RESUMO
Alcohol dehydrogenases (ADHs) are a group of zinc-binding enzymes belonging to the medium-length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH-ADH1 and apo-GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo- and holo-forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long-chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol-oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.
Assuntos
Álcool Desidrogenase , Proteínas de Arabidopsis , Arabidopsis , Oxirredução , Arabidopsis/enzimologia , Arabidopsis/genética , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Especificidade por Substrato , S-Nitrosoglutationa/metabolismo , Sequência de Aminoácidos , Etanol/metabolismoRESUMO
We have developed a straightforward, one-pot, low-temperature hydrothermal method to transform oyster shell waste particles (bCCP) from the species Crassostrea gigas (Mg-calcite, 5 wt% Mg) into hydroxyapatite (HA) micro/nanoparticles. The influence of the P reagents (H3PO4, KH2PO4, and K2HPO4), P/bCCP molar ratios (0.24, 0.6, and 0.96), digestion temperatures (25-200 °C), and digestion times (1 week-2 months) on the transformation process was thoroughly investigated. At 1 week, the minimum temperature to yield the full transformation significantly reduced from 160 °C to 120 °C when using K2HPO4 instead of KH2PO4 at a P/bCCP ratio of 0.6, and even to 80 °C at a P/bCCP ratio of 0.96. The transformation took place via a dissolution-reprecipitation mechanism driven by the favorable balance between HA precipitation and bCCP dissolution, due to the lower solubility product of HA than that of calcite at any of the tested temperatures. Both the bCCP and the derived HA particles were cytocompatible for MG-63 human osteosarcoma cells and m17.ASC murine mesenchymal stem cells, and additionally, they promoted the osteogenic differentiation of m17.ASC, especially the HA particles. Because of their physicochemical features and biological compatibility, both particles could be useful osteoinductive platforms for translational applications in bone tissue engineering.
Assuntos
Carbonato de Cálcio , Nanopartículas , Camundongos , Animais , Humanos , Durapatita/farmacologia , Osteogênese , ExoesqueletoRESUMO
Oxygenic phototrophs perform carbon fixation through the Calvin-Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme in the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant isoform is formed by A2B2 heterotetramers and the least abundant by A4 homotetramers. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A4-GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of its B subunits. Reversible inhibition of AB-GAPDH occurs via the oxidation of a cysteine pair located in the CTE and the substitution of NADP(H) with NAD(H) in the cofactor-binding site. These combined conditions lead to a change in the oligomerization state and enzyme inhibition. SEC-SAXS and single-particle cryo-EM analysis were applied to reveal the structural basis of this regulatory mechanism. Both approaches revealed that spinach (A2B2)n-GAPDH oligomers with n = 1, 2, 4 and 5 co-exist in a dynamic system. B subunits mediate the contacts between adjacent tetramers in A4B4 and A8B8 oligomers. The CTE of each B subunit penetrates into the active site of a B subunit of the adjacent tetramer, which in turn moves its CTE in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphosphoglycerate in the B subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H), by removing NADP(H) from A subunits, allows the entrance of the CTE into the active site of the B subunit, hence stabilizing inhibited oligomers.
Assuntos
NAD , Fotossíntese , NADP/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Fotossíntese/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismoRESUMO
S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP+ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases , S-Nitrosoglutationa , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , NADP/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Fotossíntese , S-Nitrosoglutationa/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Biomaterials such as seashells are intriguing due to their remarkable properties, including their hierarchical structure from the nanometer to the micro- or even macroscopic scale. Transferring this nanostructure to generate nanostructured polymers can improve their electrical conductivity. Here, we present the synthesis of polypyrrole using waste seashell powder as a template to prepare a polypyrrole/CaCO3 composite material. Various synthesis parameters were optimized to produce a composite material with an electrical conductivity of 2.1 × 10-4 ± 3.2 × 10-5 S/cm. This work presents the transformation of waste seashells into sustainable, electronically conductive materials and their application as an antistatic agent in polymers. The requirements of an antistatic material were met for a safety shoe sole.
Assuntos
Polímeros , Pirróis , Exoesqueleto , Animais , Materiais Biocompatíveis/química , Condutividade Elétrica , Polímeros/química , Pirróis/químicaRESUMO
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.
