RESUMEN

Neurotransmission is critical for brain function, allowing neurons to communicate through neurotransmitters and neuropeptides. RVD-hemopressin (RVD-Hp), a novel peptide identified in noradrenergic neurons, modulates cannabinoid receptors CB1 and CB2. Unlike hemopressin (Hp), which induces anxiogenic behaviors via transient receptor potential vanilloid 1 (TRPV1) activation, RVD-Hp counteracts these effects, suggesting that it may block TRPV1. This study investigates RVD-Hp's role as a TRPV1 channel blocker using HEK293 cells expressing TRPV1-GFP. Calcium imaging and patch-clamp recordings demonstrated that RVD-Hp reduces TRPV1-mediated calcium influx and TRPV1 ion currents. Molecular docking and dynamics simulations indicated that RVD-Hp interacts with TRPV1's selectivity filter, forming stable hydrogen bonds and van der Waals contacts, thus preventing ion permeation. These findings highlight RVD-Hp's potential as a therapeutic agent for conditions involving TRPV1 activation, such as pain and anxiety.

Asunto(s)

Endocannabinoides , Canales Catiónicos TRPV , Humanos , Calcio/metabolismo , Endocannabinoides/farmacología , Endocannabinoides/metabolismo , Endocannabinoides/química , Células HEK293 , Hemoglobinas , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/metabolismo , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores

RESUMEN

Thyroxine (T4) is a drug extensively utilized for the treatment of hypothyroidism. However, the oral absorption of T4 presents certain limitations. This research investigates the efficacy of CO2 nanobubbles in water as a potential oral carrier for T4 administration to C57BL/6 hypothyroid mice. Following 18 h of fasting, the formulation was administered to the mice, demonstrating that the combination of CO2 nanobubbles and T4 enhanced the drug's absorption in blood serum by approximately 40%. To comprehend this observation at a molecular level, we explored the interaction mechanism through which T4 engages with the CO2 nanobubbles, employing molecular simulations, semi-empirical quantum mechanics, and PMF calculations. Our simulations revealed a high affinity of T4 for the water-gas interface, driven by additive interactions between the hydrophobic region of T4 and the gas phase and electrostatic interactions of the polar groups of T4 with water at the water-gas interface. Concurrently, we observed that at the water-gas interface, the cluster of T4 formed in the water region disassembles, contributing to the drug's bioavailability. Furthermore, we examined how the gas within the nanobubbles aids in facilitating the drug's translocation through cell membranes. This research contributes to a deeper understanding of the role of CO2 nanobubbles in drug absorption and subsequent release into the bloodstream. The findings suggest that utilizing CO2 nanobubbles could enhance T4 bioavailability and cell permeability, leading to more efficient transport into cells. Additional research opens the possibility of employing lower concentrations of this class of drugs, thereby potentially reducing the associated side effects due to poor absorption.

Asunto(s)

Dióxido de Carbono , Modelos Animales de Enfermedad , Hipotiroidismo , Tiroxina , Agua , Animales , Hipotiroidismo/tratamiento farmacológico , Hipotiroidismo/metabolismo , Ratones , Dióxido de Carbono/química , Agua/química , Ratones Endogámicos C57BL , Administración Oral , Nanopartículas/química , Portadores de Fármacos/química

RESUMEN

Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd+, but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca2+ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca2+ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca2+ failed to induce cell death, and the Ca2+ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or 10Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1-/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca2+ overload that promotes cell death.

Asunto(s)

Calcio , Conexinas , Proteínas del Tejido Nervioso , Humanos , Conexinas/metabolismo , Conexinas/genética , Células HeLa , Calcio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Apoptosis , Muerte Celular , Señalización del Calcio

