RESUMEN
Model systems constituted by proteins and unsaturated lipid vesicles were used to gain more insight into the effects of the propagation of an initial radical damage on protein to the lipid compartment. The latter is based on liposome technology and allows measuring the trans unsaturated fatty acid content as a result of free radical stress on proteins. Two kinds of sulfur-containing proteins were chosen to connect their chemical reactivity with membrane lipid transformation, serum albumins and metallothioneins. Biomimetic systems based on radiation chemistry were used to mimic the protein exposure to different kinds of free radical stress and Raman spectroscopy to shed light on protein structural changes caused by the free radical attack. Among the amino acid residues, Cys is one of the most sensitive residues towards the attack of free radicals, thus suggesting that metal-Cys clusters are good interceptors of reactive species in metallothioneins, together with disulfides moieties in serum albumins. Met is another important site of the attack, in particular under reductive conditions. Tyr and Phe are sensitive to radical stress too, leading to electron transfer reactions or radical-induced modifications of their structures. Finally, modifications in protein folding take place depending on reactive species attacking the protein.
Asunto(s)
Radicales Libres/química , Lípidos de la Membrana/química , Metalotioneína/química , Albúmina Sérica/química , Aminoácidos/química , Biomimética , Isomerismo , Proteínas de Plantas/química , Isoformas de Proteínas/química , Estructura Secundaria de Proteína , Quercus , Espectrometría RamanRESUMEN
Substance P (SP) is one of the most studied peptide hormones and knowing the relationship between its structure and function may have important therapeutic applications in the treatment of a variety of stress-related illnesses. In order to obtain a deeper insight into its folding, the effects of different factors, such as pH changes, the presence of Ca2+ ions, and the substitution of the Met-NH2 moiety in the SP structure, was studied by Raman and infrared spectroscopies. SP has a pH-dependent structure. Under acidic-neutral conditions, SP possesses a prevalent ß-sheet structure although also other secondary structure elements are present. By increasing pH, a higher orderliness in the SP secondary structure is induced, as well as the formation of strongly bound intermolecular ß-strands with a parallel alignment, which favour the self-assembly of SP in ß-aggregates. The substitution of the Met-NH2 moiety with the acidic functional group in the SP sequence, giving rise to a not biologically active SP analogue, results in a more disordered folding, where the predominant contribution comes from a random coil. Conversely, the presence of Ca2+ ions affects slightly but sensitively the folding of the polypeptide chain, by favouring the α-helical content and a different alignment of ß-strands; these are structural elements, which may favour the SP biological activity. In addition, the capability of SERS spectroscopy to detect SP in its biologically active form was also tested by using different metal nanoparticles. Thanks to the use of silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride, SP can be detected at very low peptide concentration (~ 90 nM). However, the SERS spectra cannot be obtained under alkaline conditions since both the formation of SP aggregates and the lack of ion pairs do not allow a strong enough interaction of SP with silver NPs. Graphical abstract.
Asunto(s)
Espectroscopía Infrarroja por Transformada de Fourier/métodos , Sustancia P/química , Vibración , Calcio/química , Conformación Proteica , Espectrometría Raman/métodosRESUMEN
Metallothioneins are small Cys-rich peptides capable of coordinating metal ions, and proposed to be involved in radical stress. The four Zn(ii)-GmMT complexes of soybean (Glycine max) were recombinantly synthesised and exposed to oxidative (HOË) and reductive (HË atoms and eaq-) stress conditions. Gamma-irradiation was used to simulate the endogenous formation of the reactive species in both aqueous solutions and unsaturated lipid vesicle suspensions, a biomimetic model that showed that tandem protein/lipid damage occurs, in particular under reductive radical stress. This is due to the formation of diffusible sulphur-centred radicals, which migrate from the aqueous phase to the lipid bilayer and are thus able to transform the cis double bond of the oleate moiety into the trans isomer. Among the amino acid residues present in GmMTs, Cys is one of the most sensitive residues towards the attack of free radicals, thus suggesting metal-clusters to be good interceptors of free radicals. Also Met, Tyr and Phe residues are sensitive amino acid sites of attack under both oxidative and reductive conditions. The modification of the Zn(ii)-GmMT complexes, in particular isoform 2, was monitored by Raman spectroscopy and mass spectrometry. Free radical stress on the Zn(ii)-GmMT complexes is able to induce significant structural changes such as partial deconstruction and/or rearrangement of the metal clusters, but not the complete demetallation of the proteins nor breaking of the backbone, thus confirming their capability to act as protectors under free radical stress conditions.