Assuntos
Chlamydomonas reinhardtii , Oxirredutases , Aldeído Oxirredutases/genética , Chlamydomonas reinhardtii/genética , Cisteína , Óxido Nítrico , S-NitrosoglutationaRESUMO
The native structure of the ß-chitin in the gladius (squid pen) of Loligo vulgaris squid can be used as a natural plaster to entrap and release a model drug, doxorubicin, in a targeted and controlled way. Local pH determines the protonation state of the doxorubicin molecules, controlling the two phenomena. Confocal microscopy shows that doxorubicin is uniformly embedded in the ß-chitin squid pen and is not simply adsorbed on its surface. Coculture with HeLa cells reveals that the ß-chitin squid pen plaster is perfectly biocompatible, while when it is loaded with doxorubicin it shows high cytotoxicity toward the cancer cells. The drug, once released, rapidly accumulates inside the cells. In conclusion, the native structure of a ß-chitin squid pen can be potentially applied as a "green" pH-responsive drug vehicle for controlled release.
RESUMO
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.
Assuntos
Arabidopsis/metabolismo , Glutationa/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Anotação de Sequência Molecular , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico , Glutarredoxinas/metabolismo , Glutationa/química , Dissulfeto de Glutationa/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Cinética , Simulação de Dinâmica Molecular , Oxirredução , Dobramento de Proteína , Solubilidade , Tiorredoxinas/metabolismoRESUMO
In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central ß-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.
Assuntos
Arabidopsis , Chlamydomonas , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fotossíntese/fisiologia , Proteínas de Plantas/química , Arabidopsis/química , Arabidopsis/enzimologia , Chlamydomonas/química , Chlamydomonas/enzimologia , Cristalografia , Modelos Moleculares , Oxirredução , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/químicaRESUMO
Fe-EDDHSA/CaCO3 hybrid crystals are synthesized and tested in vitro to determine their effect in treating iron chlorosis in kiwifruit plants, used as a proof of concept. Under the alkaline conditions provided by the calcareous substrate, plants release protons that dissolve the hybrids and trigger Fe uptake. These CaCO3 hybrids represent a new system for active molecule delivery in agriculture.
Assuntos
Carbonato de Cálcio/uso terapêutico , Quelantes de Ferro/uso terapêutico , Ferro/uso terapêutico , Fenilacetatos/uso terapêutico , Doenças das Plantas/prevenção & controle , Actinidia/metabolismo , Carbonato de Cálcio/síntese química , Carbonato de Cálcio/química , Carbonato de Cálcio/metabolismo , Cristalização , Concentração de Íons de Hidrogênio , Ferro/química , Ferro/metabolismo , Quelantes de Ferro/química , Quelantes de Ferro/metabolismo , Deficiências de Ferro , Fenilacetatos/química , Fenilacetatos/metabolismoRESUMO
Global environmental change, in marine ecosystems, is associated with concurrent shifts in water temperature, circulation, stratification, and nutrient input, with potentially wide-ranging biological effects. Variations in seawater temperature might alter physiological functioning, reproductive efficiency, and demographic traits of marine organisms, leading to shifts in population size and abundance. Differences in temperature tolerances between organisms can identify individual and ecological characteristics, which make corals able to persist and adapt in a climate change context. Here we investigated the possible effect of temperature on the reproductive output of the solitary non-zooxanthellate temperate coral Leptopsammia pruvoti, along an 8° latitudinal gradient. Samples have been collected in six populations along the gradient and each polyp was examined using histological and cyto-histometric analyses. We coupled our results with previous studies on the growth, demography, and calcification of L. pruvoti along the same temperature gradient, and compared them with those of another sympatric zooxanthellate coral Balanophyllia europaea to understand which trophic strategy makes the coral more tolerant to increasing temperature. The non-zooxanthellate species seemed to be quite tolerant to temperature increases, probably due to the lack of the symbiosis with zooxanthellae. To our knowledge, this is the first field investigation of the relationship between reproductive output and temperature increase of a temperate asymbiotic coral, providing novel insights into the poorly studied non-zooxanthellate scleractinians.
Assuntos
Antozoários/fisiologia , Ecossistema , Animais , Água do Mar , Simbiose/fisiologia , TemperaturaRESUMO
Phenotype can express different morphologies in response to biotic or abiotic environmental influences. Mollusks are particularly sensitive to different environmental parameters, showing macroscale shell morphology variations in response to environmental parameters. Few studies concern shell variations at the different scale levels along environmental gradients. Here, we investigate shell features at the macro, micro and nanoscale, in populations of the commercially important clam Chamelea gallina along a latitudinal gradient (~400 km) of temperature and solar radiation in the Adriatic Sea (Italian cost). Six populations of clams with shells of the same length were analyzed. Shells from the warmest and the most irradiated population were thinner, with more oval shape, more porous and lighter, showing lower load fracture. However, no variation was observed in shell CaCO3 polymorphism (100% aragonite) or in compositional and textural shell parameters, indicating no effect of the environmental parameters on the basic processes of biomineralization. Because of the importance of this species as commercial resource in the Adriatic Sea, the experimentally quantified and significant variations of mass and fracture load in C. gallina shells along the latitudinal gradient may have economic implications for fisheries producing different economical yield for fishermen and consumers along the Adriatic coastline.
