ABSTRACT
Abyssal seafloor communities cover more than 60% of Earth's surface. Despite their great size, abyssal plains extend across modest environmental gradients compared to other marine ecosystems. However, little is known about the patterns and processes regulating biodiversity or potentially delimiting biogeographical boundaries at regional scales in the abyss. Improved macroecological understanding of remote abyssal environments is urgent as threats of widespread anthropogenic disturbance grow in the deep ocean. Here, we use a new, basin-scale dataset to show the existence of clear regional zonation in abyssal communities across the 5,000 km span of the Clarion-Clipperton Zone (northeast Pacific), an area targeted for deep-sea mining. We found two pronounced biogeographic provinces, deep and shallow-abyssal, separated by a transition zone between 4,300 and 4,800 m depth. Surprisingly, species richness was maintained across this boundary by phylum-level taxonomic replacements. These regional transitions are probably related to calcium carbonate saturation boundaries as taxa dependent on calcium carbonate structures, such as shelled molluscs, appear restricted to the shallower province. Our results suggest geochemical and climatic forcing on distributions of abyssal populations over large spatial scales and provide a potential paradigm for deep-sea macroecology, opening a new basis for regional-scale biodiversity research and conservation strategies in Earth's largest biome.
Subject(s)
Biodiversity , Ecosystem , Calcium Carbonate , CarbonatesABSTRACT
Cavernous hemangiomas, also known as deep hemangiomas are benign tumors of blood vessels, including normal and abnormal vascular structures, that develop in skin tissue and sometimes even in deep tissues. Its intraneural development in the peripheral nerve is very rare with less than 50 cases reported in the literature. We present a case of a cavernous hemangioma of the medial sural nerve in a patient with symptoms of severe pain and allodynia with complete resolution of symptoms with microsurgery.
Subject(s)
Hemangioma, Cavernous , Humans , Hemangioma, Cavernous/complications , Hemangioma, Cavernous/diagnostic imaging , Hemangioma, Cavernous/surgery , Peripheral Nerves/pathologyABSTRACT
La meralgia parestésica es un desorden neurológico causado por una neuropatía del nervio femorocutáneo lateral. Su etiología puede ser idiopática o iatrogénica. Se caracteriza por dolor, parestesias y entumecimiento en la cara anterolateral del muslo. Su diagnóstico es básicamente clínico, aunque pueden ser útiles pruebas de imagen o neurofisiológicas. A pesar de que el tratamiento conservador suele ser eficaz en la mayoría de los pacientes, existen casos refractarios que pueden precisar de otras formas de tratamiento. Los procedimientos quirúrgicos disponibles son la descompresión nerviosa (neurólisis) o la sección (neurectomía) y las ablaciones por radiofrecuencia. Presentamos un caso de meralgia parestésica invalidante refractaria en el cual empleamos la estimulación medular como posible técnica eficaz en el alivio del dolor y poder evitar la realización de una neurectomía del nervio femorocutáneo lateral (AU)
Meralgia paresthetica is a neurological disorder caused by a neuropathy of the lateral femoral cutaneous nerve. Its etiology can be spontaneous or iatrogenic. It is characterized by pain, paresthesia, and numbness in the anterolateral aspect of the thigh. Diagnosis is based on clinical examination, although image and neurophysiological tests can be useful as well. Despite conservative measures use to be effective in most of patients, refractory cases can benefit from alternative treatments. Available surgical procedures are: nerve decompression (neurolysis) or section (neurectomy) and radiofrequency ablation. We present a case of refractory meralgia paresthetica where spinal cord stimulation was used as a possible effective technique in pain relief and to avoid the neurectomy of the lateral femoral cutaneous nerve (AU)
Subject(s)
Humans , Female , Adult , Femoral Neuropathy/diagnosis , Nerve Compression Syndromes/diagnosis , Spinal Cord Stimulation , Femoral Neuropathy/therapy , Nerve Compression Syndromes/therapy , Treatment OutcomeABSTRACT
