Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.

Polycaps: Reversibly formed polymeric capsules

Described are assemblies consisting of polymeric capsules, “polycaps,” formed from two calix[4]arene tetraureas covalently connected at their lower rims. In these structures self-assembly leads to reversibly formed capsule sites along a chain, reminiscent of beads on a string. Their dynamic behavior is characterized by 1H NMR spectroscopy through encapsulation of guest species, reversible polymerization, and the formation of sharply defined hybrid capsules.

Mitochondrial Ca2+-induced Ca2+ Release Mediated by the Ca2+ Uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca2+ during cell stimulation. The present study focuses on the pathways for mitochondrial Ca2+ efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca2+ uptake and increased the cytosolic [Ca2+] ([Ca2+]c) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca2+]c kinetics and inhibited Ca2+ release from Ca2+-loaded mitochondria. This effect was due to inhibition of mitochondrial Na+/Ca2+ exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca2+]c triggered fast Ca2+ release from these depolarized Ca2+-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca2+-induced Ca2+ release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca2+ uniporter. This novel kind of mitochondrial Ca2+-induced Ca2+ release might contribute to Ca2+ clearance from mitochondria that become depolarized during Ca2+ overload.

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles1

The initial rate of Ca2+ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca2+-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca2+-selective minielectrodes to determine pCa values. The initial rate of Ca2+ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca2+ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K+. In addition, Ca2+ was shown to move across the membrane vesicles in the presence of a K+ diffusion potential gradient. The potential-stimulated rate of Ca2+ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl3, verapamil, and nifedipine had little or no effect. These results indicate that Ca2+ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga1

The divalent cation Sr2+ induced repetitive transient spikes of the cytosolic Ca2+ activity [Ca2+]cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca2+]cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr2+-induced [Ca2+]cy oscillations. Sr2+ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca2+]cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca2+-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr2+-induced repetitive [Ca2+]cy spikes, whereas the compartmentalization of Sr2+ was not influenced. A repetitive Ca2+ release and Ca2+ re-uptake by the ER probably generated repetitive [Ca2+]cy spikes in E. viridis in the presence of Sr2+. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr2+-induced Ca2+ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca2+ channel. The blockage of Sr2+-induced repetitive [Ca2+]cy spikes by La3+ or Gd3+ indicated the necessity of a certain influx of divalent cations for sustained [Ca2+]cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca2+]cy oscillations in E. viridis.

Redox transitions between oxygen intermediates in cytochrome-c oxidase.

Some intermediates in the reduction of O2 to water by cytochrome-c oxidase have been characterized by optical, Raman, and magnetic circular dichroism spectroscopy. The so-called "peroxy" (P) and "ferryl" (F) forms of the enzyme, which have been considered to be intermediates of the oxygen reaction, can be generated when the oxidized enzyme reacts with H2O2, or when the two-electron reduced ("CO mixed-valence") enzyme reacts with O2. The structures as well as the overall redox states of P and F have recently been controversial. We show here, using tris(2,2'-bipyridyl)ruthenium(II) as a photoinducible reductant, that one-electron reduction of P yields F, and that one-electron reduction of F yields the oxidized enzyme. This confirms that the overall redox states of P and F differ from the oxidized enzyme by two and one electron equivalents, respectively. The structures of the P and F states are discussed.

Photochemical energy conversion in a helical oligoproline assembly.

A general method is described for constructing a helical oligoproline assembly having a spatially ordered array of functional sites protruding from a proline-II helix. Three different redox-active carboxylic acids were coupled to the side chain of cis-4-amino-L-proline. These redox modules were incorporated through solid-phase peptide synthesis into a 13-residue helical oligoproline assembly bearing in linear array a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor. Upon transient 460-nm irradiation in acetonitrile, this peptide triad formed with 53% efficiency an excited state containing a phenothiazine radical cation and an anthraquinone radical anion. This light-induced redox-separated state had a lifetime of 175 ns and stored 1.65 eV of energy.

Synthesis and assembly of self-complementary calix[4]arenes.

A calix[4]arene was designed to reversibly dimerize and form an egg-shaped enclosure. Adhesive interactions in the assembly were provided by four self-associating ureas, which form a cyclic array containing 16 hydrogen bonds. The synthesis was completed in four steps from the previously described O,O',O",O"'-tetrabenzylcalix[4]arene. Evidence for dimerization of the calixarene tetraurea was provided by H NMR, mass spectrometry, and the observation of encapsulated molecules. The resulting cavity was of sufficient size to capture guests such as ethyl benzene and p-xylene.

Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode.

