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Fil: Cisneros, Héctor. Universidad Juan Agustín Maza; Universidad Nacional de Cuyo; Universidad Nacional de San Luis. República Argentina Fil: Carini, F. Universidad Nacional de San Luis. República Argentina Fil: Fermani, S. Universidad de Congreso. Mendoza. República Argentina. Fil: Ahumada, María Nélida. Universidad Juan Agustín Maza. Mendoza. República Argentina Fil: Torres, Jorge. Universidad Tecnológica Argentina Regional Mendoza. Maestría en Desarrollo Sustentable del hábitat Humano. Mendoza. República Argentina La comarca ubicada en el sector centro-norte de la provincia de Mendoza, conocida como “Gran Mendoza”, con una población aproximada de 1.000.000 de habitantes, es considerada como una de las regiones del país con mayor vulnerabilidad ante eventos destructivos de origen geológico e hidrológico. Respecto al primero, en la zona pedemontana de Mendoza y San juan se concentran más del 80% de las estructuras sismogénicas activas del sector continental argentino. Por otro lado, los aluviones han demostrado ser el fenómeno catastrófico que más daños ha causado a la región metropolitana de esta provincia. Por estas razones se hace evidente la necesidad de estudiar las características de estos eventos, el peligro sísmico y aluvional, y de realizar efectivas evaluaciones de riesgo. La metodología usada incluye la sistematización de los datos y su presentación cartográfica con geotecnologías SIG y de Teledetección. Se presentan en esta contribución los resultados del estudio realizado en el marco del proyecto acreditado por la Universidad Juan Agustín Maza.

In order to understand the cutaneous water loss in the desert-adapted and venomous lizard Heloderma suspectum, the microscopic structure and lipid composition of epidermal molts have been examined using microscopic, spectroscopic and chemical analysis techniques. The molt is formed by a variably thick, superficial beta-layer, an extensive mesos-region and few alpha-cells in its lowermost layers. The beta-layer contains most corneous beta proteins while the mesos-region is much richer in lipids. The proteins in the mesos-region are more unstructured than those located in the beta-layer. Most interestingly, among other lipids, high contents of cholesteryl-β-glucoside and cholesteryl sulfate were detected, molecules absent or present in traces in other species of squamates. These cholesterol derivatives may be involved in the stabilization and compaction of the mesos-region, but present a limited permeability to water movements. The modest resistance to cutaneous water-loss of this species is compensated by adopting other physiological strategies to limit thermal damage and water transpiration as previous eco-physiological studies have indicated. The increase of steroid derivatives may also be implicated in the heat shock response, influencing the relative behavior in this desert-adapted lizard.

Control over the shape and morphology of single crystals is a theme of great interest in fundamental science and for technological application. Many synthetic strategies to achieve this goal are inspired by biomineralization processes. Indeed, organisms are able to produce crystals with high fidelity in shape and morphology utilizing macromolecules that act as modifiers. An alternative strategy can be the recovery of crystals from biomineralization products, in this case, seashells. In particular, waste mussel shells from aquaculture are considered. They are mainly built up of single crystals of calcite fibers and aragonite tablets forming an outer and an inner layer, respectively. A simple mechanochemical treatment has been developed to separate and recover these two typologies of single crystals. The characterization of these single crystals showed peculiar properties with respect to the calcium carbonate from quarry or synthesis. We exploited these biomaterials in the water remediation field using them as substrate adsorbing dyes. We found that these substrates show a high capability of adsorption for anionic dye, such as Eosin Y, but a low capability of adsorption for cationic dyes, such as Blue Methylene. The adsorption was reversible at pH 5.6. This application represents just an example of the potential use of these biogenic single crystals. We also envision potential applications as reinforcing fillers and optical devices. published

This study investigates the effects of long-term exposure to OA on skeletal parameters of four tropical zooxanthellate corals naturally living at CO2 seeps and adjacent control sites from two locations (Dobu and Upa Upasina) in the Papua New Guinea underwater volcanic vent system. The seeps are characterized by seawater pH values ranging from 8.0 to about 7.7. The skeletal porosity of Galaxea fascicularis, Acropora millepora, massive Porites, and Pocillopora damicornis was higher (up to ~ 40%, depending on the species) at the seep sites compared to the control sites. Pocillopora damicornis also showed a decrease of micro-density (up to ~ 7%). Thus, further investigations conducted on this species showed an increase of the volume fraction of the larger pores (up to ~ 7%), a decrease of the intraskeletal organic matrix content (up to ~ 15%), and an increase of the intraskeletal water content (up to ~ 59%) at the seep sites. The organic matrix related strain and crystallite size did not vary between seep and control sites. This multi-species study showed a common phenotypic response among different zooxanthellate corals subjected to the same environmental pressures, leading to the development of a more porous skeletal phenotype under OA.