The exploration of therapies combining antimicrobial lung proteins and conventional antibiotics is important due to the growing problem of multidrug-resistant bacteria. The aim of this study was to investigate whether human SP-A and a recombinant trimeric fragment (rfhSP-A) have cooperative antimicrobial activity with antibiotics against pathogenic Gram-negative bacteria. We found that SP-A bound the cationic peptide polymyxin B (PMB) with an apparent dissociation constant (K D) of 0.32 ± 0.04 µM. SP-A showed synergistic microbicidal activity with polymyxin B and E, but not with other antibiotics, against three SP-A-resistant pathogenic bacteria: Klebsiella pneumoniae, non-typable Haemophilus influenzae (NTHi), and Pseudomonas aeruginosa. SP-A was not able to bind to K. pneumoniae, NTHi, or to mutant strains thereof expressing long-chain lipopolysaccharides (or lipooligosaccharides) and/or polysaccharide capsules. In the presence of PMB, SP-A induced the formation of SP-A/PMB aggregates that enhance PMB-induced bacterial membrane permeabilization. Furthermore, SP-A bound to a molecular derivative of PMB lacking the acyl chain (PMBN) with a K D of 0.26 ± 0.02 µM, forming SP-A/PMBN aggregates. PMBN has no bactericidal activity but can bind to the outer membrane of Gram-negative bacteria. Surprisingly, SP-A and PMBN showed synergistic bactericidal activity against Gram-negative bacteria. Unlike native supratrimeric SP-A, the trimeric rfhSP-A fragment had small but significant direct bactericidal activity against K. pneumoniae, NTHi, and P. aeruginosa. rfhSP-A did not bind to PMB under physiological conditions but acted additively with PMB and other antibiotics against these pathogenic bacteria. In summary, our results significantly improve our understanding of the antimicrobial actions of SP-A and its synergistic action with PMB. A peptide based on SP-A may aid the therapeutic use of PMB, a relatively cytotoxic antibiotic that is currently being reintroduced into clinics due to the global problem of antibiotic resistance.
Subject(s)
Polymyxin B , Polymyxins , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antibiotics, Antineoplastic , Bacteria , Gram-Negative Bacteria/metabolism , Humans , Klebsiella pneumoniae , Polymyxin B/metabolism , Polymyxin B/pharmacology , Polymyxins/chemistry , Polymyxins/metabolism , Polymyxins/pharmacology , Pseudomonas aeruginosa , Pulmonary Surfactant-Associated Protein AABSTRACT
Meralgia paresthetica is a neurological disorder caused by a neuropathy of the lateral femoral cutaneous nerve. Its aetiology can be spontaneous or iatrogenic. It is characterized by pain, paresthesia, and numbness in the anterolateral aspect of the thigh. Diagnosis is based on clinical examination, although image and neurophysiological tests can be useful as well. Despite conservative measures use to be effective in most of patients, refractory cases can benefit from alternative treatments. Available surgical procedures are: nerve decompression (neurolysis) or section (neurectomy) and radiofrequency ablation. We present a case of refractory meralgia paresthetica where spinal cord stimulation was used as a possible effective technique in pain relief and to avoid the neurectomy of the lateral femoral cutaneous nerve.
Subject(s)
Femoral Neuropathy , Nerve Compression Syndromes , Spinal Cord Stimulation , Humans , Femoral Neuropathy/therapy , Femoral Neuropathy/complications , Spinal Cord Stimulation/adverse effects , Nerve Compression Syndromes/etiology , Nerve Compression Syndromes/therapy , Paresthesia/etiology , Pain/complicationsABSTRACT
Pulmonary fungal infections lead to damage of the endogenous lung surfactant system. However, the molecular mechanism underlying surfactant inhibition is unknown. ß-D-glucan is the major component of pathogenic fungal cell walls and is also present in organic dust, which increases the risk of respiratory diseases. The objective of this study was to characterize the interaction of this D-glucopyranose polymer with pulmonary surfactant. Our results show that ß-D-glucan induced a concentration-dependent inhibition of the surface adsorption, respreading, and surface tension-lowering activity of surfactant preparations containing surfactant proteins SP-B and SP-C. Our data support a new mechanism of surfactant inhibition that consists in the extraction of phospholipid molecules from surfactant membranes by ß-D-glucan. As a result, surfactant membranes became more fluid, as demonstrated by fluorescence anisotropy, and showed decreased Tm and transition enthalpy. Surfactant preparations containing surfactant protein A (SP-A) were more resistant to ß-D-glucan inhibition. SP-A bound to different ß-D-glucans with high affinity (Kd = 1.5⯱â¯0.1â¯nM), preventing and reverting ß-D-glucan inhibitory effects on surfactant interfacial adsorption and partially abrogating ß-D-glucan inhibitory effects on surfactant's reduction of surface tension. We conclude that ß-D-glucan inhibits the biophysical function of surfactant preparations lacking SP-A by subtraction of phospholipids from surfactant bilayers and monolayers. The increased resistance of SP-A-containing surfactant preparations to ß-D-glucan reinforces its use in surfactant replacement therapy.