Asunto(s)
Complejos de Coordinación/química , Glycine max/metabolismo , Membrana Dobles de Lípidos/química , Metalotioneína/química , Estrés Oxidativo , Procesamiento Proteico-Postraduccional , Zinc/metabolismo , Complejos de Coordinación/metabolismo , Radicales Libres , Membrana Dobles de Lípidos/metabolismo , Metalotioneína/metabolismo , Oxidación-Reducción , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Radiólisis de Impulso , Zinc/químicaRESUMEN
Seaweed extracts can be employed as biostimulants during crop cultivation owing to their positive effects on plant performance. Therefore, in this study one extract from Laminaria (A) and five extracts from Ascophyllum nodosum (B-F) were assayed on maize (Zea mays L.) plants supplied for 2 days with 0.5 mL L-1 of single products to evaluate their capacity to stimulate root growth and morphology, nutrition, and sugars accumulation. Firstly, extracts were chemically characterized via Fourier transform infrared (FT-IR) and FT-Raman spectroscopies, and their content in carbon, nitrogen, phenolic acids and hormones (indole-3-acetic acid, IAA, and Isopentenyladenosine, IPA) was quantified. The auxin like- and gibberellic acid -like activities of all extracts were also determined. FT-IR and FT-Raman spectra provided complementary information depicting distinct spectral pattern for each extract. Bands assigned to alginic and uronic acids were dominant in FT-IR spectra, while those corresponding to polyaromatic rings were evident in FT-Raman spectra. In general, extracts stimulated root growth, nutrition, esterase activity, and sugar content. However, they showed high variation in chemical features, which may explain their different capacity in triggering physiological responses in maize. Among A. nodosum extracts for instance, E was the most efficient in promoting root morphology traits, likely because of its elevate content in IAA (32.43 nM), while F extract was the highest in phenol content (1,933 mg L-1) and the most successful in improving plant nutrition. On the other hand, C extract was very effective in stimulating root elongation, but did not influence plant nutrition. B and D extracts induced similar positive effects on plants, although they greatly varied in chemical composition. Laminaria extract (A) differed from A. nodosum extracts, because of its low content in total phenols and the presence of both IAA- and GA-like activity. We conclude that all seaweed extracts acted as biostimulants in maize, but their chemical properties appeared crucial in predicting the physiological response preferentially elicited by individual seaweed extracts.
RESUMEN
The influence of vegetal extracts derived from red grape, blueberry fruits and hawthorn leaves on Zea mays L. plant growth and the activity of phenylalanine ammonia-lyase (PAL), a key enzyme of the phenylpropanoid pathway, was investigated in laboratory experiments. The extracts were characterized using FT-IR and Raman spectroscopies in order to obtain a pattern of the main functional groups. In addition, phenols content was determined by HPLC, whereas the content of indoleacetic acid and isopentenyladenosine hormones was determined by ELISA test and the auxin and gibberellin-like activities by plant-bioassays. The treated maize revealed increased root and leaf biomass, chlorophyll and sugars content with respect to untreated plants. Hawthorn, red grape skin and blueberry at 1.0 mL/L induced high p-coumaric content values, whilst hawthorn also showed high amounts of gallic and p-hydroxybenzoic acids. PAL activity induced by hawthorn at 1.0 mL/L had the highest values (11.1-fold UNT) and was strongly and linearly related with the sum of leaf phenols. Our results suggest that these vegetal extracts contain more than one group of plant-promoting substances.