The three-dimensional structures of two type 1 RIPs, bouganin and lychnin, has been solved. Their adenine polynucleotide glycosylase activity was also determined together with other known RIPs: dianthin 30, PAP-R, momordin I, ricin A chain and saporin-S6. Saporin-S6 releases the highest number of adenine molecules from rat ribosomes, and poly(A), while its efficiency is similar to dianthin 30, bouganin and PAP-R on herring sperm DNA. Measures of the protein synthesis inhibitory activity confirmed that saporin-S6 is the most active. The overall structure of bouganin and lychnin is similar to the other considered RIPs and the typical RIP fold is conserved. The superimpositioning of their Cα atoms highlights some differences in the N-terminal and C-terminal domains. A detailed structural analysis indicates that the efficiency of saporin-S6 on various polynucleotides can be ascribed to a negative electrostatic surface potential at the active site and several exposed positively charged residues in the region around that site. These two conditions, not present at the same time in other examined RIPs, could guarantee an efficient interaction with the substrate and an efficient catalysis. © 2009 Elsevier Inc. All rights reserved.

Boc-L-DOPA(OBn)2-OH is a simple synthetic molecule that promotes hydrogelation through electrostatic and π-πstacking interactions. Hydrogelation can occur in alkaline conditions by the use of triggers. Four hydrogels were prepared varying the base, NaOH or Na2CO3, and the trigger, GdL or CaCl2. When the hydrogel formed in the presence of Na2CO3 and CaCl2, the concomitant production of CaCO3 crystals occurred, generating an organic/inorganic composite material. It was observed that the hydrogel once self-assembled preserved its status even if the trigger, the calcium ions, was removed. The viscoelastic behavior of the hydrogels was analyzed through rheological experiments, which showed a solid-like behavior of the hydrogels. The corresponding xerogels were analyzed mainly by scanning electron microscopy (SEM) and synchrotron X-ray diffraction analysis (XRD). They showed differences in structure, morphology, and fiber organization according to their source. This research presents a hydrogel system that can be applied as a soft biomaterial for tissue engineering, cosmetics, food, and environmental science. Moreover, it represents a model for biomineralization studies in which the hydrogel structure can act as an analogue of the insoluble matrix that confines the calcification site, provides Ca2+, and preserves its structure.

The Scleractinia coral biomineralization process is a representative example of a heterogeneous process of nudeation and growth of biogenic CaCO3 over a mineral phase. Indeed, even if the biomineralization process starts before settlement, the bulk formation of the skeleton takes place only when the larvae attach to a solid substrate, which can be Mg-calcite from coralline algae, and the following growth proceeds on the Mg-calcite surface of the formed baseplate of the planula. Despite this peculiarity and central role of the Mg-calcite substrate, the in vitro overgrowth of CaCO3 on single crystals of Mg-calcite, or calcite, in the presence of magnesium ions and the soluble organic matrix (SOM) extracted from coral skeletons has not been performed until now. In this study, the SOMs from Stylophora pistillata and Oculina patagonica skeletons were used in a set of overgrowth experiments. The overgrown CaCO3 was characterized by microscopic, diffractometric, and spectroscopic techniques. Our results showed that CaCO3 overgrowth in the presence of S. pistillata or O. patagonica SOM produces different effects. However, there appears to be a minor distinction between samples when magnesium ions are present in solution. Moreover, the Mg-calcite substrate appears to be a favorable substrate for the overgrowth of aragonite, differently from calcite. These observations fit with the observed settling of coral larvae on Mg-calcite-based substrates and with the in vivo observation that in the planula aragonite forms on first-formed Mg-calcite crystals. The overall results of this study highlight the importance of magnesium ions, either in the solution or in the substrate, in defining the shape, morphology, and polymorphism of biodeposited CaCO3. They also suggest a magnesium-dependent biological control on the deposition of coral skeletons.

The biomineralization of corals occurs under conditions of high and low supersaturation with respect to aragonite, which corresponds to day- or night-time periods of their growth, respectively. Here, in vitro precipitation of aragonite in artificial seawater was investigated at a high supersaturation, allowing spontaneous nucleation and growth, as well as at low supersaturation conditions, which allowed only the crystal growth on the deliberately introduced aragonite seeds. In either chemical systems, soluble organic matrix (SOM) extracted from Balanophyllia europaea (light sensitive) or Leptopsammia pruvoti (light insensitive) was added. The analyses of the kinetic and thermodynamic data of aragonite precipitation and microscopic observations showed that, at high supersaturation, the SOMs increased the induction time, did not affect the growth rate and were incorporated within aggregates of nanoparticles. At low supersaturation, the SOMs affected the aggregation of overgrowing crystalline units and did not substantially change the growth rate. On the basis of the obtained results we can infer that at high supersaturation conditions the formation of nanoparticles, which is typically observed in the skeleton's early mineralization zone may occur, whereas at low supersaturation the overgrowth on prismatic seeds observed in the skeleton fiber zone is a predominant process. In conclusion, this research brings insight on coral skeletogenesis bridging physicochemical (supersaturation) and biological (role of SOM) models of coral biomineralization and provides a source of inspiration for the precipitation of composite materials under different conditions of supersaturation.