Subject(s)
Pulmonary Surfactants , Glucans , Phospholipids , Pulmonary Surfactant-Associated Protein A , Pulmonary Surfactant-Associated Protein BABSTRACT
Introducción. La afectación de la columna vertebral en la gota es una complicación extremadamente infrecuente. La dorsalgia y la cuadriplejía son algunas manifestaciones que se pueden presentar, aunque estos síntomas se ven con más frecuencia en otras patologías más prevalentes, como los tumores medulares. Caso clínico. Se presenta un caso inusual de compresión medular dorsal en D10-D11 causado por el depósito extradural de material tofáceo en una paciente de 52 años con gota tofácea crónica incontrolada. Además de un tratamiento médico intensivo, la paciente requirió cirugía (hemilaminectomía y descompresión medular) y rehabilitación posterior. La evolución general y neurológica fue satisfactoria (AU)
Introduction. Spine involvement in gout is an extremely uncommon complication. Dorsalgia and quadriplegia are some manifestations that may occur, although these symptoms are seen more frequently in other more prevalent pathologies, such as spinal tumors. Case report. We present an unusual case of thoracic spinal cord compression at T10-T11 level caused by the extradural deposit of tophaceous material in a 52-year-old woman with uncontrolled chronic tophaceous gout. In addition to intensive medical treatment, the patient required surgery (hemilaminectomy and spinal decompression) and subsequent rehabilitation. Overall and neurological evolution were satisfactory (AU)
Subject(s)
Humans , Female , Middle Aged , Spinal Cord Compression/etiology , Gout/complications , Decompression, Surgical/methods , Laminectomy/methods , Paraplegia/surgery , Spinal Cord Compression/surgeryABSTRACT
Introducción. El mutismo acinético se considera una alteración del estado motivacional de la persona, por el cual el paciente es incapaz de iniciar respuestas verbales o motoras de carácter voluntario, aun teniendo preservadas las funciones sensomotoras y de vigilancia. Caso clínico. Varón de 43 años, intervenido de una fístula arteriovenosa del cerebelo complicada con hidrocefalia, que respondió espectacularmente al tratamiento con bromocriptina. Conclusión. Típicamente se ha descrito el mutismo acinético como una complicación transitoria de las cirugías de la fosa posterior. Sin embargo, también puede aparecer tras múltiples fallos valvulares en pacientes con hidrocefalia (AU)
Introduction. Akinetic mutism is considered as an alteration of the motivational state of the person, which the patient is unable to initiate verbal or motor responses voluntary, even with preserved sensorimotor and surveillance functions. Case report. A 43 year-old male involved in a cerebellum arteriovenous fistula complicated with hydrocephalus, who responded dramatically to treatment with bromocriptine. Conclusion. Typically, akinetic mutism is described as a transient surgeries posterior fossa. However, it can also occur after multiple valvular failure in patients with hydrocephalus (AU)
Subject(s)
Humans , Male , Adult , Bromocriptine/therapeutic use , Akinetic Mutism/drug therapy , Postoperative Complications/drug therapy , Arteriovenous Fistula/surgery , Hydrocephalus/etiology , Tomography, X-Ray ComputedABSTRACT