Amperometry at a carbon fiber microelectrode modified with a composite of ruthenium oxide and cyanoruthenate was used to monitor chemical secretions of single pancreatic beta cells from rats and humans. When the insulin secretagogues glucose, tolbutamide, and K+ were applied to the cell, a series of randomly occurring current spikes was observed. The current spikes were shown to be due to the detection of chemical substances secreted from the cell. Chromatography showed that the primary secreted substance detected by the electrode was insulin. The current spikes were strongly dependent on external Ca2+, had an average area that was independent of the stimulation method, and had an area distribution which corresponded to the distribution of vesicle sizes in beta cells. It was concluded that the spikes were due to the detection of concentration pulses of insulin secreted by exocytosis.

Effects of mechanical signaling on plant cell cytosolic calcium.

Mechanical signals are important influences on the development and morphology of higher plants. Using tobacco transformed with the Ca(2+)-sensitive luminescent protein aequorin, we recently reported the effects of mechanical signals of touch and wind on the luminescence and thus intracellular calcium of young seedlings. When mesophyll protoplasts are isolated from these transgenic tobacco plants and mechanically stimulated by swirling them in solution, cytoplasmic Ca2+ increases immediately and transiently up to 10 microM, and these transients are unaffected by an excess of EGTA in the medium. The size of the transient effect is related to the strength of swirling. Epidermal strips isolated from transgenic tobacco leaves and containing only viable guard cells and trichomes also respond to the strength of swirling in solution and can increase their cytoplasmic Ca2+ transiently up to 10 microM. Finally, the moss Physcomitrella patens containing recombinant aequorin exhibits transient increases in cytoplasmic Ca2+ up to 5 microM when swirled in solution. This effect is strongly inhibited by ruthenium red. Our data indicate that the effect of mechanical stimulation can be found in a number of different cell types and in a lower plant as well as tobacco and suggest that mechanoperception and the resulting increase in cytoplasmic Ca2+ may be widespread.

Activation of Ca2+ signaling in neutrophils by the mast cell-released immunophilin FKBP12.

The immunophilins of the FK506-binding protein (FKBP) family are intracellular proteins that bind the immunosuppresants FK506 and rapamycin. In this study we show that HMC-1 mast cells sensitized with IgE release FKBP12 upon stimulation with anti-IgE. The release is rapid and not affected by actinomycin D or cycloheximide, suggesting that it is due to exocytosis from a storage compartment. FKBP12 from HMC-1 mast cells exhibits biological activity. When applied extracellularly to human neutrophils, it induces transient changes in the intracellular Ca2+ concentration ([Ca2+]i) due to Ca2+ release from intracellular stores. Inhibition of [Ca2+]i changes by ruthenium red and ryanodine indicates that ryanodine receptor/Ca2+ release channels are involved in FKBP12-induced Ca2+ signaling. Neutrophil activation by mast cell-derived FKBP12 is prevented by complexing FKBP12 with FK506 or rapamycin. These results demonstrate that extracellular FKBP12 functions as a cytokine in cell-to-cell communication. They further suggest a pathophysiological role for FKBP12 as a mediator in immediate or type I hypersensitivity and may have implications for novel therapeutic strategies in the treatment of allergic disorders with FK506 and rapamycin.

Synthesis of new calixarene-based lubricant additives

The lubricants are normally composed by base oils and a number of additives which are added to improve the performances of the final product. In this work, which is due to the collaboration between ENI S.p.A. and Prof. Casnati’s group, significant results in the application of calixarene structures to two classes of lubricant additives (viscosity index improvers and detergents) were shown. In particular, several calix[8]arene derivatives were synthesized to use as core precursors in the “arm-first" synthetic processes of star polymers for viscosity index improver applications. The use of calixarene derivatives enable the production of star polymers with a high and well-defined number of branches and endowed with a very low dispersivity of molecular weight which can originate better performances than the current commercially available viscosity index improvers of the major competitor. Several functional groups were considered to prepare reactive p-tert-butylcalix[8]arene cores to be used in living anionic polymerization. n-butyllithium was used as model of the living anionic polymer to test the outcome of the reaction of polymer insertion on the calixarene core, facilitating the analyses of the products. The calixarene derivative, which easier reacts with n-BuLi, was selected for the preparation of star polymers by using a isoprene/styrene living anionic polymer. Finally, the lubricant formulations, which include the calixarene-based star polymers or commercially available products as viscosity index improvers, were prepared and comparatively tested. In the last part of Thesis, the use of calixarenes as polycarboxylic acids to synthetize new sulfur-free detergents as lubricant additives was carried out. In this way, these calcium-based detergents can be used for the formulation of new automotive lubricants with low content of ash, phosphorus and sulfur (low SAPS). To increase the low deprotonation degree of OH groups and their capacity to complex calcium ions, a complete functionalization of the calixarene mixtures with acetic acid groups was required. Futhermore, the “one-step” synthesis of new calixarenes with alkyl chains in para positions longer than the ones already known was necessary to improve the oil solubility and stability of reverse micelles formed by the detergents. Moreover, the separation and characterization of the calixarenes were carried out to optimize their synthetic process, also on pilot scale. For our purpose, the use of p-tert-octylcalixarenes for the preparation of detergents was carried out to compare the properties of the final detergents respect to the use of the p-dodecyl calixarenes. Once achieved the functionalization of both calixarene mixtures with carboxylic acid groups, the syntheses of new calixarene-based detergents were carried out to identify the best calixarene derivative for our research goals. The synthetic process for the preparation of calixarene-based detergent having very high basicity (TBN 400) was also investigated for applications in lubricants for marine engines. In addition, with the aim of testing the calixarene-based detergents in automotive lubricants, several additive packages (concentrated mixture of additives) containing our detergents were prepared. Using these packages the corresponding automotive lubricants can be formulated. Besides, a lubricant containing commercial calcium alkylbenzene-sulfonates detergents was prepared to compare its detergency properties with those of the calixarene-based oils.

