RESUMO
Background: A common manifestation of organophosphorus insecticide self-poisoning is prolonged respiratory failure due to neuromuscular junction dysfunction and likely nicotinic receptor overstimulation. We aimed at collecting preliminary data on whether addition of the competitive nicotinic antagonist rocuronium to standard early therapy might be clinically feasible and associated with reduced duration of ventilation.Methods: A pilot three-arm dose-response phase II trial was set up to compare bolus doses of rocuronium bromide titrated to produce initial >95% or 50% inhibition of neuromuscular function, measured using acceleromyography, plus standard treatment, versus standard treatment alone. After attaining inhibition, patients receiving bolus rocuronium then received rocuronium infusions for a maximum of 120 h. Primary outcome was duration of intubation; secondary outcome was case fatality. Plasma butyrylcholinesterase activity was measured throughout the inpatient stay. Blood was analysed to confirm the organophosphorus insecticide ingested.Results: Forty-five patients were randomised to receive: rocuronium to initially attain 95% inhibition (Roc>95, n = 15), rocuronium to initially attain 50% inhibition (Roc50, n = 14), or no rocuronium (control, n = 16). The most commonly ingested pesticide was profenofos (29/45, 64.4%). Butyrylcholinesterase activity remained severely inhibited for the duration of the study for most patients. Case fatality was 9/45 (20%) and similar across study arms: control 3/16 (18.8%), Roc50 4/14 (28.6%) and Roc>95 2/15 (13.3%) (p = .5842). When excluding patients who died, median [IQR] duration of intubation was significantly longer in the Roc50 (259.5 [176-385] h) and Roc>95 (226.8 [186-355] h) groups compared to controls (88.5 [47-160] h, p = .0162 and p = .0016, respectively).Conclusions: In this pilot dose-response study, we found no evidence that rocuronium in addition to standard therapy reduced the duration of intubation. It is possible that it worsened neuromuscular junction function. Further clinical research, including testing of shorter duration regimens, needs to be performed before nicotinic antagonists can be used in the clinical management of OP poisoning.
Assuntos
Inseticidas/intoxicação , Intoxicação por Organofosfatos/complicações , Respiração Artificial/estatística & dados numéricos , Insuficiência Respiratória/induzido quimicamente , Rocurônio/administração & dosagem , Adulto , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fármacos Neuromusculares não Despolarizantes/administração & dosagem , Intoxicação por Organofosfatos/terapia , Projetos Piloto , Insuficiência Respiratória/terapia , Comportamento Autodestrutivo/complicações , Fatores de Tempo , Adulto JovemRESUMO
Alexandrium ostenfeldii is a toxic dinoflagellate that has recently bloomed in Ouwerkerkse Kreek, The Netherlands, and which is able to cause a serious threat to shellfish consumers and aquacultures. We used a large set of 68 strains to the aim of fully characterizing the toxin profiles of the Dutch A. ostenfeldii in consideration of recent reports of novel toxins. Alexandrium ostenfeldii is known as a causative species of paralytic shellfish poisoning, and consistently in the Dutch population we determined the presence of several paralytic shellfish toxins (PST) including saxitoxin (STX), GTX2/3 (gonyautoxins), B1 and C1/C2. We also examined the production of spiroimine toxins by the Dutch A. ostenfeldii strains. An extensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed a high intraspecific variability of spirolides (SPX) and gymnodimines (GYM). Spirolides included 13-desMethyl-spirolide C generally as the major compound and several other mostly unknown SPX-like compounds that were detected and characterized. Besides spirolides, the presence of gymnodimine A and 12-Methyl-gymnodimine A was confirmed, together with two new gymnodimines. One of these was tentatively identified as an analogue of gymnodimine D and was the most abundant gymnodimine (calculated cell quota up to 274 pg cell-1, expressed as GYM A equivalents). Our multi-clonal approach adds new analogues to the increasing number of compounds in these toxin classes and revealed a high strain variability in cell quota and in toxin profile of toxic compounds within a single population.
RESUMO
Gymnodimines are lipophilic toxins produced by the marine dinoflagellates Karenia selliformis and Alexandrium ostenfeldii. Currently four gymnodimine analogues are known and characterized. Here we describe a novel gymnodimine and a range of gymnodimine related compounds found in an A. ostenfeldii isolate from the northern Baltic Sea. Gymnodimine D (1) was extracted and purified from clonal cultures, and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS), nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography-high resolution mass spectrometry (LC-HRMS) experiments. The structure of 1 is related to known gymnodimines (2-5) with a six-membered cyclic imine ring and several other fragments typical of gymnodimines. However, the carbon chain in the gymnodimine macrocyclic ring differs from the known gymnodimines in having two tetrahydrofuran rings in the macrocyclic ring.