Lung surfactant protein A (SP-A) plays an important function in modulating inflammation in the lung. However, the exact role of SP-A and the mechanism by which SP-A affects IFN-γ-induced activation of alveolar macrophages (aMÏs) remains unknown. To address these questions, we studied the effect of human SP-A on rat and human aMÏs stimulated with IFN-γ, LPS, and combinations thereof and measured the induction of proinflammatory mediators as well as SP-A's ability to bind to IFN-γ or IFN-γR1. We found that SP-A inhibited (IFN-γ + LPS)-induced TNF-α, iNOS, and CXCL10 production by rat aMÏs. When rat macrophages were stimulated with LPS and IFN-γ separately, SP-A inhibited both LPS-induced signaling and IFN-γ-elicited STAT1 phosphorylation. SP-A also decreased TNF-α and CXCL10 secretion by ex vivo-cultured human aMÏs and M-CSF-derived macrophages stimulated by either LPS or IFN-γ or both. Hence, SP-A inhibited upregulation of IFN-γ-inducible genes (CXCL10, RARRES3, and ETV7) as well as STAT1 phosphorylation in human M-CSF-derived macrophages. In addition, we found that SP-A bound to human IFN-γ (KD = 11 ± 0.5 nM) in a Ca(2+)-dependent manner and prevented IFN-γ interaction with IFN-γR1 on human aMÏs. We conclude that SP-A inhibition of (IFN-γ + LPS) stimulation is due to SP-A attenuation of both inflammatory agents and that the binding of SP-A to IFN-γ abrogates IFN-γ effects on human macrophages, suppressing their classical activation and subsequent inflammatory response.
Subject(s)
Interferon-gamma/immunology , Macrophage Activation/immunology , Macrophages, Alveolar/immunology , Pulmonary Surfactant-Associated Protein A/immunology , Receptors, Interferon/immunology , Animals , Blotting, Western , Cytokines/biosynthesis , Humans , Interferon-gamma/metabolism , Male , Pulmonary Surfactant-Associated Protein A/metabolism , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Receptors, Interferon/metabolismABSTRACT
The anionic antimicrobial peptide SP-B(N), derived from the N-terminal saposin-like domain of the surfactant protein (SP)-B proprotein, and SP-A are lung anti-infective proteins. SP-A-deficient mice are more susceptible than wild-type mice to lung infections, and bacterial killing is enhanced in transgenic mice overexpressing SP-B(N). Despite their potential anti-infective action, in vitro studies indicate that several microorganisms are resistant to SP-A and SP-B(N). In this study, we test the hypothesis that these proteins act synergistically or cooperatively to strengthen each other's microbicidal activity. The results indicate that the proteins acted synergistically in vitro against SP-A- and SP-B(N)-resistant capsulated Klebsiella pneumoniae (serotype K2) at neutral pH. SP-A and SP-B(N) were able to interact in solution (Kd = 0.4 µM), which enabled their binding to bacteria with which SP-A or SP-B(N) alone could not interact. In vivo, we found that treatment of K. pneumoniae-infected mice with SP-A and SP-B(N) conferred more protection against K. pneumoniae infection than each protein individually. SP-A/SP-B(N)-treated infected mice showed significant reduction of bacterial burden, enhanced neutrophil recruitment, and ameliorated lung histopathology with respect to untreated infected mice. In addition, the concentrations of inflammatory mediators in lung homogenates increased early in infection in contrast with the weak inflammatory response of untreated K. pneumoniae-infected mice. Finally, we found that therapeutic treatment with SP-A and SP-B(N) 6 or 24 h after bacterial challenge conferred significant protection against K. pneumoniae infection. These studies show novel anti-infective pathways that could drive development of new strategies against pulmonary infections.