The diversity in the skeletal features of coral species is an outcome of their evolution, distribution and habitat. Here, we explored, from macro- to nano-scale, the skeletal structural and compositional characteristics of three coral species belonging to the genus Balanophyllia having different trophic strategies. The goal is to address whether the onset of mixotrophy influenced the skeletal features of B. elegans, B. regia, and B. europaea. The macroscale data suggest that the presence of symbiotic algae in B. europaea can lead to a surplus of energy input that increases its growth rate and skeletal bulk density, leading to larger and denser corals compared to the azooxanthellate ones, B. regia and B. elegans. The symbiosis would also explain the higher intra-skeletal organic matrix (OM) content, which is constituted by macromolecules promoting the calcification, in B. europaea compared to the azooxanthellate species. The characterization of the soluble OM also revealed differences between B. europaea and the azooxanthellate species, which may be linked to diverse macromolecular machineries responsible for skeletal biosynthesis and final morphology. Differently, the crystallographic features were homogenous among species, suggesting that the basic building blocks of skeletons remained a conserved trait in these related species, regardless of the trophic strategy. These results show changes in skeletal phenotype that could be triggered by the onset of mixotrophy, as a consequence of the symbiotic association, displaying remarkable plasticity of coral skeletons which repeatedly allowed this coral group to adapt to a range of changing environments throughout its geological history.

The presence of non-hexagonal rings in the honeycomb carbon arrangement of graphene produces rippled graphene layers with valuable chemical and physical properties. In principle, a bottom-up approach to introducing distortion from planarity of a graphene sheet can be achieved by careful insertion of curved polyaromatic hydrocarbons during the growth of the lattice. Corannulene, the archetype of such non-planar polyaromatic hydrocarbons, can act as an ideal wrinkling motif in 2D carbon nanostructures. Herein we report an electrochemical bottom-up method to obtain egg-box shaped nanographene structures through a polycondensation of corannulene that produces a new conducting layered material. Characterization of this new polymeric material by electrochemistry, spectroscopy, electron microscopy (SEM and TEM), scanning probe microscopy, and laser desorption-ionization time of flight mass spectrometry provides strong evidence that the anodic polymerization of corannulene, combined with electrochemically induced oxidative cyclodehydrogenations (Scholl reactions), leads to polycorannulene with a wavy graphene-like structure., A bottom-up synthesis of wavy graphene structures obtained through an anodic polymerization process, combined with an electrochemically triggered oxidative cyclodehydrogenation, of the bowl-shaped polyaromatic hydrocarbon corannulene.

Seashells are a calcium-carbonate-based material that can be converted into valuable advanced functional materials. Seashells are also a waste material from aquaculture. They are produced in millions of tonnes per year and represent an environmental issue. They uniquely contain an intraskeletal organic matrix rich in carboxylate groups that so far has not been exploited or has been even removed, when they were used as calcium carbonate substitutes. The intraskeletal organic matrix allows for a so far never reported covalent functionalization. Such a process strengthens the surface functionalization with respect to adsorption and, most importantly, opens up the possibility for the functionalization of the biogenic calcium carbonate with a wide variety of molecules by means of organic chemistry reactions. As a proof of concept, powdered waste oyster shells were covalently functionalized with a fluorescent probe. The impact of this research can be terrific in the valorization of CaCO3 from biogenic wastes providing advanced functional products tailored for individual applications. Moreover, its consequences on the environment and society will epitomize a perfect example of a circular economy.

Oxygenic phototrophs use the Calvin–Benson cycle to fix CO2 during photosynthesis. In the dark, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), two enzymes of the Calvin–Benson cycle, form an inactive complex with the regulatory protein CP12, mainly under the control of thioredoxins and pyridine nucleotides. In the light, complex dissociation allows GAPDH and PRK reactivation. The GAPDH/CP12/PRK complex is conserved from cyanobacteria to angiosperms and coexists in land plants with an autoassembling GAPDH that is analogously regulated. With the recently described 3D structures of PRK and GAPDH/CP12/PRK, the structural proteome of this ubiquitous regulatory system has been completed. This outcome opens a new avenue for understanding the regulatory potential of photosynthetic carbon fixation by laying the foundation for its knowledge-based manipulation.

