclusions: Primary open-angle glaucoma eyes have a higher elevation of the posterior corneal surface than that in central corneal thickness–matched nonglaucomatous eyes.

Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyta) in a rockpool-scenario, link to supplementary data

Rising atmospheric CO2-concentrations will have severe consequences for a variety of biological processes. We investigated the responses of the green alga Ulva lactuca (Linnaeus) to rising CO2-concentrations in a rockpool scenario. U. lactuca was cultured under aeraton with air containing either preindustrial pCO2 (280µatm) or for the end of the 21st century predicted (700µatm) pCO2 for 31 days. We addressed the following question: Will elevated CO2-concentrations affect photosynthesis (net photosynthesis, rETR(max), Fv/Fm, pigment composition) and growth of U. lactuca in rockpools with limited water exchange? Two phases of the experiment were distinguished: In the initial phase (day 1-4) the Seawater Carbonate System (SWCS) of the culture medium could be adjusted to the selected atmospheric pCO2 condition by continuous aeration with target pCO2 values. In the second phase (day 4-31) the SWCS was largely determined by the metabolism of the growing U. lactuca biomass. In the initial phase, Fv/Fm and rETR(max) were only slightly elevated at high CO2-concentrations whereas growth was significantly enhanced. After 31 days the Chl a content of the thalli was significantly lower under future conditions and the photosynthesis of thalli grown under preindustrial conditions was not dependent on external carbonic anhydrase. Biomass increased significantly at high CO2-concentrations. At low CO2-concentrations most adult thalli disintegrated between day 14 and 21, whereas at high CO2-concentrations most thalli remained integer until day 31. Thallus disintegration at low CO2-concentrations was mirrored in a drastic decline in seawater DIC and HCO3-. Accordingly, the SWCS differed significantly between the treatments. Our results indicated a slight enhancement of photosynthetic performance and significantly elevated growth of U. lactuca at future CO2-concentrations. The accelerated thallus disintegration at high CO2-concentrations under conditions of limited water exchange indicates additional CO2 effects on the life cycle of U. lactuca when living in rockpools.

Acetazolamide reduces exercise capacity and increases leg fatigue under hypoxic conditions

Acetazolamide (Acz) is used at altitude to prevent acute mountain sickness, but its effect on exercise capacity under hypoxic conditions is uncertain. Nine healthy men completed this double-blind, randomized, crossover study. All subjects underwent incremental exercise to exhaustion with an inspired O-2 fraction of 0.13, hypoxic ventilatory responses, and hypercapnic ventilatory responses after Acz (500 mg twice daily for 5 doses) and placebo. Maximum power of 203 +/- 38 (SD) Won Acz was less than the placebo value of 225 +/- 40 W (P < 0.01). At peak exercise, arterialized capillary pH was lower and PO2 higher on Acz (P < 0.01). Ventilation was 118.6 +/- 20.0 l/min at the maximal power on Acz and 102.4 +/- 20.7 l/min at the same power on placebo (P < 0.02), and Borg score for leg fatigue was increased on Acz (P < 0.02), with no difference in Borg score for dyspnea. Hypercapnic ventilatory response on Acz was greater (P < 0.02), whereas hypoxic ventilatory response was unchanged. During hypoxic exercise, Acz reduced exercise capacity associated with increased perception of leg fatigue. Despite increased ventilation, dyspnea was not increased.

Molecular crowding effects of linear polymers in protein solutions

Measurement of protein-polymer second virial coefficients (B-AP) by sedimentation equilibrium studies of carbonic anhydrase and cytochrome c in the presence of dextrans (T10-T80) has revealed an inverse dependence of B-AP upon dextran molecular mass that conforms well with the behaviour predicted for the excluded-volume interaction between a spherical protein solute A and a random-flight representation of the polymeric cosolute P. That model of the protein-polymer interaction is also shown to provide a reasonable description of published gel chromatographic and equilibrium dialysis data on the effect of polymer molecular mass on BAP for human serum albumin in the presence of polyethylene glycols, a contrary finding from analysis of albumin solubility measurements being rejected on theoretical grounds. Inverse dependence upon polymer chainlength is also the predicted excluded-volume effect on the strength of several types of macromolecular equilibria-protein isomerization, protein dimerization, and 1 : 1 complex formation between dissimilar protein reactants. It is therefore concluded that published experimental observations of the reverse dependence, preferential reaction enhancement within DNA replication complexes by larger polyethylene glycols, must reflect the consequences of cosolute chemical interactions that outweigh those of thermodynamic nonideality arising from excluded-volume effects. (c) 2005 Elsevier B.V. All rights reserved.