New multivalent calixarene-based ligands for protein recognition and inhibition

One of the challenges that concerns chemistry is the design of molecules able to modulate protein-protein and protein-ligand interactions, since these are involved in many physiological and pathological processes. The interactions occurring between proteins and their natural counterparts can take place through reciprocal recognition of rather large surface areas, through recognition of single contact points and single residues, through inclusion of the substrates in specific, more or less deep binding sites. In many cases, the design of synthetic molecules able to interfere with the processes involving proteins can benefit from the possibility of exploiting the multivalent effect. Multivalency, widely spread in Nature, consists in the simultaneous formation between two entities (cell-cell, cell-protein, protein-protein) of multiple equivalent ligand-recognition site complexes. In this way the whole interaction results particularly strong and specific. Calixarenes furnish a very interesting scaffold for the preparation of multivalent ligands and in the last years calixarene-based ligands demonstrated their remarkable capability to recognize and inhibit or restore the activity of different proteins, with a high efficiency and selectivity in several recognition phenomena. The relevance and versatility of these ligands is due to the different exposition geometries of the binding units that can be explored exploiting the conformational properties of these macrocycles, the wide variety of functionalities that can be linked to their structure at different distances from the aromatic units and to their intrinsic multivalent nature. With the aim of creating new multivalent systems for protein targeting, the work reported in this thesis regards the synthesis and properties of glycocalix[n]arenes and guanidino calix[4]arenes for different purposes. Firstly, a new bolaamphiphile glycocalix[4]arene in 1,3-alternate geometry, bearing cellobiose, was synthesized for the preparation of targeted drug delivery systems based on liposomes. The formed stable mixed liposomes obtained by mixing the macrocycle with DOPC were shown to be able of exploiting the sugar units emerging from the lipid bilayer to agglutinate Concanavalin A, a lectin specific for glucose. Moreover, always thanks to the presence of the glycocalixarene in the layer, the same liposomes demonstrated through preliminary experiments to be uptaken by cancer cells overexpressing glucose receptors on their exterior surface more efficiently respect to simple DOPC liposomes lacking glucose units in their structure. Then a small library of glycocalix[n]arenes having different valency and geometry was prepared, for the creation of potentially active immunostimulants against Streptococcus pneumoniae, particularly the 19F serotype, one of the most virulent. These synthesized glycocalixarenes bearing β-N-acetylmannosamine as antigenic unit were compared with the natural polysaccharide on the binding to the specific anti-19F human polyclonal antibody, to verify their inhibition potency. Among all, the glycocalixarene based on the conformationally mobile calix[4]arene resulted the more efficient ligand, probably due its major possibility to explore the antibody surface and dispose the antigenic units in a proper arrangement for the interaction process. These results pointed out the importance of how the different multivalent presentation in space of the glycosyl units can influence the recognition phenomena. At last, NMR studies, using particularly 1H-15N HSQC experiments, were performed on selected glycocalix[6]arenes and guanidino calix[4]arenes blocked in the cone geometry, in order to better understand protein-ligand interactions. The glycosylated compounds were studied with Ralstonia solanacearum lectin, in order to better understand the nature of the carbohydrate‐lectin interactions in solution. The series of cationic calixarene was employed with three different acidic proteins: GB1, Fld and alpha synuclein. Particularly GB1 and Fld were observed to interact with all five cationic calix[4]arenes but showing different behaviours and affinities.