Streptococcus suis is an encapsulated bacterium and one of the most important swine pathogens and a zoonotic agent for which no effective vaccine exists. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. In addition to the previously known serotypes 2 and 14, that are non-immunogenic, we have recently purified and described the CPS structures for serotypes 1, 1/2, 3, 7, 8, and 9. Here, we aimed to elucidate how these new structurally diverse CPSs interact with the immune system to generate anti-CPS antibody responses. CPS-stimulated dendritic cells produced significant levels of C–C motif chemokine ligand (CCL) 3, partially via Toll-like receptor (TLR) 2- and myeloid differentiation factor 88-dependent pathways, and CCL2, via TLR-independent mechanisms. Mice immunized with purified serotype 3 CPS adjuvanted with TiterMax Gold® produced an opsonizing IgG response, whereas other CPSs or adjuvants were negative. Mice hyperimmunized with heat-killed S. suis serotypes 3 and 9 both produced anti-CPS type 1 IgGs, whereas serotypes 7 and 8 remained negative. Also, mice infected with sublethal doses of S. suis serotype 3 produced primary anti-CPS IgM and IgG responses, of which only IgM were boosted after a secondary infection. In contrast, mice sublethaly infected with S. suis serotype 9 produced weak anti-CPS IgM and IgG responses following a secondary infection. This study provides important information on the divergent evolution of CPS serotypes with highly different structural and/or biochemical properties within S. suis and their interaction with the immune system.

In this study the multi-scale hierarchical structure of the β-chitin matrix from squid pen of Loligo vulgaris was used as substrate to synthesize new bio-inspired materials. Aiming to mimic the byssus peculiar mechanical properties, we chemically functionalized the β-chitin matrix with catechols, one of the main functional groups of the byssus. The obtained matrix preserved its multi-scale structural organization and was able to chelate reversibly Fe(III). Thus, it behaved as the byssus, acting as a metal cross-linkable matrix that upon metalation increased its Young‘s modulus, E (>10 times). The functionalized matrix was also cross-linked by oxidation provoking an increase of the E (>10 times) and first failure stress (>5 times). The oxidation of the functionalized matrix followed by metalation slightly increased the material mechanical properties. In conclusion, we added specific bio-functionalities in a natural matrix tuning its mechanical properties without altering its multi-scale organization.

Biogenic crystals produced by organisms have been known for several decades to exhibit intracrystalline organic macromolecules. Here, using a reductionist approach, we tackle the question of whether the incorporation of single amino acids is driven by kinetics or by thermodynamics. We show that when calcite is grown in the presence of amino acids under nonambient conditions, extremely high loading levels of up to 6.12 mol % of aspartic acid (Asp) are achieved. This incorporation leads to marked changes in the host calcite crystal's structure and expansion coefficient. The latter is as much as twice as high as that of pure calcite. This is the first example showing that an organic molecule incorporated into an inorganic host can strongly affect the expansion coefficient. Most importantly, we show that the incorporation of amino acids in calcite is controlled by their thermodynamic solubility in calcite rather than kinetically and that hybrid amino acid-calcite crystals can indeed be considered a solid solution.

Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. Its kinetic properties were determined and the three-dimensional structures of the apo-, NAD+- and NAD+/GSNO-forms were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity and folding, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed., Highlights • Chlamydomonas protein extracts catalyze NAD(P)H-dependent GSNO degradation. • Chlamydomonas GSNOR1 is a zinc-containing protein strictly relying on GSNO and NADH. • The 3D-structure of CrGSNOR1 revealed a conserved folding with other plant GSNORs. • CrGSNOR1 contains only two solvent-exposed/reactive cysteines. • Oxidizing and nitrosylating treatments have limited effects on CrGSNOR1 activity.

Understanding how marine taxa will respond to near-future climate changes is one of the main challenges for management of coastal ecosystem services. Ecological studies that investigate relationships between the environment and shell properties of commercially important marine species are commonly restricted to latitudinal gradients or small-scale laboratory experiments. This paper aimed to explore the variations in shell features and growth of the edible bivalve Chamelea gallina from the Holocene sedimentary succession to present-day thanatocoenosis of the Po Plain-Adriatic Sea system (Italy). Comparing the Holocene sub-fossil record to modern thanatocoenoses allowed obtaining an insight of shell variations dynamics on a millennial temporal scale. Five shoreface-related assemblages rich in C. gallina were considered: two from the Middle Holocene, when regional sea surface temperatures were higher than today, representing a possible analogue for the near-future global warming, one from the Late Holocene and two from the present-day. We investigated shell biometry and skeletal properties in relation to the valve length of C. gallina. Juveniles were found to be more porous than adults in all horizons. This suggested that C. gallina promoted an accelerated shell accretion with a higher porosity and lower density at the expense of mechanically fragile shells. A positive correlation between sea surface temperature and both micro-density and bulk density were found, with modern specimens being less dense, likely due to lower aragonite saturation state at lower temperature, which could ultimately increase the energetic costs of shell formation. Since no variation was observed in shell CaCO3 polymorphism (100% aragonite) or in compositional parameters among the analyzed horizons, the observed dynamics in skeletal parameters are likely not driven by a diagenetic recrystallization of the shell mineral phase. This study contributes to understand the response of C. gallina to climate-driven environmental shifts and offers insights for assessing anthropogenic impacts on this economic relevant species.