RESUMEN

The occurrence of intercellular channels formed by pannexin1 has been challenged for more than a decade. Here, we provide an electrophysiological characterization of exogenous human pannexin1 (hPanx1) cell­cell channels expressed in HeLa cells knocked out for connexin45. The observed hPanx1 cell­cell channels show two phenotypes: O-state and S-state. The former displayed low transjunctional voltage (Vj) sensitivity and single-channel conductance of ∼175 pS, with a substate of ∼35 pS; the latter showed a peculiar dynamic asymmetry in Vj dependence and single-channel conductance identical to the substate conductance of the O-state. S-state hPanx1 cell­cell channels were also identified between TC620 cells, a human oligodendroglioma cell line that endogenously expresses hPanx1. In these cells, dye and electrical coupling increased with temperature and were strongly reduced after hPanx1 expression was knocked down by small interfering RNA or inhibited with Panx1 mimetic inhibitory peptide. Moreover, cell­cell coupling was augmented when hPanx1 levels were increased with a doxycycline-inducible expression system. Application of octanol, a connexin gap junction (GJ) channel inhibitor, was not sufficient to block electrical coupling between HeLa KO Cx45-hPanx1 or TC620 cell pairs. In silico studies suggest that several arginine residues inside the channel pore may be neutralized by hydrophobic interactions, allowing the passage of DAPI, consistent with dye coupling observed between TC620 cells. These findings demonstrate that endogenously expressed hPanx1 forms intercellular cell­cell channels and their unique properties resemble those described in innexin-based GJ channels. Since Panx1 is ubiquitously expressed, finding conditions to recognize Panx1 cell­cell channels in different cell types might require special attention.

Asunto(s)

Conexinas , Proteínas del Tejido Nervioso , Animales , Conexinas/metabolismo , Humanos , Canales Iónicos , Mamíferos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo

RESUMEN

Large-pore channels, including those formed by connexin, pannexin, innexin proteins, are part of a broad family of plasma membrane channels found in vertebrates and invertebrates, which share topology features. Despite their relevance in parasitic diseases such as Chagas and malaria, it was unknown whether these large-pore channels are present in unicellular organisms. We identified 14 putative proteins in Trypanosomatidae parasites as presumptive homologs of innexin proteins. All proteins possess the canonical motif of the innexin family, a pentapeptide YYQWV, and 10 of them share a classical membrane topology of large-pore channels. A sequence similarity network analysis confirmed their closeness to innexin proteins. A bioinformatic model showed that a homolog of Trypanosoma cruzi (T. cruzi) could presumptively form a stable octamer channel with a highly positive electrostatic potential in the internal cavities and extracellular entrance due to the notable predominance of residues such as Arg or Lys. In vitro dye uptake assays showed that divalent cations-free solution increases YO-PRO-1 uptake and hyperosmotic stress increases DAPI uptake in epimastigotes of T. cruzi. Those effects were sensitive to probenecid. Furthermore, probenecid reduced the proliferation and transformation of T. cruzi. Moreover, probenecid or carbenoxolone increased the parasite sensitivity to antiparasitic drugs commonly used in therapy against Chagas. Our study suggests the existence of innexin homologs in unicellular organisms, which could be protein subunits of new large-pore channels in unicellular organisms.

Asunto(s)

Parásitos , Trypanosoma cruzi , Trypanosomatina , Animales , Conexinas/metabolismo , Parásitos/metabolismo , Probenecid/farmacología , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Trypanosomatina/metabolismo

RESUMEN

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation-measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

Asunto(s)

Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Conexinas/química , Conexinas/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Conexinas/genética , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Indoles/farmacocinética , Canales Iónicos/genética , Canales Iónicos/metabolismo , Simulación de Dinámica Molecular , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Fosforilación , Serina/genética , Serina/metabolismo

RESUMEN

NOD1 is an intracellular receptor that, when activated, induces gene expression of pro-inflammatory factors promoting macrophages and neutrophils recruitment at the infection site. However, iE-DAP, the dipeptide agonist that promotes this receptor's activation, cannot permeate cell membranes. To develop a nanocarrier capable of achieving a high and prolonged activation over time, iE-DAP was encapsulated in nanoparticles (NPs) made of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The physicochemical properties, colloidal stability, encapsulation efficiency, and cellular uptake of iE-DAP-loaded PHVB NPs were analyzed. Results evidenced that physicochemical properties of iE-DAP-loaded NPs remained stable over time, and NPs were efficiently internalized into cells, a process that depends on time and concentration. Moreover, our results showed that NPs elicited a controlled cargo release in vitro, and the encapsulated agonist response was higher than its free form, suggesting the possibility of activating intracellular receptors triggering an immune response through the release of NOD1 agonist.