Caracterização e aplicação analítica de eletrodos modificados com sistemas porfirínicos supramoleculares

Estudos com eletrodos modificados foram conduzidos utilizando dois sistemas porfirínicos supramoleculares diferentes. O primeiro foi baseado na modificação de eletrodo de carbono vítreo com uma porfirina de níquel tetrarrutenada, [NiIITPyP{RuII(bipy)2Cl}4]4+. A modificação do eletrodo foi realizada por meio de sucessivos ciclos voltamétricos em meio alcalino (pH 13), gerando um eletrodo com característica similar a eletrodos modificados com α-Ni(OH)2. A caracterização química do filme formado foi realizada através das técnicas de voltametria cíclica, ressonância paramagnética eletrônica, espectroscopia eletrônica por reflectância e espectroscopia Raman com ensaio espectro-eletroquímico. Os resultados sugerem a formação de um polímero de coordenação, [µ-O2-NiIITPyP{RuII(bipy)2Cl}4]n, composto por subunidades porfirínicas ligadas entre si por pontes µ-peroxo axialmente coordenadas aos átomos de níquel (Ni-O-O-Ni). O crescimento do filme apresentou dependência da alcalinidade do meio pela formação do precursor octaédrico [Ni(OH)2TRPyP]2+ em solução, pela coordenação de OH- nas posições axiais do átomo de níquel. O processo de eletropolimerização indicou a participação de radical hidroxil, gerado por oxidação eletrocatalítica da água nos sítios periféricos da porfirina contendo o complexo de rutênio. O mesmo eletrodo foi aplicado como sensor eletroquímico para análise amperométrica de ácido fólico em comprimidos farmacêuticos. O sensor foi associado a um sistema de Batch Injection Analysis (BIA) alcançando considerável rapidez e baixo limite de detecção. Para as análises das amostras também foi proposto um método para a remoção da lactose, que agia como interferente. O segundo estudo envolveu a modificação de eletrodos de carbono vítreo com diferentes hemoglobinas, naturais (HbA0, HbA2 e HbS) e sintéticas (Hb-PEG5K2, αα-Hb-PEG5K2 e BT-PEG5K4), para a avaliação da eficiência na redução eletrocatalítica de nitrito mediada por FeI-heme. Os filmes foram produzidos pela mistura de soluções das hemoglobinas com brometo de didodecildimetiltrimetilamônio (DDAB), aplicados nas superfícies com consecutiva evaporação, formando filmes estáveis. Os valores de potencial redox para os processos do grupo heme e a sua associação com a disponibilidade do grupo na proteína foram avaliados por voltametria cíclica. Os valores das constantes de velocidade, k, para redução de nitrito foram obtidos por cronoamperometria em -1,1 V (vs Ag/AgCl(KCl 3M)) que foram utilizados para estudo comparativo entre as espécies sintéticas para eventual aplicação clínica.

Novel Photochemical Methodologies For Diversity Oriented Synthesis And Screening Of Combinatorial Libraries

The main goal of this project was to develop an efficient methodology allowing rapid access to structurally diverse scaffolds decorated with various functional groups. Initially, we discovered and subsequently developed an experimentally straightforward, high-yielding photoinduced conversion of readily accessible diverse starting materials into polycyclic aldehydes and their (hemi)acetals decorated by various pendants. The two step sequence, involving the Diels-Alder addition of heterocyclic chalcones and other benzoyl ethylenes to a variety of dienes, followed by the Paternò-Büchi reaction, was described as an alkene-carbonyl oxametathesis. This methodology offers a rapid increase in molecular complexity and diversity of the target scaffolds. To develop this novel methodology further and explore its generality, we directed our attention to the Diels-Alder adducts based on various chromones. We discovered that the Diels-Alder adducts of chromones are capable of photoinduced alkene-arene [2+2] cycloaddition producing different dienes, which can either dimerize or be introduced into a double-tandem [4π+2π]·[2π+2π]·[4π+2π]·[2π+2π] synthetic sequence, followed by an acid-catalyzed oxametathesis, leading to a rapid expansion of molecular complexity over a few experimentally simple steps. In view of the fact that oxametathesis previously was primarily observed in aromatic oxetanes, we decided to prepare model aliphatic oxetanes with a conformationally unconstrained or "flexible" methyl group based on the Diels-Alder adducts of cyclohexadiene or cyclopentadiene with methyl vinyl ketone. Upon addition of an acid, the expected oxametathesis occurred with results similar to those observed in the aromatic series proving the generality of this approach. Also we synthesized polycyclic oxetanes resulting from the Diels-Alder adducts of cyclic ketones. This not only gave us access to remarkably strained oxetane systems, but also the mechanism for their protolytic ring opening provided a great deal of insight to how the strain affects the reactivity. Additionally, we discovered that although the model Hetero-Diels-Alder adducts did not undergo [2+2] cycloaddition, both exo- and endo-Sulfa-Diels-Alder products, nonetheless, were photochemically active and various products with defined stereochemistry could be produced upon photolysis. In conclusion, we have developed an approach to the encoding and screening of solution phase libraries based on the photorelease of externally sensitized photolabile tags. The encoding tags can be released into solution only when a binding event occurs between the ligand and the receptor, equipped with an electron transfer sensitizer. The released tags are analyzed in solution revealing the identity of the lead ligand or narrowing the range of potential leads.