Protein aggregation is a complex physiological process, primarily determined by stress-related factors revealing the hidden aggregation propensity of proteins that otherwise are fully soluble. Here we report a mechanism by which glycolytic glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana (AtGAPC1) is primed to form insoluble aggregates by the glutathionylation of its catalytic cysteine (Cys149). Following a lag phase, glutathionylated AtGAPC1 initiates a self-aggregation process resulting in the formation of branched chains of globular particles made of partially misfolded and totally inactive proteins. GSH molecules within AtGAPC1 active sites are suggested to provide the initial destabilizing signal. The following removal of glutathione by the formation of an intramolecular disulfide bond between Cys149 and Cys153 reinforces the aggregation process. Physiological reductases, thioredoxins and glutaredoxins, could not dissolve AtGAPC1 aggregates but could efficiently contrast their growth. Besides acting as a protective mechanism against overoxidation, S-glutathionylation of AtGAPC1 triggers an unexpected aggregation pathway with completely different and still unexplored physiological implications.

Recent research studies have shown that the intra-skeletal organic matrix of corals contains lipids. This communication reports their characterization and their influence on calcium carbonate precipitation. In addition, their potential role in coral's biomineralization is discussed.

Anthropogenic CO2 is a major driver of current environmental change in most ecosystems1, and the related ocean acidification (OA) is threatening marine biota2. With increasing pCO2, calcification rates of several species decrease3, although cases of up-regulation are observed4. Here, we show that biological control over mineralization relates to species abundance along a natural pH gradient. As pCO2 increased, the mineralogy of a scleractinian coral (Balanophyllia europaea) and a mollusc (Vermetus triqueter) did not change. In contrast, two calcifying algae (Padina pavonica and Acetabularia acetabulum) reduced and changed mineralization with increasing pCO2, from aragonite to the less soluble calcium sulphates and whewellite, respectively. As pCO2 increased, the coral and mollusc abundance was severely reduced, with both species disappearing at pH < 7.8. Conversely, the two calcifying and a non-calcifying algae (Lobophora variegata) showed less severe or no reductions with increasing pCO2, and were all found at the lowest pH site. The mineralization response to decreasing pH suggests a link with the degree of control over the biomineralization process by the organism, as only species with lower control managed to thrive in the lowest pH.

In this study, the influence of Mg2+ on the aggregation and morphology of precipitated aragonite crystals is investigated at different temperatures. Different from some investigations described in the literature, aragonite precipitates in chemical systems in which Mg2+ acts specifically as crystal habit modifiers and not as polymorphic selectors. The results show that at increased Mg2+ concentration and temperature the aragonite crystals are less aggregated and that the amount of crystals sharing {; ; 110}; ; faces and having larger extension of {; ; 001}; ; faces increases. These outcomes may be relevant in biological, geochemical and technological contexts.

Among protein residues, cysteines are one of the prominent candidates to ROS- and RNS-mediated post-translational modifications, and hydrogen peroxide (H2 O2 ) is the main ROS candidate for inducing cysteine oxidation. The reaction with H2 O2 is not common to all cysteine residues, being their reactivity an utmost prerequisite for the sensitivity towards H2 O2 . Indeed, only deprotonated Cys (i.e. thiolate form, -S-) can react with H2 O2 leading to sulphenic acid formation (-SOH), which is considered as a major/central player of ROS sensing pathways. However, cysteine sulphenic acids are generally unstable since they can be further oxidized to irreversible forms (sulphinic and sulphonic acids, -SO2 H and -SO3 H, respectively) or alternatively, they can proceed toward further modifications including disulphide bond formation (-SS-), S-glutathionylation (-SSG) and sulphenamide formation (-SN=). To understand why and how cysteine residues undergo primary oxidation to sulphenic acid, and to explore the stability of cysteine sulphenic acids, a combination of biochemical, structural and computational studies are required. Here, we will discuss the current knowledge of the structural determinants for cysteine reactivity and sulphenic acid stability within protein microenvironments.