Asunto(s)

Ácido Diaminopimélico/análogos & derivados , Nanopartículas/química , Proteína Adaptadora de Señalización NOD1/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Química Farmacéutica , Ácido Diaminopimélico/administración & dosificación , Ácido Diaminopimélico/farmacología , Relación Dosis-Respuesta a Droga , Portadores de Fármacos/química , Liberación de Fármacos , Estabilidad de Medicamentos , Ratones , Poliésteres/química , Células RAW 264.7

RESUMEN

Most of the computational tools involved in drug discovery developed during the 1980s were largely based on computational chemistry, quantitative structure-activity relationship (QSAR) and cheminformatics. Subsequently, the advent of genomics in the 2000s gave rise to a huge number of databases and computational tools developed to analyze large quantities of data, through bioinformatics, to obtain valuable information about the genomic regulation of different organisms. Target identification and validation is a long process during which evidence for and against a target is accumulated in the pursuit of developing new drugs. Finally, the drug delivery system appears as a novel approach to improve drug targeting and releasing into the cells, leading to new opportunities to improve drug efficiency and avoid potential secondary effects. In each area: target discovery, drug discovery and drug delivery, different computational strategies are being developed to accelerate the process of selection and discovery of new tools to be applied to different scientific fields. Research on these three topics is growing rapidly, but still requires a global view of this landscape to detect the most challenging bottleneck and how computational tools could be integrated in each topic. This review describes the current state of the art in computational strategies for target discovery, drug discovery and drug delivery and how these fields could be integrated. Finally, we will discuss about the current needs in these fields and how the continuous development of databases and computational tools will impact on the improvement of those areas. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Asunto(s)

Biología Computacional , Sistemas de Liberación de Medicamentos , Descubrimiento de Drogas , Estudio de Asociación del Genoma Completo , Humanos , Secuenciación del Exoma

RESUMEN

Amphiphilic Janus dendrimers (JDs) are repetitively branched molecules with hydrophilic and hydrophobic components that self-assemble in water to form a variety of morphologies, including vesicles analogous to liposomes with potential pharmaceutical and medical application. To date, the self-assembly of JDs has not been fully investigated thus it is important to gain insight into its mechanism and dependence on JDs' molecular structure. In this study, the aggregation behavior in water of a second-generation bis-MPA JD was evaluated using experimental and computational methods. Dispersions of JDs in water were carried out using the thin-film hydration and ethanol injection methods. Resulting assemblies were characterized by dynamic light scattering, confocal microscopy, and atomic force microscopy. Furthermore, a coarse-grained molecular dynamics (CG-MD) simulation was performed to study the mechanism of JDs aggregation. The obtaining of assemblies in water with no interdigitated bilayers was confirmed by the experimental characterization and CG-MD simulation. Assemblies with dendrimersome characteristics were obtained using the ethanol injection method. The results of this study establish a relationship between the molecular structure of the JD and the properties of its aggregates in water. Thus, our findings could be relevant for the design of novel JDs with tailored assemblies suitable for drug delivery systems.

Asunto(s)

Dendrímeros/química , Simulación de Dinámica Molecular , Agua/química , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular

RESUMEN

BACKGROUND: Antifreeze proteins (AFPs) production is a survival strategy of psychrophiles in ice. These proteins have potential in frozen food industry avoiding the damage in the structure of animal or vegetal foods. Moreover, there is not much information regarding the interaction of Antarctic bacterial AFPs with ice, and new determinations are needed to understand the behaviour of these proteins at the water/ice interface. RESULTS: Different Antarctic places were screened for antifreeze activity and microorganisms were selected for the presence of thermal hysteresis in their crude extracts. Isolates GU1.7.1, GU3.1.1, and AFP5.1 showed higher thermal hysteresis and were characterized using a polyphasic approach. Studies using cucumber and zucchini samples showed cellular protection when samples were treated with partially purified AFPs or a commercial AFP as was determined using toluidine blue O and neutral red staining. Additionally, genome analysis of these isolates revealed the presence of genes that encode for putative AFPs. Deduced amino acids sequences from GU3.1.1 (gu3A and gu3B) and AFP5.1 (afp5A) showed high similarity to reported AFPs which crystal structures are solved, allowing then generating homology models. Modelled proteins showed a triangular prism form similar to ß-helix AFPs with a linear distribution of threonine residues at one side of the prism that could correspond to the putative ice binding side. The statistically best models were used to build a protein-water system. Molecular dynamics simulations were then performed to compare the antifreezing behaviour of these AFPs at the ice/water interface. Docking and molecular dynamics simulations revealed that gu3B could have the most efficient antifreezing behavior, but gu3A could have a higher affinity for ice. CONCLUSIONS: AFPs from Antarctic microorganisms GU1.7.1, GU3.1.1 and AFP5.1 protect cellular structures of frozen food showing a potential for frozen food industry. Modeled proteins possess a ß-helix structure, and molecular docking analysis revealed the AFP gu3B could be the most efficient AFPs in order to avoid the formation of ice crystals, even when gu3A has a higher affinity for ice. By determining the interaction of AFPs at the ice/water interface, it will be possible to understand the process of adaptation of psychrophilic bacteria to Antarctic ice.