Assuntos
Bivalves/fisiologia , Luz Solar , Exoesqueleto/anatomia & histologia , Exoesqueleto/química , Animais , Bivalves/anatomia & histologia , Bivalves/efeitos da radiação , Carbonato de Cálcio/análise , Módulo de Elasticidade , Microscopia Eletrônica de Varredura , Porosidade , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Difração de Raios XRESUMO
AIMS: Cysteines and H2O2 are fundamental players in redox signaling. Cysteine thiol deprotonation favors the reaction with H2O2 that generates sulfenic acids with dual electrophilic/nucleophilic nature. The protein microenvironment surrounding the target cysteine is believed to control whether sulfenic acid can be reversibly regulated by disulfide formation or irreversibly oxidized to sulfinates/sulfonates. In this study, we present experimental oxidation kinetics and a quantum mechanical/molecular mechanical (QM/MM) investigation to elucidate the reaction of H2O2 with glycolytic and photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana (cytoplasmic AtGAPC1 and chloroplastic AtGAPA, respectively). RESULTS: Although AtGAPC1 and AtGAPA have almost identical 3D structure and similar acidity of their catalytic Cys149, AtGAPC1 is more sensitive to H2O2 and prone to irreversible oxidation than AtGAPA. As a result, sulfenic acid is more stable in AtGAPA. INNOVATION: Based on crystallographic structures of AtGAPC1 and AtGAPA, the reaction potential energy surface for Cys149 oxidation by H2O2 was calculated by QM. In both enzymes, sulfenic acid formation was characterized by a lower energy barrier than sulfinate formation, and sulfonate formation was prevented by very high energy barriers. Activation energies for both oxidation steps were lower in AtGAPC1 than AtGAPA, supporting the higher propensity of AtGAPC1 toward irreversible oxidation. CONCLUSIONS: QM/MM calculations coupled to fingerprinting analyses revealed that two Arg of AtGAPA (substituted by Gly and Val in AtGAPC1), located at 8-15 Å distance from Cys149, are the major factors responsible for sulfenic acid stability, underpinning the importance of long-distance polar interactions in tuning sulfenic acid stability in native protein microenvironments.
Assuntos
Arabidopsis/metabolismo , Cisteína/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Ácidos Sulfênicos/metabolismo , Sítios de Ligação , Domínio Catalítico , Cloroplastos/metabolismo , Citoplasma/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/química , Peróxido de Hidrogênio/metabolismo , Cinética , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação ProteicaRESUMO
The variety of reproductive processes and modes among coral species reflects their extraordinary regeneration ability. Scleractinians are an established example of clonal animals that can exhibit a mixed strategy of sexual and asexual reproduction to maintain their populations. This study provides the first description of the annual reproductive cycle and embryogenesis of the temperate species Caryophyllia inornata. Cytometric analyses were used to define the annual development of germ cells and embryogenesis. The species was gonochoric with three times more male polyps than female. Polyps were sexually mature from 6 to 8 mm length. Not only females, but also sexually inactive individuals (without germ cells) and males were found to brood their embryos. Spermaries required 12 months to reach maturity, while oogenesis seemed to occur more rapidly (5-6 months). Female polyps were found only during spring and summer. Furthermore, the rate of gamete development in both females and males increased significantly from March to May and fertilization was estimated to occur from April to July, when mature germ cells disappeared. Gametogenesis showed a strong seasonal influence, while embryos were found throughout the year in males and in sexually inactive individuals without a defined trend. This unusual embryogenesis suggests the possibility of agamic reproduction, which combined with sexual reproduction results in high fertility. This mechanism is uncommon and only four other scleractinians (Pocillopora damicornis, Tubastraea diaphana, T. coccinea and Oulastrea crispata) have been shown to generate their broods asexually. The precise nature of this process is still unknown.
Assuntos
Antozoários/embriologia , Antozoários/fisiologia , Desenvolvimento Embrionário , Reprodução , Animais , Feminino , Fertilidade , Masculino , Mar Mediterrâneo , Estações do Ano , Razão de Masculinidade , Comportamento SexualRESUMO
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.