Corals are among the most important calcium carbonate mineralizers and form the main structures of the reefs, which provide an important socio-economical support. Despite this, and the fact the is quite generally accepted that coral mineralization is a biological controlled process, few studied have so far addressed the role of the intra-skeletal organic matrix in the calcifi cation process. This chapter makes a scientifi c path on what is known on the biological control of coral mineralization describing the more relevant studies. The sections are sequenced with the aim to guide the readers to be conscious of the importance of the organic matrix in the mineralization process that is fi nally illustrated through a series of experiments in vivo and in vitro. Accordingly the chapter presents an overview on coral biomineralization, anatomy and physiology, skeleton microsctructure, tissue-skeleton, minor element distribution, organic matrix, biomineralization proteins and fi nally calcium carbonate precipitation in the presence of coral organic matrix

Doxorubicin (DOX)/CaCO3 single crystals act as pH responsive drug carrier. A biomimetic approach demonstrates that calcite single crystals are able, during their growth in the presence of doxorubicin, to entrap drug molecules inside their lattice along specific crystallographic directions. Alterations in lattice dimensions and microstructural parameters are determined by means of high-resolution synchrotron powder diffraction measurements. Confocal microscopy confirms that doxorubicin is uniformly embedded in the crystal and is not simply adsorbed on the crystal surface. A slow release of DOX was obtained preferentially in the proximity of the crystals, targeting cancer cells.

Ribosome inactivating proteins (RIPs) are plant proteins with enzymatic activity identified as rRNA N-glycosidase (EC 3.2.2.22), which cleaves the N-glycosidic bond of a specific adenine on the ricin/sarcin region of rRNA, thus causing inhibition of protein synthesis. They also depurinate extensively DNA and other polynucleotides. The three-dimensional structure of dianthin 30, a type 1 (single-chain) RIP of Dianthus caryophyllus (leaves), is now described at 1.4 angstroms, a resolution never achieved before for any RIP. The fold typical of RIPs is conserved, despite some differences in the loop regions. The general structure comparison by superimposed alpha-carbon (249 atoms) and the sequence alignment by structure for dianthin 30 and saporin-S6 give a root mean square deviation of 0.625 angstroms. Despite the differences reported for the biological activities of the two RIPs, their structures fit quite well and both show a protein segment containing strands beta7, beta8, and beta9 shorter than other RIPs. However, the surface electrostatic potential in the active site region neatly distinguishes dianthin 30 from saporin-S6. The possible relationship between the charge distribution and the behavior of the proteins toward different substrates is discussed.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2′-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat (NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2′-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.

Synthesis of inorganic particles using routes inspired by biomineralization is a goal of growing interest. Recently it was demonstrated that the size and geometry of crystallization sites are as important as the structure of charged templating surfaces to obtain particles with controlled features. Most biominerals are formed inside restricted, constrained or confined spaces where at least parts of the boundaries are cell membranes containing phospholipids. In this study, we used a gas diffusion method to determine the effect of different lecithin media on the crystallization of CaCO3 and to evaluate the influence of the spatial arrangement of lecithin molecules on templating CaCO3 crystal formation. By using inorganic synthesis, Raman spectroscopy, dynamic light scattering, electrochemical methods and scanning electron microscopy, we showed that the occurrence of surface-modified calcite crystals and diverse textured vaterite crystals reflects the geometry and spatial distribution of aqueous constrained spaces due to the lecithin assembly controlled by lecithin concentration in an ionized calcium chloride solution under a continuous CO2 diffusion atmosphere. This research shows that by tailoring the assembly of lecithin molecules, as micelles or reversed micelles, it is possible to modulate the texture, polymorphism, size and shape of calcium carbonate crystals. published

The squid pen (gladius) from the Loligo vulgaris was used for preparation of β-chitin materials characterized by different chemical, micro- and nano-structural properties that preserved, almost completely the macrostructural and the mechanical ones. The β-chitin materials obtained by alkaline treatment showed porosity, wettability and swelling that are a function of the duration of the treatment. Microscopic, spectroscopic and synchrotron X-ray diffraction techniques showed that the chemical environment of the N-acetyl groups of the β-chitin chains changes after the thermal alkaline treatment. As a consequence, the crystalline packing of the β-chitin is modified, due to the intercalation of water molecules between β-chitin sheets. Potential applications of these β-chitin materials range from the nanotechnology to the regenerative medicine. The use of gladii, which are waste products of the fishing industry, has also important environmental implications.

In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (-335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys(227) and Cys(361). Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view.