Asunto(s)

Proteínas Anticongelantes/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Aminoácidos , Regiones Antárticas , Proteínas Anticongelantes/química , Proteínas Anticongelantes/genética , Bacterias/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Cucurbita/metabolismo , Cucurbitaceae/metabolismo , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de ADN , Secuenciación Completa del Genoma

RESUMEN

Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies.

Asunto(s)

Dendrímeros/química , Receptor 1 de Folato/química , Receptor 1 de Folato/metabolismo , Ácido Fólico/química , Polietilenglicoles/química , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Solventes/química

RESUMEN

BACKGROUND: Nanotechnology is a science that involves imaging, measurement, modeling and a manipulation of matter at the nanometric scale. One application of this technology is drug delivery systems based on nanoparticles obtained from natural or synthetic sources. An example of these systems is synthetized from poly(3-hydroxybutyrate-co-3-hydroxyvalerate), which is a biodegradable, biocompatible and a low production cost polymer. The aim of this work was to investigate the uptake mechanism of PHBV nanoparticles in two different epithelial cell lines (HeLa and SKOV-3). RESULTS: As a first step, we characterized size, shape and surface charge of nanoparticles using dynamic light scattering and transmission electron microscopy. Intracellular incorporation was evaluated through flow cytometry and fluorescence microscopy using intracellular markers. We concluded that cellular uptake mechanism is carried out in a time, concentration and energy dependent way. Our results showed that nanoparticle uptake displays a cell-specific pattern, since we have observed different colocalization in two different cell lines. In HeLa (Cervical cancer cells) this process may occur via classical endocytosis pathway and some internalization via caveolin-dependent was also observed, whereas in SKOV-3 (Ovarian cancer cells) these patterns were not observed. Rearrangement of actin filaments showed differential nanoparticle internalization patterns for HeLa and SKOV-3. Additionally, final fate of nanoparticles was also determined, showing that in both cell lines, nanoparticles ended up in lysosomes but at different times, where they are finally degraded, thereby releasing their contents. CONCLUSIONS: Our results, provide novel insight about PHBV nanoparticles internalization suggesting that for develop a proper drug delivery system is critical understand the uptake mechanism.

Asunto(s)

Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos , Nanopartículas/metabolismo , Neoplasias/tratamiento farmacológico , Poliésteres/metabolismo , Transporte Biológico , Línea Celular Tumoral , Endocitosis , Células HeLa , Humanos , Nanopartículas/ultraestructura

RESUMEN

An ideal nucleic-acid transfection system should combine the physical and chemical characteristics of cationic lipids and linear polymers to decrease cytotoxicity and uptake limitations. Previous research described new types of carriers termed amphiphilic dendrimers (ADs), which are based on polyamidoamine dendrimers (PAMAM). These ADs display the cell membrane affinity advantage of lipids and preserve the high affinity for DNA possessed by cationic dendrimers. These lipid/dendrimer hybrids consist of a low-generation, hydrophilic dendron (G2, G1, or G0) bonded to a hydrophobic tail. The G2-18C AD was reported to be an efficient siRNA vector with significant gene silencing. However, shorter tail ADs (G2-15C and G2-13C) and lower generation (G0 and G1) dendrimers failed as transfection carriers. To date, the self-assembly phenomenon of this class of amphiphilic dendrimers has not been molecularly explored using molecular simulation methods. To gain insight into these systems, the present study used coarse-grained molecular dynamics simulations to describe how ADs are able to self-assemble into an aggregate, and, specifically, how tail length and generation play a key role in this event. Finally, explanations are given for the better efficiency of G2/18-C as gene carrier in terms of binding of siRNA. This knowledge could be relevant for the design of novel, safer ADs with well-optimized affinity for siRNA.