Asunto(s)
Arándanos Azules (Planta)/química , Crataegus/química , Fenilanina Amoníaco-Liasa/metabolismo , Extractos Vegetales/farmacología , Vitis/química , Zea mays/crecimiento & desarrollo , Biomasa , Ácidos Indolacéticos/química , Ácidos Indolacéticos/farmacología , Isopenteniladenosina/química , Isopenteniladenosina/farmacología , Fenoles/análisis , Fenoles/farmacología , Extractos Vegetales/análisis , Reguladores del Crecimiento de las Plantas/química , Reguladores del Crecimiento de las Plantas/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/crecimiento & desarrollo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Zea mays/efectos de los fármacosRESUMEN
Four Metallothioneins (MTs) from soybean (Glycine max) were heterologously synthesized and comparatively analysed by Raman spectroscopy. The participation of protein donor groups (S-thiol and N-imidazol) in Zn(II) chelation, as well as the presence of secondary structure elements was comparatively analysed. Metal clusters with different geometry can be hypothesised for the four GmMTs: a cubane-like or an adamantane-like metal cluster in Zn-GmMT1, and dinuclear Zn-S clusters in Zn-GmMT2, Zn-GmMT3 and Zn-GmMT4. The latter have also a similar average Cys/Zn content, whereas a lower ratio is present in Zn-GmMT1. This is possible thanks to the involvement in metal coordination of a greater number of bridging Cys, as well as of some carboxylate groups. As regards secondary structure elements, a large content of ß-turn segments is present in all four Zn-GmMTs, especially for isoforms 1 and 4. ß-strands give a contribution to the folding of three GmMTs isoforms, and the highest percentage was found in Zn-GmMT2 (~45%). Conversely, the α-helix content is negligible in all the GmMTs except in Zn-GmMT3, where this peculiar feature coincides with the possible involvement of the two His residues in metal coordination. Conversely, His is predominantly free and present as tautomer I in Zn-GmMT4. In conclusion, this work illustrates the attractive potential of Raman spectroscopy, combined with other techniques, to be a very informative tool for evidencing structural differences among in vivo synthesized metal-MT complexes.
Asunto(s)
Metalotioneína/metabolismo , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/metabolismo , Espectrometría Raman/métodos , Zinc/metabolismo , Secuencia de Aminoácidos , Metalotioneína/química , Datos de Secuencia Molecular , Proteínas de Plantas/química , Conformación Proteica , Isoformas de Proteínas/química , Homología de Secuencia de AminoácidoRESUMEN
Lipid geometry is an important issue in biology and medicine. The cis-trans geometry conversion of double bonds in lipids is an endogenous process that can be mediated by sulfur-centered free radicals. Trans isomers of polyunsaturated fatty acids can be used as biological markers of free radical stress, and their presence in biological samples can be determined by synthesis and characterization of appropriate reference compounds. Fractions of plasma lipids, such as cholesteryl linoleate and arachidonate esters, are interesting targets because of their connection with membrane phospholipid turnover and their roles in cardiovascular health. In this context, Raman spectroscopy can provide a useful contribution, since Raman analysis can be performed directly on the lipid extracts without any derivatization reaction, is nondestructive, and can rapidly supply biochemical information. This study focused on the build up of Raman spectral libraries of different cis and trans isomers of cholesteryl esters to be used as references for the examination of complex biological samples and to facilitate isomer recognition. Unsaturated cholesteryl esters obtained by chemical synthesis and with different alkyl chain lengths, double bond numbers, or both, were analyzed. The potential of Raman analysis for trans isomer detection in biological samples was successfully tested on some cholesteryl ester lipid fractions from human serum. The data suggest promising applications of Raman spectroscopy in metabolomics and lipidomics.
Asunto(s)
Ésteres del Colesterol/química , Espectrometría Raman/métodos , Ésteres del Colesterol/sangre , Cromatografía de Gases , Humanos , IsomerismoRESUMEN
Molecular basis of the role played by Cu(2+) and Zn(2+) ions during the thermal aggregation processes of beta-lactoglobulin (BLG) was studied by using a joint application of different techniques. In particular, Raman spectroscopy was very useful in identifying the different effects caused by the two metals at molecular level (i.e. changes in His protonation state, disulfides bridge conformation, and micro-environment of aromatic residues), evidencing the primary importance of the protein charge distribution during the aggregation process. Both metal ions are able to act on this factor and favor the protein aggregation, but Zn(2+) is able to alter the natural conformational state of BLG, causing a slight unfolding, whereas Cu(2+) ions play a role only during the thermal treatment. Thus, Zn(2+) ions favor the formation of bigger aggregates and branched fibril-like structures, whereas for Cu(2+) ions a greater number of cross-beta structures during thermal incubation and finally, fibrillar structures. The aggregation process occurs in two phases, as suggested by the measurements on the time evolution of the BLG aggregates: the first one is characterized by a partial unfolding of the protein and aggregate growth, forming oligomers and protofibrils, whereas the second one is characterized by further supramolecular assembly, leading to the formation of fibrils.