Scleractinian coral skeletons are composed mainly of aragonite in which a small percentage of organic matrix (OM) molecules is entrapped. It is well known that in corals the mineral deposition occurs in a biological confined nucleation site, but it is still unclear to what extent the calcification is controlled by OM molecules. Hence, the shape, size and organization of skeletal crystals from the fiber level through the colony architecture, were also attributed to factors as diverse as nucleation site mineral supersaturation and environmental factors in the habitat. In this work the OMs were extracted from the skeleton of three colonial corals, Acropora digitifera , Lophelia pertusa and Montipora caliculata . A. digitifera has a higher calcification rate than the other two species. OM molecules were characterized and their CaCO 3 mineralization activity was evaluated by experiments of overgrowth on coral skeletons and of precipitation from solutions containing OM soluble and insoluble fractions and magnesium ions. The precipitates were characterized by spectroscopic and microscopic techniques. The results showed that the OM molecules of the three coral share similar features, but differ from those associated with mollusk shells. However, A. digitifera OM shows peculiarities from those from L. pertusa and M. caliculata . The CaCO 3 overgrowth and precipitation experiments confirm the singularity of A. digitifera OM molecules as mineralizers. Moreover, their comparison indicates that only specific molecules are involved in the polymorphism control and suggests that when the whole extracted materials are used the OM’s main effect is on the control of particles’ shape and morphology.

Carbon assimilation in plants is regulated by the reduction of specific protein disulfides by light and their re-oxidation in the dark. The redox switch CP12 is an intrinsically disordered protein that can form two disulfide bridges. In the dark oxidized CP12 forms an inactive supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase, two enzymes of the carbon assimilation cycle. Here we show that binding of CP12 to GAPDH, the first step of ternary complex formation, follows an integrated mechanism that combines conformational selection with induced folding steps. Initially, a CP12 conformation characterized by a circular structural motif including the C-terminal disulfide is selected by GAPDH. Subsequently, the induced folding of the flexible C-terminal tail of CP12 in the active site of GAPDH stabilizes the binary complex. Formation of several hydrogen bonds compensates the entropic cost of CP12 fixation and terminates the interaction mechanism that contributes to carbon assimilation control.

The crystal structure of the A(4) isoform of photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana, expressed in recombinant form and complexed with NAD, is reported. The crystals, which were grown in 2.4 M ammonium sulfate and 0.1 M sodium citrate, belonged to space group I222. The asymmetric unit includes ten subunits, i.e. two independent tetramers plus a dimer that generates a third tetramer by a crystallographic symmetry operation. The crystal structure was solved by molecular replacement and refined to an R factor of 23.7% and an R(free) factor of 28.9% at 2.6 A resolution. In the final model, each subunit binds one NAD(+) molecule and two sulfates, which occupy the P(s) and the P(i) anion-binding sites. Detailed knowledge of this structure is instrumental for structural investigation of supramolecular complexes of A(4)-GAPDH, phosphoribulokinase and CP12, which are involved in the regulation of photosynthesis in the model plant A. thaliana.

Ribosome-inactivating proteins (RIPs) inhibit protein synthesis and induce cell death by removing a single adenine from a specific rRNA loop. They can be divided into two main groups: type 1 and type 2 RIPs. Type 1 RIPs are single-chain enzymes with N-glycosidase activity. Type 2 RIPs contain two chains (A and B) linked by a disulfide bond. The A chain has RIP enzymatic activity, whereas the B chain shows lectin activity and is able to bind to glycosylated receptors on the cell surface. Stenodactylin is a type 2 RIP from the caudex of Adenia stenodactyla from the Passifloraceae family that has been recently purified and characterized. It shows a strong enzymatic activity towards several substrates and is more cytotoxic than other toxins of the same type. Here, the crystallization and preliminary X-ray diffraction data analysis of stenodactylin are reported. This RIP forms crystals that diffract to high resolution (up to 2.15 A). The best data set was obtained by merging data collected from two crystals. Stenodactylin crystals belonged to the centred monoclinic space group C2 and contained two molecules in the asymmetric unit.

We analyzed the crystallographic relationships during hydrothermal conversion of a calcitic sea urchin spine into apatite. We identified a pseudomorphic mineral replacement mechanism involving a superficial dissolution of calcite and a subsequent overgrowth of oriented carbonated hydroxylapatite (HA) nanocrystals. Cross-section images of these converted spines show that the dimensions of the HA crystals increase the further they are from the outer surface. This replacement process is favored by an increase in porosity, which enables both fluid and mass to be transported by diffusion, thereby allowing the replacement reaction to progress toward the interior of the spine. These recrystallization reactions take place on the surface of the calcite single crystal, which acts as a substrate for the epitaxial nucleation of HA crystals. The epitaxial relationship observed between the parent calcite crystal and the newly formed apatite crystals can be defined as (0001) apatite//(0118) calcite and [10.0] apatite//...