Subject(s)
Pulmonary Surfactant-Associated Proteins/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Cytokines/metabolism , Disease Models, Animal , Drug Synergism , Humans , Hydrogen-Ion Concentration , Klebsiella Infections/immunology , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/immunology , Lung/drug effects , Lung/immunology , Lung/microbiology , Lung/pathology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Knockout , Neutrophil Infiltration , Protein Binding , Pulmonary Surfactant-Associated Protein A/genetics , Pulmonary Surfactant-Associated Protein A/metabolism , Pulmonary Surfactant-Associated Protein A/pharmacology , Pulmonary Surfactant-Associated Protein B/genetics , Pulmonary Surfactant-Associated Protein B/metabolism , Pulmonary Surfactant-Associated Protein B/pharmacology , Pulmonary Surfactant-Associated Proteins/genetics , Pulmonary Surfactant-Associated Proteins/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacologyABSTRACT
Surfactant protein C (SP-C) is a novel amyloid protein found in the lung tissue of patients suffering from interstitial lung disease (ILD) due to mutations in the gene of the precursor protein pro-SP-C. SP-C is a small α-helical hydrophobic protein with an unusually high content of valine residues. SP-C is prone to convert into ß-sheet aggregates, forming amyloid fibrils. Nature's way of solving this folding problem is to include a BRICHOS domain in pro-SP-C, which functions as a chaperone for SP-C during biosynthesis. Mutations in the pro-SP-C BRICHOS domain or linker region lead to amyloid formation of the SP-C protein and ILD. In this study, we used an in vitro transcription/translation system to study translocon-mediated folding of the WT pro-SP-C poly-Val and a designed poly-Leu transmembrane (TM) segment in the endoplasmic reticulum (ER) membrane. Furthermore, to understand how the pro-SP-C BRICHOS domain present in the ER lumen can interact with the TM segment of pro-SP-C, we studied the membrane insertion properties of the recombinant form of the pro-SP-C BRICHOS domain and two ILD-associated mutants. The results show that the co-translational folding of the WT pro-SP-C TM segment is inefficient, that the BRICHOS domain inserts into superficial parts of fluid membranes, and that BRICHOS membrane insertion is promoted by poly-Val peptides present in the membrane. In contrast, one BRICHOS and one non-BRICHOS ILD-associated mutant could not insert into membranes. These findings support a chaperone function of the BRICHOS domain, possibly together with the linker region, during pro-SP-C biosynthesis in the ER.
Subject(s)
Protein Precursors/chemistry , Pulmonary Surfactant-Associated Protein C/chemistry , Amyloidogenic Proteins/chemistry , Amyloidogenic Proteins/genetics , Amyloidogenic Proteins/metabolism , Animals , Endoplasmic Reticulum/metabolism , Humans , In Vitro Techniques , Lung Diseases, Interstitial/genetics , Lung Diseases, Interstitial/metabolism , Membrane Lipids/metabolism , Mutagenesis, Site-Directed , Protein Binding , Protein Folding , Protein Interaction Domains and Motifs , Protein Precursors/genetics , Protein Precursors/metabolism , Pulmonary Surfactant-Associated Protein C/genetics , Pulmonary Surfactant-Associated Protein C/metabolism , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
INTRODUCTION: Ring chromosome 20 syndrome is a rare genetic disorder, with a late diagnosis. CASE REPORT: A 43-year-old woman who had had refractory epilepsy since the age of six years, for which she was treated with deep brain stimulation of the centromedian nucleus, and also a ring chromosome 20. CONCLUSIONS: From the findings of the study it can be concluded that deep brain stimulation of the centromedian nucleus is ineffective in patients with ring chromosome, but note must be taken of the importance of genetic characterisation for the management of refractory epilepsy.
TITLE: Estimulacion del nucleo centromediano en la epilepsia farmacorresistente asociada al cromosoma 20 en anillo.Introduccion. El sindrome del cromosoma 20 en anillo es una alteracion genetica infrecuente, con un diagnostico tardio. Caso clinico. Mujer de 43 años con epilepsia farmacorresistente desde los 6 años, tratada mediante estimulacion cerebral profunda del nucleo centromediano y con un cromosoma 20 en anillo. Conclusiones. Del estudio se extrae la conclusion de la inefectividad de la estimulacion cerebral profunda del nucleo centromediano en pacientes con cromosoma en anillo, pero se apunta la importancia de la caracterizacion genetica para el manejo de la epilepsia farmacorresistente.