Asunto(s)
Cobre/química , Iones/química , Lactoglobulinas/química , Zinc/química , Lactoglobulinas/metabolismo , Agregado de Proteínas , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , TemperaturaRESUMEN
Prevention of iron chlorosis with Fe synthetic chelates is a widespread agronomical practice but implies high costs and environmental risks. Blood meal is one of the main fertilizers allowed to be used in organic farming. Through this work a novel blood meal fertilizer was audited. Measurements such as FTIR, Raman, electron paramagnetic resonance, and Mössbauer spectroscopy, UV-visible properties, stability against pH, and batch experiments were performed to characterize and assess the reactivity on soil constituents and agronomic soils. The spectroscopy findings give clear indications that Fe is in the ferric oxidation state, is hexacoordinated, and has a low-spin form suggesting a similar structure to hemin and hematin. A spectrophotometric method at 400 nm was validated to quantify blood meal concentration at low electrolyte concentrations. Batch experiments demonstrated high reactivity of blood meal fertilizer with soil constituents, mainly in the presence of calcium, where aggregation processes are predominant, and its ability to take Fe from synthetic Fe (hydr)oxides. The beneficial profile of blood meal by a providing nitrogen source together with the capability to keep the Fe bound to porphyrin organic compounds makes it a good candidate to be used as Fe fertilizer in organic farming.
Asunto(s)
Compuestos Férricos/química , Fertilizantes/análisis , Agricultura Orgánica/métodos , Sangre , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Suelo/química , Espectroscopía Infrarroja por Transformada de Fourier , Espectroscopía de MossbauerRESUMEN
Acute and chronic Plasmodium falciparum infections alter the immune competence of the host possibly through changes in dendritic cell (DC) functionality. DCs are the most potent activators of T cells, and migration is integral to their function. Mature DCs express lymphoid chemokine receptors (CCRs), expression of which enables them to migrate to the lymph nodes, where they encounter naïve T cells. The present study aimed to investigate the impact of the synthetic analog to malaria parasite pigment hemozoin, i.e., ß-hematin, or infected erythrocytes (iRBCs) on the activation status of human monocyte-derived DCs and on their expression of CCRs. Human monocyte-derived DCs partially matured upon incubation with ß-hematin as indicated by an increased expression of CD80 and CD83. Both ß-hematin and iRBCs provoked the release of proinflammatory and anti-inflammatory cytokines, such as interleukin-6 (IL-6), IL-10, and tumor necrosis factor alpha, but not IL-12, and induced upregulation of the lymphoid chemokine receptor CXCR4, which was coupled to an increased migration to lymphoid ligands. Taken together, these results suggest that the partial and transient maturation of human myeloid DCs upon stimulation with malaria parasite-derived products and the increased IL-10 but lack of IL-12 secretion may lead to suboptimal activation of T cells. This may in turn lead to impaired adaptive immune responses and therefore insufficient clearance of the parasites.
Asunto(s)
Quimiocinas/inmunología , Células Dendríticas/inmunología , Eritrocitos/metabolismo , Eritrocitos/parasitología , Hemoproteínas/metabolismo , Plasmodium falciparum/fisiología , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígeno B7-1/biosíntesis , Antígeno B7-1/genética , Movimiento Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Humanos , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/genética , Interleucina-10/biosíntesis , Interleucina-12/deficiencia , Interleucina-6/biosíntesis , Activación de Linfocitos , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Plasmodium falciparum/inmunología , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Antígeno CD83RESUMEN
Metallobiomolecules are highly elaborated coordination complexes, and their fundamental metal-ligand interactions are critical components of metalloprotein folding, assembly, stability, electrochemistry, and catalytic function. Herein, we have described the benefits in using Raman spectroscopy to define the metal-ion binding properties of MTs toward metal ions such as Zn(ii) and Cd(ii). In particular, this vibrational technique can shed light on the secondary structures eventually present in MTs and the ligands involved in metal coordination. The oxidation state of Cys residues and their participation in the metal chelation can be clearly defined, as well as the eventual involvement of His residues. With regards to exogenous metal ligands such as sulfide anions, their presence can be identified by some marker bands whose intensity is linearly correlated with sulfide/metal molar ratio. Finally, Raman can be also an useful tool for providing information on the favourite sites of the radical attack and radical-induced modification in protein folding. In conclusion, many advantages such as the capability of defining local regions in large complexes and detecting several structural features at the same time, the ability in supporting mechanisms, as well as the requirement of low sample amount, make to propose Raman spectroscopy, in coupling with analytical techniques such as atomic emission spectroscopy, gas chromatography, and circular dichroism, as one of the most promising experimental strategies in the research on structure-activity relationships in MTs.