Assuntos
Carbonato de Cálcio/química , Doxorrubicina/química , Portadores de Fármacos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalização , Doxorrubicina/toxicidade , Humanos , Microscopia Confocal , Microscopia Eletrônica de VarreduraRESUMO
Recently, the results of experimental and theoretical investigations have revealed that, in vaterite, two or even more crystalline structures coexist. In this communication we report evidence of diverse vaterite structures in biogenic samples of different origin. In addition, it is shown that the synthetic vaterite precipitated in the presence of poly-l-aspartate has structures similar to those of biogenic samples.
Assuntos
Carbonato de Cálcio/química , Animais , Carbonato de Cálcio/síntese química , Cloreto de Cálcio/química , Varredura Diferencial de Calorimetria , Carbonatos/química , Precipitação Química , Cristalização , Cyprinidae , Microscopia Eletrônica de Varredura , Estrutura Molecular , Peptídeos/química , Bicarbonato de Sódio/química , Espectroscopia de Infravermelho com Transformada de Fourier , Unionidae , Urocordados , Difração de Raios XRESUMO
Upon slow evaporation of a 1:1 diastereoisomeric mixture of Boc-(L-Phe-L-Oxd)2 -OBn (1; Boc=tert-butyloxycarbonyl; L-Oxd=trans-(4S,5R)-4-carboxy 5-methyloxazolidin-2-one, Bn=benzyloxycarbonyl) and Boc-L-Phe-L-Oxd-D-Phe-L-Oxd-OBn (2) in methyl tert-butyl ether, single crystals suitable for an X-ray diffraction study were obtained. In contrast, the two pure oligomers lead to the formation of amorphous solids under any crystallization conditions. The preferential conformation of both oligomers was fully elucidated in the solid phase and compared with the known conformation of Boc-(L-Phe-D-Oxd)2 -OBn (3). The preferred conformation of 1 ranges from a polyprolineâ II (PPII) helix to ß strands and we can gather that longer and more structured oligomers will form PPII helices. In contrast, compound 3 forms infinite antiparallel ß-sheet structures; thus showing the strong effect of the reversal of the absolute configuration of the Oxd moieties on the secondary structure of these hybrid foldamers. The same outcome was retained in solution, as demonstrated by vibrational circular dichroism analysis. Finally, we have demonstrated that a 1:1 mixture of 1 and 2 leads to the formation of new materials with interesting properties that are missing from the two pure compounds, such as the tendency to form crystals, fibers, and globules, depending on the solvent.
RESUMO
Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES) pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC). A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A). Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Diferenciação Celular , Polpa Dentária/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Biomarcadores/metabolismo , Criança , Pré-Escolar , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fator 4 Semelhante a Kruppel , Fator 3 de Transcrição de Octâmero/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
While knowledge of the reproductive biology of tropical scleractinian corals is extensive, information from temperate zones is limited. The aim of this study is to describe the reproductive biology of Caryophyllia inornata, a temperate species, and to increase the understanding of the reproductive strategies of Mediterranean corals. Samples of C. inornata were collected during SCUBA surveys at Elba island. Sexually active individuals displayed either male or female germ cells, showing a gonochoric sexuality. C. inornata exhibited an unusual pattern of embryogenesis. Embryos appeared throughout the whole year in males and in sexually inactive individuals, and they did not show a seasonal pattern of development, as usually expected for sexual reproduction. This observation suggests the possibility of asexual origin. These embryogenetic sexually inactive individuals were larger in size than the embryogenetic sexually active ones, and they might be senile polyps that preserve the ability to produce embryos only by agamic reproduction.
Assuntos
Antozoários/embriologia , Desenvolvimento Embrionário , Reprodução Assexuada , Animais , Tamanho Corporal , Feminino , Masculino , Mar Mediterrâneo , ReproduçãoRESUMO
Periosteum contains mesenchymal stem cells (Pe-MSCs) that contribute to normal bone growth, healing, and turnover; understanding Pe-MSC capabilities may shed light over the treatment of bone defects using tissue engineering. Bone tissue regeneration needs in vitro bone precursors or stem cell coculture onto specific scaffolds but, despite extensive research in the field, very little is known about the matrix structure of the tissue-engineered tissues and the scaffold's effects on cell differentiation. To this purpose we have selected a clonal population (murine Pe-MSCs) that was seeded and differentiated onto an acellular bone scaffold. Cell differentiation was assessed after 3 months and 1 year by molecular, histological, biochemical, and biophysical analyses and results were compared with the same osteoinduced clonal cells cultured as cellular aggregates. Our data show that Pe-MSCs cultured onto acellular bone scaffold develop a complex three-dimensional matrix and an osteoblastic phenotype but do not produce hydroxyapatite (HA); moreover, they seem able to reabsorb the colonized bone scaffold. On the contrary, cells cultured as three-dimensional aggregates differentiate and produce osteoblastic markers and HA nanocrystals.