Subject(s)
Deep Brain Stimulation , Drug Resistant Epilepsy/therapy , Intralaminar Thalamic Nuclei , Adult , Drug Resistant Epilepsy/genetics , Female , Humans , Ring ChromosomesABSTRACT
Introducción. El síndrome del cromosoma 20 en anillo es una alteración genética infrecuente, con un diagnóstico tardío. Caso clínico. Mujer de 43 años con epilepsia farmacorresistente desde los 6 años, tratada mediante estimulación cerebral profunda del núcleo centromediano y con un cromosoma 20 en anillo. Conclusiones. Del estudio se extrae la conclusión de la inefectividad de la estimulación cerebral profunda del núcleo centromediano en pacientes con cromosoma en anillo, pero se apunta la importancia de la caracterización genética para el manejo de la epilepsia farmacorresistente (AU)
Introduction. Ring chromosome 20 syndrome is a rare genetic disorder, with a late diagnosis. Case report. A 43-year-old woman who had had refractory epilepsy since the age of six years, for which she was treated with deep brain stimulation of the centromedian nucleus, and also a ring chromosome 20. Conclusions. From the findings of the study it can be concluded that deep brain stimulation of the centromedian nucleus is ineffective in patients with ring chromosome, but note must be taken of the importance of genetic characterisation for the management of refractory epilepsy (AU)
Subject(s)
Adult , Female , Humans , Intralaminar Thalamic Nuclei/physiology , Electric Stimulation/methods , Epilepsy/therapy , Chromosomes, Human, Pair 20 , Drug Resistance , Anticonvulsants/therapeutic use , Genetic Predisposition to Disease , Epilepsy/geneticsABSTRACT
BRICHOS domains are encoded in > 30 human genes, which are associated with cancer, neurodegeneration, and interstitial lung disease (ILD). The BRICHOS domain from lung surfactant protein C proprotein (proSP-C) is required for membrane insertion of SP-C and has anti-amyloid activity in vitro. Here, we report the 2.1 Å crystal structure of the human proSP-C BRICHOS domain, which, together with molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry, reveals how BRICHOS domains may mediate chaperone activity. Observation of amyloid deposits composed of mature SP-C in lung tissue samples from ILD patients with mutations in the BRICHOS domain or in its peptide-binding linker region supports the in vivo relevance of the proposed mechanism. The results indicate that ILD mutations interfering with proSP-C BRICHOS activity cause amyloid disease secondary to intramolecular chaperone malfunction.
Subject(s)
Amyloid/antagonists & inhibitors , Lung/metabolism , Molecular Chaperones/metabolism , Pulmonary Surfactant-Associated Protein C/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Chaperones/chemistry , Molecular Sequence Data , Protein Conformation , Pulmonary Surfactant-Associated Protein C/chemistryABSTRACT
Intratracheal administration of immunosuppressive agents to the lung is a novel treatment after lung transplantation. Nanoparticles of tacrolimus (FK506) might interact with human SP-A, which is the most abundant lipoprotein in the alveolar fluid. This study was undertaken to determine whether the formation of FK506/SP-A complexes interferes with FK506 immunosuppressive actions on stimulated human macrophage-like U937 cells. We found that SP-A was avidly bound to FK506 (K(d) = 35 ± 4nM), as determined by solid phase-binding assays and dynamic light scattering. Free FK506, at concentrations ≤ 1 µM, had no effect on the inflammatory response of LPS-stimulated U937 macrophages. However, coincubation of FK506 and SP-A, at concentrations where each component alone did not affect LPS-stimulated macrophage response, significantly inhibited LPS-induced NF-κB activation and TNF-alpha secretion. Free FK506, but not FK506/SP-A, functioned as substrate for the efflux transporter P-glycoprotein. FK506 bound to SP-A was delivered to macrophages by endocytosis, since several endocytosis inhibitors blocked FK506/SP-A anti-inflammatory effects. This process depended partly on SP-A binding to its receptor, SP-R210. These results indicate that FK506/SP-A complexes have a greater anti-inflammatory effect than either FK506 or SP-A alone and suggest that SP-A strengthened FK506 anti-inflammatory activity by facilitating FK506 entrance into the cell, overcoming P-glycoprotein.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Immunosuppressive Agents/pharmacology , Macrophages/drug effects , Pulmonary Surfactant-Associated Protein A/pharmacology , Tacrolimus/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Cell Survival/drug effects , Dose-Response Relationship, Drug , Endocytosis/drug effects , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/metabolism , Lipopolysaccharides/pharmacology , Macrophages/immunology , Macrophages/metabolism , Models, Molecular , Molecular Structure , Protein Binding , Pulmonary Surfactant-Associated Protein A/administration & dosage , Pulmonary Surfactant-Associated Protein A/metabolism , Receptors, Cell Surface/metabolism , Tacrolimus/administration & dosage , Tacrolimus/metabolism , U937 CellsABSTRACT