Asunto(s)
Metalotioneína/química , Espectrometría Raman/métodos , Cobre/química , Cobre/metabolismo , Metalotioneína/metabolismo , Relación Estructura-Actividad , Zinc/química , Zinc/metabolismoRESUMEN
The effect of ageing in phosphate-containing solution of bioactive calcium-silicate cements on the chemistry, morphology and topography of the surface, as well as on in vitro human marrow stromal cells viability and proliferation was investigated. A calcium-silicate cement (wTC) mainly based on dicalcium-silicate and tricalcium-silicate was prepared. Alpha-TCP was added to wTC to obtain wTC-TCP. Bismuth oxide was inserted in wTC to prepare a radiopaque cement (wTC-Bi). A commercial calcium-silicate cement (ProRoot MTA) was tested as control. Cement disks were aged in DPBS for 5 h ('fresh samples'), 14 and 28 days, and analyzed by ESEM/EDX, SEM/EDX, ATR-FTIR, micro-Raman techniques and scanning white-light interferometry. Proliferation, LDH release, ALP activity and collagen production of human marrow stromal cells (MSC) seeded for 1-28 days on the cements were evaluated. Fresh samples exposed a surface mainly composed of calcium-silicate hydrates CSH (from the hydration of belite and alite), calcium hydroxide, calcium carbonate, and ettringite. Apatite nano-spherulites rapidly precipitated on cement surfaces within 5 h. On wTC-TCP the Ca-P deposits appeared thicker than on the other cements. Aged cements showed an irregular porous calcium-phosphate (Ca-P) coating, formed by aggregated apatite spherulites with interspersed calcite crystals. All the experimental cements exerted no acute toxicity in the cell assay system and allowed cell growth. Using biochemical results, the scores were: fresh cements>aged cements for cell proliferation and ALP activity (except for wTC-Bi), whereas fresh cementsAsunto(s)
Apatitas/farmacología
, Materiales Biocompatibles/química
, Células de la Médula Ósea/efectos de los fármacos
, Compuestos de Calcio/química
, Cemento de Silicato/química
, Silicatos/química
, Apatitas/síntesis química
, Materiales Biocompatibles/farmacología
, Bismuto/farmacología
, Compuestos de Calcio/farmacología
, Fosfatos de Calcio/farmacología
, Proliferación Celular/efectos de los fármacos
, Supervivencia Celular/efectos de los fármacos
, Células Cultivadas
, Colágeno/biosíntesis
, Medios de Contraste/farmacología
, Almacenaje de Medicamentos
, Humanos
, Interacciones Hidrofóbicas e Hidrofílicas
, Interferometría
, Ensayo de Materiales
, Nanosferas
, Espectroscopía de Fotoelectrones
, Cemento de Silicato/farmacología
, Silicatos/farmacología
, Espectroscopía Infrarroja por Transformada de Fourier
, Espectrometría Raman
, Células del Estroma/efectos de los fármacos
, Propiedades de Superficie/efectos de los fármacos
RESUMEN
The bioactivity of calcium silicate mineral trioxide aggregate (MTA) cements has been attributed to their ability to produce apatite in presence of phosphate-containing fluids. This study evaluated surface morphology and chemical transformations of an experimental accelerated calcium-silicate cement as a function of soaking time in different phosphate-containing solutions. Cement discs were immersed in Dulbecco's phosphate-buffered saline (DPBS) or Hank's balanced salt solution (HBSS) for different times (1-180 days) and analysed by scanning electron microscopy connected with an energy dispersive X-ray analysis (SEM-EDX) and micro-Raman spectroscopy. SEM-EDX revealed Ca and P peaks after 14 days in DPBS. A thin Ca- and P-rich crystalline coating layer was detected after 60 days. A thicker multilayered coating was observed after 180 days. Micro-Raman disclosed the 965-cm(-1) phosphate band at 7 days only on samples stored in DPBS and later the 590- and 435-cm(-1) phosphate bands. After 60-180 days, a layer approximately 200-900 µm thick formed displaying the bands of carbonated apatite (at 1,077, 965, 590, 435 cm(-1)) and calcite (at 1,088, 713, 280 cm(-1)). On HBSS-soaked, only calcite bands were observed until 90 days, and just after 180 days, a thin apatite-calcite layer appeared. Micro-Raman and SEM-EDX demonstrated the mineralization induction capacity of calcium-silicate cements (MTAs and Portland cements) with the formation of apatite after 7 days in DPBS. Longer time is necessary to observe bioactivity when cements are immersed in HBSS.