Asunto(s)

Dendrímeros/química , Lípidos/química , ARN Interferente Pequeño/metabolismo , Dendrímeros/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Transfección

RESUMEN

Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

RESUMEN

Biomimetics, or the use of principles of Nature for developing new materials, is a paradigm that could help Nanomedicine tremendously. One of the current challenges in Nanomedicine is the rational design of new efficient and safer gene carriers. Poly(amidoamine) (PAMAM) dendrimers are a well-known class of nanoparticles, extensively used as non-viral nucleic acid carriers, due to their positively charged end-groups. Yet, there are still several aspects that can be improved for their successful application in in vitro and in vivo systems, including their affinity for nucleic acids as well as lowering their cytotoxicity. In the search of new functional groups that could be used as new dendrimer-reactive groups, we followed a biomimetic approach to determine the amino acids with highest prevalence in protein-DNA interactions. Then we introduced them individually as terminal groups of dendrimers, generating a new class of nanoparticles. Molecular dynamics studies of two systems: PAMAM-Arg and PAMAM-Lys were also performed in order to describe the formation of complexes with DNA. Results confirmed that the introduction of amino acids as terminal groups in a dendrimer increases their affinity for DNA and the interactions in the complexes were characterized at atomic level. We end up by briefly discussing additional modifications that can be made to PAMAM dendrimers to turned them into promising new gene carriers.

Asunto(s)

Biomimética , Dendrímeros/química , Técnicas de Transferencia de Gen , Nanopartículas , Supervivencia Celular , Simulación de Dinámica Molecular

RESUMEN

Potassium channels exhibit a large diversity of single-channel conductances. Shaker is a low-conductance K-channel in which Pro475→Asp, a single-point mutation near the internal pore entrance, promotes 6- to 8-fold higher unitary current. To assess the mechanism for this higher conductance, we measured Shaker-P475D single-channel current in a wide range of symmetrical K(+) concentrations and voltages. Below 300 mM K(+), the current-to-voltage relations (i-V) showed inward rectification that disappeared at 1000 mM K(+). Single-channel conductance reached a maximum of ∼190 pS at saturating [K(+)], a value 4- to 5-fold larger than that estimated for the native channel. Intracellular Mg(2+) blocked this variant with ∼100-fold higher affinity. Near zero voltage, blockade was competitively antagonized by K(+); however, at voltages >100 mV, it was enhanced by K(+). This result is consistent with a lock-in effect in a single-file diffusion regime of Mg(2+) and K(+) along the pore. Molecular-dynamics simulations revealed higher K(+) density in the pore, especially near the Asp-475 side chains, as in the high-conductance MthK bacterial channel. The molecular dynamics also showed that K(+) ions bound distally can coexist with other K(+) or Mg(2+) in the cavity, supporting a lock-in mechanism. The maximal K(+) transport rate and higher occupancy could be due to a decrease in the electrostatic energy profile for K(+) throughout the pore, reducing the energy wells and barriers differentially by ∼0.7 and ∼2 kT, respectively.

Asunto(s)

Conductividad Eléctrica , Magnesio/farmacología , Mutación Puntual , Bloqueadores de los Canales de Potasio/farmacología , Potasio/metabolismo , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Canales de Potasio de la Superfamilia Shaker/metabolismo , Animales , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Simulación de Dinámica Molecular , Porosidad , Conformación Proteica , Canales de Potasio de la Superfamilia Shaker/química , Canales de Potasio de la Superfamilia Shaker/genética , Electricidad Estática , Xenopus laevis

RESUMEN

El análisis de las dimensiones y proporciones faciales es necesario en distintos ámbitos de la odontoestomatología y de la antropología física. En este informe presentamos el software Antropmeter, diseñado para realizar análisis de dimensiones y proporciones faciales, en base a fotografías estandarizadas, de fácil manejo por parte del clínico y de utilidad en análisis faciales estéticos y antropológicos.

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The dimensions and facial proportions analysis are necessary in different areas of the odontostomatology and physical anthropology practice. In this report we present the Antropmeter software, designed to carry out analysis of dimensions and facial proportions, based on standardized pictures, of easy handling on the part of the clinical one and of utility in aesthetic and anthropological facial analysis.

Asunto(s)

Humanos , Antropometría/instrumentación , Cara/anatomía & histología , Fotogrametría , Programas Informáticos