The purpose of this study was to investigate how surfactant membranes can be perturbed by C-reactive protein (CRP) and whether surfactant protein A (SP-A) might overcome CRP-induced surfactant membrane alterations. The effect of CRP on surfactant surface adsorption was evaluated in vivo after intratracheal instillation of CRP into rat lungs. Insertion of CRP into surfactant membranes was investigated through monolayer techniques. The effect of CRP on membrane structure was studied through differential scanning calorimetry and fluorescence spectroscopy and microscopy using large and giant unilamellar vesicles. Our results indicate that CRP inserts into surfactant membranes and drastically increases membrane fluidity, resulting in surfactant inactivation. At 10% CRP/phospholipid weight ratio, CRP causes disappearance of liquid-ordered/liquid-disordered phase coexistence distinctive of surfactant membranes. SP-A, the most abundant surfactant lipoprotein structurally similar to C1q, binds to CRP (K(d)=56±8 nM), as determined by solid-phase binding assays and dynamic light scattering. This novel SP-A/CRP interaction reduces CRP insertion and blocks CRP effects on surfactant membranes. In addition, intratracheal coinstillation of SP-A+CRP into rat lungs prevents surfactant inhibition induced by CRP, indicating that SP-A/CRP interactions might be an important factor in vivo in controlling harmful CRP effects in the alveolus.
Subject(s)
C-Reactive Protein/pharmacology , Membrane Fluidity/drug effects , Pulmonary Surfactant-Associated Protein A/pharmacology , Animals , Bronchoalveolar Lavage Fluid , C-Reactive Protein/administration & dosage , Calorimetry, Differential Scanning , Cell Membrane/metabolism , Male , Microscopy, Fluorescence , Pulmonary Surfactant-Associated Protein A/administration & dosage , Rats , Rats, Sprague-Dawley , Spectrometry, FluorescenceABSTRACT
Nature's high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider's spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 A resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider's silk extrusion duct.
Subject(s)
Silk/chemistry , Silk/metabolism , Spiders/chemistry , Amino Acid Sequence , Animals , Circular Dichroism , Conserved Sequence , Crystallography, X-Ray , Hydrogen-Ion Concentration , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Sequence Alignment , Silk/ultrastructure , Static ElectricityABSTRACT
Surfactant protein C (SP-C) constitutes the transmembrane part of prosurfactant protein C (proSP-C) and is alpha-helical in its native state. The C-terminal part of proSP-C (CTC) is localized in the endoplasmic reticulum lumen and binds to misfolded (beta-strand) SP-C, thereby preventing its aggregation and amyloid fibril formation. In this study, we investigated the structure of recombinant human CTC and the effects of CTC-membrane interaction on protein structure. CTC forms noncovalent trimers and supratrimeric oligomers. It contains two intrachain disulfide bridges, and its secondary structure is significantly affected by urea or heat only after disulfide reduction. The postulated Brichos domain of CTC, with homologs found in proteins associated with amyloid and proliferative disease, is up to 1000-fold more protected from limited proteolysis than the rest of CTC. The protein exposes hydrophobic surfaces, as determined by CTC binding to the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate). Fluorescence energy transfer experiments further reveal close proximity between bound 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate) and tyrosine residues in CTC, some of which are conserved in all Brichos domains. CTC binds to unilamellar phospholipid vesicles with low micromolar dissociation constants, and differential scanning calorimetry and CD analyses indicate that membrane-bound CTC is less structurally ordered than the unbound protein. The exposed hydrophobic surfaces and the structural disordering that result from interactions with phospholipid membranes suggest a mechanism whereby CTC binds to misfolded SP-C in the endoplasmic reticulum membrane.