Asunto(s)
Compuestos de Aluminio/química , Apatitas/química , Materiales Biocompatibles/química , Compuestos de Calcio/química , Óxidos/química , Fosfatos/química , Materiales de Obturación del Conducto Radicular/química , Silicatos/química , Tampones (Química) , Calcio/análisis , Carbonato de Calcio/química , Precipitación Química , Cristalización , Combinación de Medicamentos , Microanálisis por Sonda Electrónica , Humanos , Concentración de Iones de Hidrógeno , Soluciones Isotónicas/química , Cinética , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Microespectrofotometría , Fósforo/análisis , Porosidad , Obturación Retrógrada , Cloruro de Sodio/química , Espectrometría Raman , Propiedades de Superficie , Temperatura , Factores de TiempoRESUMEN
Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies. In this work the effects of Cu(II) and Zn(II) ions on heat-induced structural modifications of bovine serum albumin (BSA) were studied, with the aim of delineating the role of these ions in the early stages of proteins aggregation kinetics. A joint application of different techniques was used. The aggregate growth was followed by dynamic light scattering measurements, whereas the conformational changes occurring in the protein structure were monitored by Raman and IR spectroscopy. Both in absence and in presence of metal ions, heating treatment gave rise to beta-structures to the detriment of alpha-helix conformation of BSA. The temperature of protein unfolding was not sensitively affected by the presence of Zn(II) or Cu(II) ions; on the contrary, only Zn(II) ions slightly promoted the heat-induced aggregation of the protein, since bigger aggregates were formed in their presence. The different efficacy of the Cu(II) and Zn(II) ions in promoting the BSA aggregation were highlighted by Raman measurements, assessing the role of His residues in metal binding. A distinct polypeptide folding of the two metal-BSA systems takes place, since the predominant mode of metal binding depends on metal. In particular, in Zn-BSA the metal coordination involves the imidazole N(tau) atom of His which can promote inter-molecular cross-linking.
Asunto(s)
Cobre/metabolismo , Histidina/metabolismo , Albúmina Sérica Bovina/metabolismo , Zinc/metabolismo , Animales , Bovinos , Cinética , Unión Proteica , Conformación Proteica , Albúmina Sérica Bovina/química , TemperaturaRESUMEN
Metallothioneins (MTs) are metal-chelating peptides that play an active role in zinc homeostasis. The participation of metal ligands other than cysteines and the presence of secondary structure elements in metal-MT complexes are fairly unknown, especially in nonvertebrate MTs. Here, four Zn(II) complexes of invertebrate MTs (mollusc, insect, nematode, and echinoderm) and the Zn(II)-MT complex of the mammalian MT1 isoform, heterologously synthesized in E. coli, were studied by analytic and spectroscopic techniques. By Raman and circular dichroism spectroscopy, new structural informations were obtained. The five analyzed MT isoforms consist largely of beta-turns with the near exclusion of alpha-helical segments. Raman spectroscopy was revealed as an useful tool, providing information about the state of the cysteine sulfur atoms (metal coordinated and oxidized), the participation of histidine in metal coordination, and the molecular environment of tyrosine residues. In all the five Zn(II)-MT studied samples, acid-labile sulfide anions were found as nonproteic ligands, since sulfide-containing and sulfide-devoid species coexisted in the corresponding preparations. Significantly, Raman bands useful as markers of sulfide bridging ligands were identified. Overall, this work illustrates how the combination of analytical and spectroscopic techniques can be a very informative approach for the analysis of in vivo-synthesized metal-MT complexes, providing new data on the metal binding behavior of MTs from the most diverse organisms.