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in plant photosynthetic carbon reduction. Unlike lower photosynthetic organisms, which only contain A 4 –GAPDH, the major GAPDH isoform of land plants is made up of A and B subunits, the latter containing a C-terminal extension (CTE) with fundamental regulatory functions. Light-activation of AB–GAPDH depends on the redox state of a pair of cysteines of the CTE, which can form a disulfide bond under control of thioredoxin f , leading to specific inhibition of the NADPH-dependent activity. The tridimensional structure of A 2 B 2 –GAPDH from spinach chloroplasts, crystallized in the oxidized state, shows that each disulfide-containing CTE is docked into a deep cleft between a pair of A and B subunits. The structure of the CTE was derived from crystallographic data and computational modeling and confirmed by site-specific mutagenesis. Structural analysis of oxidized A 2 B 2 –GAPDH and chimeric mutant [A+CTE] 4 –GAPDH revealed that Arg-77, which is essential for coenzyme specificity and high NADPH-dependent activity, fails to interact with NADP in these kinetically inhibited GAPDH tetramers and is attracted instead by negative residues of oxidized CTE. Other subtle changes in catalytic domains and overall conformation of the tetramers were noticed in oxidized A 2 B 2 –GAPDH and [A+CTE] 4 –GAPDH, compared with fully active A 4 –GAPDH. The CTE is envisioned as a redox-sensitive regulatory domain that can force AB–GAPDH into a kinetically inhibited conformation under oxidizing conditions, which also occur during dark inactivation of the enzyme in vivo .

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in plant photosynthetic carbon reduction. At difference with lower photosynthetic organisms which only contain A4-GAPDH, the major GAPDH isoform of land plants is made up of A and B subunits, the latter containing a C-terminal extension (CTE) with fundamental regulatory functions. Light-activation of AB-GAPDH depends on the redox state of a pair of CTE cysteines forming a disulfide bond under control of thioredoxin f, leading to specific inhibition of the NADPH-dependent activity. The crystallographic structural model of oxidized A2B2-GAPDH complexed with NADP is composed of one tetramer and a dimer, which generates a second tetramer using a crystallographic two-fold axis coincident with the molecular axis P. A2B2- GAPDH showed an overall structural organization similar to thioredoxin-independent A4-GAPDH [2]. These two isoforms almost identical along P and Q axes, are differently sized along R axis, where the A2B2 tetramer (75.7 Å) appeared shorter than A4-GAPDH (78.1 Å). Each A or B subunit consists of a coenzyme-binding domain and a catalytic domain. One NADP molecule was bound to each coenzyme domain, and one or two sulphate ions were found in each catalytic domain. The CTE of B-subunits was only partially detectable by x-ray diffraction experiments. The inspection of the electron density maps showed an elongated electron density region not continuous with any subunit, but interpretable as a protein chain slipping into the cleft bordered by a pair of A and B-subunits. This cleft features a positive electrostatic surface potential due to six exposed arginines and two histidines (Arg77, Arg183, Arg191 and His190), conserved in both A and B-subunits. The CTE contains negatively charged residues and could be attracted by the strongly cationic cleft. Present data support the view that A2B2-GAPDH inhibition by thioredoxin depends on the docking of the oxidized CTE into the cleft delimited by A/B-subunits. In this location, the CTE appears to interfere with the recognition of bound NADP by the crucial residues Arg77 and Ser188, thus leaving the tetramer in a kinetically inhibited conformation, unable to efficiently use NADPH as the preferred coenzyme.

Regulation of the Calvin-Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin-Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A(2)B(2)-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A(4)-GAPDH. GapB subunits of A(2)B(2)-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A(2)B(2)-GAPDH into strongly inactivated A(8)B(8)-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A(4)-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A(2)B(2)-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.

Although both tooth enamel and bone are composed of organized assemblies of carbonated apatite crystals, enamel is unusual in that it does not contain collagen nor does it remodel. Self-assembly of amelogenin protein into nanospheres has been recognized as a key factor in controlling the oriented and elongated growth of carbonated apatite crystals during dental enamel biomineralization. We report the in vitro formation of birefringent microribbon structures that were generated through the supramolecular assembly of amelogenin nanospheres. These microribbons have diffraction patterns that indicate a periodic structure of crystalline units along the long axis. The growth of apatite crystals orientated along the c axis and parallel to the long axes of the microribbons was observed in vitro. The linear arrays (chains) of nanospheres observed as intermediate states before the microribbon formation give an important indication as to the function of amelogenin in controlling the oriented growth of apatite crystals during enamel mineralization.

The structural, morphological and chemical–physical characterization of films constituted of self-assembled non-helical region free type I collagen molecules has been investigated. The results indicate the presence of information at the molecular level which allow collagen I molecules to self-assemble. In the fiber formation process the collagen molecules re-establish the greater part of the native intermolecular cross-links. The films, obtained by air drying the fiber suspension, are water insoluble and characterized by a high mechanical performance. The mechanical and thermal properties of the films increase strongly as a function of the collagen fiber orientation induced in the films by uniaxial stretching.