Subject(s)
Endoplasmic Reticulum/metabolism , Pulmonary Surfactant-Associated Protein C/chemistry , Pulmonary Surfactant-Associated Protein C/metabolism , Amino Acid Sequence , Anilino Naphthalenesulfonates/chemistry , Calorimetry , Circular Dichroism , Humans , Hydrophobic and Hydrophilic Interactions , Liposomes/chemistry , Molecular Sequence Data , Phospholipids/chemistry , Protein Folding , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Precursors/metabolism , Pulmonary Surfactant-Associated Protein C/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Temperature , Ultracentrifugation , Urea/chemistryABSTRACT
SP-A, the major protein component of pulmonary surfactant, is absent in exogenous surfactants currently used in clinical practice. However, it is thought that therapeutic properties of natural surfactants improve after enrichment with SP-A. The objective of this study was to determine SP-A effects on physical properties and surface activity of a new synthetic lung surfactant based on a cationic and hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK, KL(4). We have analyzed the interaction of SP-A with liposomes consisting of DPPC/POPG/PA (28:9:5.6, w/w/w) with and without 0.57 mol % KL(4) peptide. We found that SP-A had a concentration-dependent effect on the surface activity of KL(4)-DPPC/POPG/PA membranes but not on that of an animal-derived LES. The surface activity of KL(4)-surfactant significantly improved after enrichment with 2.5-5 wt % SP-A. However, it worsened at SP-A concentrations > or =10 wt %. This was due to the fluidizing effect of supraphysiological SP-A concentrations on KL(4)-DPPC/POPG/PA membranes as determined by fluorescence anisotropy measurements, calorimetric studies, and confocal fluorescence microscopy of GUVs. High SP-A concentrations caused disappearance of the solid/fluid phase coexistence of KL(4)-surfactant, suggesting that phase coexistence might be important for the surface adsorption process.
Subject(s)
Lipid Bilayers/chemistry , Liposomes/chemistry , Peptides/chemistry , Pulmonary Surfactant-Associated Protein A/chemistry , Animals , Intercellular Signaling Peptides and Proteins , Molecular Conformation , Surface Properties , SwineABSTRACT
Surfactant-like membranes containing the 21-residue peptide KLLLLKLLLLKLLLLKLLLLK (KL4), have been clinically tested as a therapeutic agent for respiratory distress syndrome in premature infants. The aims of this study were to investigate the interactions between the KL4 peptide and lipid bilayers, and the role of both the lipid composition and KL4 structure on the surface adsorption activity of KL4-containing membranes. We used bilayers of three-component systems [1,2-dipalmitoyl-phosphatidylcholine/1-palmitoyl-2-oleoyl-phosphatidylglycerol/palmitic acid (DPPC/POPG/PA) and DPPC/1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/PA] and binary lipid mixtures of DPPC/POPG and DPPC/PA to examine the specific interaction of KL4 with POPG and PA. We found that, at low peptide concentrations, KL4 adopted a predominantly alpha-helical secondary structure in POPG- or POPC-containing membranes, and a beta-sheet structure in DPPC/PA vesicles. As the concentration of the peptide increased, KL4 interconverted to a beta-sheet structure in DPPC/POPG/PA or DPPC/POPC/PA vesicles. Ca2+ favored alpha<-->beta interconversion. This conformational flexibility of KL4 did not influence the surface adsorption activity of KL4-containing vesicles. KL4 showed a concentration-dependent ordering effect on POPG- and POPC-containing membranes, which could be linked to its surface activity. In addition, we found that the physical state of the membrane had a critical role in the surface adsorption process. Our results indicate that the most rapid surface adsorption takes place with vesicles showing well-defined solid/fluid phase co-existence at temperatures below their gel to fluid phase transition temperature, such as those of DPPC/POPG/PA and DPPC/POPC/PA. In contrast, more fluid (DPPC/POPG) or excessively rigid (DPPC/PA) KL4-containing membranes fail in their ability to adsorb rapidly onto and spread at the air-water interface.