Asunto(s)
Metalotioneína/química , Metalotioneína/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Mamíferos , Metalotioneína/síntesis química , Metales/análisis , Metales/química , Ratones , Datos de Secuencia Molecular , Mytilus edulis/química , Mytilus edulis/metabolismo , Pliegue de Proteína , Isoformas de Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría Raman , Strongylocentrotus purpuratus/metabolismoRESUMEN
Tetrahymena pyriformis MT1 (TpyMT1) is a model among ciliate metallothioneins (MTs). Here, we report on the analytic (ICP-AES, GC-FPD), spectroscopic (CD, UV-Vis, Raman) and spectrometric (ESI-MS) characterization of its recombinant Cd(II)-, Zn(II)- and Cu(I)-complexes, and of those formed during in vitro Zn/Cd and Zn/Cu replacement. In the presence of Cd(II), TpyMT1 renders a major Cd 11-TpyMT1 species, which is also the final step reached in the in vitro Zn/Cd exchange process in Zn 11-TpyMT1. Spectroscopic data supports a different folding of the isostoichiometric Cd 11- and Zn 11-TpyMT1 complexes. Unexpectedly, TpyMT1 biosynthesis in Zn(II)-rich cultures was sensitive to the aeration degree, so that high oxygenation rendered undermetalated, partially-oxidized, complexes (Zn9-TpyMT1). Biosynthesis in Cu(I)-rich media rendered extremely heterogeneous mixtures of CuxZny-species (x+y=8-20), where the higher the aeration, the higher the Zn(II) content. The complexity of these samples was reproduced during the Zn/Cu replacement, as the number of generated species increased gradually with the addition of copper to Zn(11)-TpyMT1. According to our results, a clear preference of TpyMT1 for Cd(II) binding, rather than for Zn(II), and especially Cu(I) can be postulated. This character is totally consistent with the induction pattern of the TpyMT1 gene and the postulated role of TpyMT1 in Cd-detoxification.
Asunto(s)
Metalotioneína/química , Metales Pesados/metabolismo , Proteínas Protozoarias/química , Tetrahymena pyriformis/metabolismo , Animales , Cadmio/metabolismo , Dicroismo Circular , Cobre/metabolismo , Metalotioneína/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/metabolismo , Espectrometría Raman , Zinc/metabolismoRESUMEN
Zn- and Cd-complexes of Quercus suber metallothionein (QsMT) were obtained by in vivo-synthesis, in order to obtain physiologically representative aggregates, and characterized by spectrometric and spectroscopic methods. The secondary structure elements and the coordination environments of the metal binding sites of the two aggregates were determined, as well as the main metal-containing species formed. The results obtained from the analysis of the Raman and IR spectra reveal that these metal-MT complexes predominantly contain beta-sheet elements (about 60%), whereas they lack alpha-helices. These structural features slightly depend on the divalent metal bound. In particular, Cd(II) binding to QsMT induces a slight increase of the beta-sheet percentage, as well as a decrease in beta-turn elements with respect to Zn(II) binding. Conversely, the in vivo capability of QsMT to inglobe metal and sulfide ions is metal-depending. Spectroscopic vibrational data also confirm the presence of sulfide ligands in the metal clusters of both Zn- and Cd-QsMT, while the participation of the spacer His residue in metal coordination was only found in Cd-QsMT, in agreement with the CD results. Overall data suggest different coordination environments for Zn(II) and Cd(II) ions in QsMT.
Asunto(s)
Cadmio/metabolismo , Metalotioneína/química , Metalotioneína/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Quercus/química , Análisis Espectral/métodos , Zinc/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Histidina/química , Ligandos , Modelos Químicos , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Espectrofotometría Infrarroja , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Estereoisomerismo , Azufre/químicaRESUMEN
BACKGROUND: Surface characteristics play a major role in determining tissue response to implants and therefore their clinical outcome. The aim of the present study was to compare two commercially available titanium surfaces: plasma sprayed (TPS) and sand-blasted, acid-etched surface (SLA). METHODS: The surfaces were characterized by roughness testing, scanning electronic microscopy (SEM), Raman spectroscopy, and protein adsorption to determine their microtopographic and chemical properties. The effect of the surfaces on human mandibular osteoblasts was then studied in terms of cell morphology, adhesion, proliferation, and differentiation. Human osteoblasts from the mandible were cultured on these two surfaces and evaluated at 3, 6, 24, and 48 hours to determine cell attachment and morphology. Growth and differentiation kinetics were subsequently investigated by evaluating cell growth, alkaline phosphatase activity, osteocalcin and osteoprotegerin production at 7, 14, and 21 days. RESULTS: Although roughness was quite similar, the two surfaces presented strong differences in their topography, and cell morphology varied as a consequence. Osteoblasts on SLA appeared more elongated and spindle shaped than those on TPS, and their adhesion at 3 and 6 hours was weaker, but reached that of cells on TPS at hour 24. Cell proliferation was greater on SLA surfaces but differentiation parameters; i.e., alkaline phosphatase and osteocalcin, provided better results on TPS surfaces. Osteoprotegerin production was enhanced on TPS surfaces at days 14 and 21. CONCLUSION: Although cells grown on both surfaces exhibited good adhesion capabilities, a well-differentiated osteoblastic phenotype, and maintained a clear proliferation potential, our study suggests that plasma-sprayed treatment offers a better performance than SLA by creating, at least in the early phases, better conditions for tissue healing.
Asunto(s)
Implantes Dentales , Materiales Dentales/química , Mandíbula/citología , Osteoblastos/citología , Titanio/química , Grabado Ácido Dental , Adsorción , Abrasión Dental por Aire , Fosfatasa Alcalina/análisis , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Forma de la Célula , Materiales Biocompatibles Revestidos/química , Glicoproteínas/análisis , Humanos , Microscopía Electrónica de Rastreo , Osteocalcina/análisis , Osteoprotegerina , Proteínas/química , Receptores Citoplasmáticos y Nucleares/análisis , Receptores del Factor de Necrosis Tumoral/análisis , Espectrometría Raman , Propiedades de Superficie , Factores de TiempoRESUMEN
The enzymatic in vitro degradation of a commercial biodegradable hydroxyapatite (HA)-polymer (poly(epsilon-caprolactone)-poly(oxyethylene)(POE)-poly(epsilon-caprolactone) block copolymer) composite membrane was investigated by Raman and IR spectroscopies in two enzymatic solutions at 37 degrees C: esterase and alpha-chymotrypsin in saline phosphate buffer (SPB, pH 7.4). The degradation was found to be faster in the enzymatic medium than in SPB and alkaline solutions. The fastest degradation rate was observed in esterase solution. The trend of properly chosen Raman and IR intensity ratios was evaluated to go deeper inside the degradation mechanism: both polymeric and apatitic components were found to be involved in degradation. The former underwent preferential degradation of POE blocks, while HA is removed by the degradation medium faster than the polymer. Vibrational spectroscopy proved a valid tool for investigating the degradation of the membrane.
Asunto(s)
Implantes Absorbibles , Ligamento Periodontal/química , Espectrofotometría/métodos , Membrana Celular/metabolismo , Quimotripsina/química , Durapatita/química , Esterasas/química , Inflamación , Cinética , Polímeros/química , Espectrofotometría Infrarroja , Espectrometría Raman , Temperatura , Factores de TiempoRESUMEN
BACKGROUND: Six titanium disks with six different surface treatments were examined: SS: smooth (polished) surface; TPS: plasma spray; C100: sand blasting by aluminum oxide (Al2O3) diameter 100 microm and acid etching; C150: sand blasting by Al2O3 diameter 150 microm and acid etching; B60: sand blasting by zirconium oxide (ZrO2) diameter 60 microm and acid etching; and B120: sand blasting by ZrO2 diameter 120 microm and acid etching. METHODS: The surface characteristics were determined by scanning electron microscopy (SEM) observation and a roughness tester. Raman spectroscopy was used to determine the presence of residual substances on the samples. Cells were seeded onto the disk and after 24 hours, 6 days, and 12 days were observed under SEM and growth curves generated with a cell counter. Some samples were used to determine alkaline phosphatase activity (ALP), using a colorimetric assay. RESULTS: SEM observation revealed drastic differences in surface microtopography, with a higher cell density on sand-blasted and acid-etched (SLA) samples than SS and TPS, and more regularly aligned cells on B60 and B120 surfaces than on the others. The growth curves showed a greater adhesion of cells on the etched/blasted surfaces compared to the SS and TPS surfaces. The number of cells increased on all the SLA samples, especially B60, throughout the experiment. At the same time, there was considerable ALP activity on the B60 sample, while it remained at extremely low levels on SS and TPS surfaces. Raman analyses revealed Al2O3 debris on C100 and C150, partly explaining the poorer performances of these two surface treatments, since this substance was shown to be toxic for cultured osteoblasts. CONCLUSIONS: Surface treatments influence the growth and the metabolic activity of cultured osteoblasts, and B60 seems to be the most favorable surface inducing a more pronounced proliferation of cells together with a high differentiation degree.