Assuntos
Dinoflagellida/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Hidrocarbonetos Cíclicos/metabolismo , Toxinas Marinhas/metabolismo , Oceano Atlântico , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Células Clonais , Dinoflagellida/química , Dinoflagellida/citologia , Dinoflagellida/crescimento & desenvolvimento , Proliferação Nociva de Algas , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/isolamento & purificação , Compostos Heterocíclicos com 3 Anéis/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/isolamento & purificação , Hidrocarbonetos Cíclicos/química , Hidrocarbonetos Cíclicos/isolamento & purificação , Iminas/química , Iminas/isolamento & purificação , Iminas/metabolismo , Toxinas Marinhas/química , Toxinas Marinhas/isolamento & purificação , Metilação , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por Electrospray , Estereoisomerismo , Suécia , Espectrometria de Massas em TandemRESUMO
Many marine dinoflagellates, including several species of the genus Alexandrium, Gymnodinium catenatum, and Pyrodinium bahamense are known for their capability to produce paralytic shellfish toxins (PST), which can cause severe, most often food-related poisoning. The recent discovery of the first PST biosynthesis genes has laid the foundation for the development of molecular detection methods for monitoring and study of PST-producing dinoflagellates. In this study, a probe-based qPCR method for the detection and quantification of the sxtA4 gene present in Alexandrium spp. and Gymnodinium catenatum was designed. The focus was on Alexandrium ostenfeldii, a species which recurrently forms dense toxic blooms in areas within the Baltic Sea. A consistent, positive correlation between the presence of sxtA4 and PST biosynthesis was observed, and the species was found to maintain PST production with an average of 6 genomic copies of sxtA4. In August 2014, A. ostenfeldii populations were studied for cell densities, PST production, as well as sxtA4 and species-specific LSU copy numbers in Föglö, Åland, Finland, where an exceptionally dense bloom, consisting of 6.3×106cellsL-1, was observed. Cell concentrations, and copy numbers of both of the target genes were positively correlated with total STX, GTX2, and GTX3 concentrations in the environment, the cell density predicting toxin concentrations with the best accuracy (Spearman's ρ=0.93, p<0.01). The results indicated that all A. ostenfeldii cells in the blooms harbored the genetic capability of PST production, making the detection of sxtA4 a good indicator of toxicity.
Assuntos
Dinoflagellida/genética , Dinoflagellida/metabolismo , Saxitoxina/genética , Saxitoxina/metabolismo , Dinoflagellida/química , Finlândia , Densidade Demográfica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
A saxitoxin (STX) proficiency test (PT) was organized as part of the Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox) project. The aim of this PT was to provide an evaluation of existing methods and the European laboratories' capabilities for the analysis of STX and some of its analogues in real samples. Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning (PSP) toxins were produced as reference sample matrices. The reference material was characterized using various analytical methods. Acidified algal extract samples at two concentration levels were prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose. Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries. The PT offered the participating laboratories the possibility to assess their performance regarding the qualitative and quantitative detection of PSP toxins. Various techniques such as official Association of Official Analytical Chemists (AOAC) methods, immunoassays, and liquid chromatography-mass spectrometry were used for sample analyses.
Assuntos
Toxinas Marinhas/análise , Animais , Cromatografia Líquida/métodos , Dinoflagellida , Ensaio de Proficiência Laboratorial/normas , Camundongos , Mytilus , Padrões de Referência , Intoxicação por Frutos do Mar , Espectrometria de Massas em TandemRESUMO
Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of ±1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD).
Assuntos
Toxinas Marinhas/análise , Animais , Cromatografia Líquida , Ensaio de Proficiência Laboratorial , Mytilus , Intoxicação por Frutos do Mar , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Trk receptor tyrosine kinases regulate multiple important neuronal processes during the development and in the adulthood. Tyrosine phosphorylation of Trk serves as the initial step in the Trk signaling pathway and indicates receptor' autocatalytic activity. However, methods allowing simple and large-scale Trk phosphorylation analyses in cultured cells are lacking. NEW METHOD: We describe an in situ phospho-Trk ELISA (enzyme-linked immunosorbent assay) method where cell culture, receptor stimulation and Trk phosphorylation analysis are all performed on the same multiwell plate. RESULTS: In situ phospho-Trk ELISA readily and specifically detects neurotrophin-induced Trk phosphorylation in cultured cells. A proof-of-concept small molecule screening of a library composed of 2000 approved drugs and other bioactive compounds was carried out using this novel method. COMPARISON WITH EXISTING METHODS: In situ phospho-Trk ELISA utilizes the principles and advantages of conventional sandwich ELISA in an in situ context. CONCLUSIONS: We describe a novel method that can be efficiently used to examine Trk receptor phosphorylation in cultured cells. Principally similar methods can be developed to examine the levels and signaling of any intracellular protein.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Células Cultivadas , Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Fosforilação , Ratos , Receptor trkB/genética , Receptor trkB/metabolismoRESUMO
Acetaldehyde is a known mutagenic substance and has been classified as a group-one carcinogen by the WHO. It is possible to bind acetaldehyde locally in the gastrointestinal (GI) tract with the semi-essential amino acid l-cysteine, which reacts covalently with acetaldehyde and forms compound 2-methyl-thiozolidine-4-carboxylic acid (MTCA). The Caco-2 cell line was used to determine the permeation of l-cysteine and MTCA, as well as the possible cell toxicity of both substances. Neither of the substances permeated through the Caco-2 cells at the concentrations used in this study, and only the highest concentration of MTCA affected the viability of the cells in the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) test. These results showed that when l-cysteine is administered in formulations releasing it locally in the lower parts of GI tract, it is not absorbed but can react with acetaldehyde, and that neither l-cysteine nor MTCA is harmful to the cells when present locally in the upper parts of GI tract. This study also shows that MTCA is sensitive at a lower pH of 5.5. Since stable MTCA is desired in different parts of the GI tract, this observation raises concern over the influence of lower pH on l-cysteine-containing product ability to bind and eliminate carcinogenic acetaldehyde.
Assuntos
Cisteína/farmacocinética , Cisteína/toxicidade , Tiazolidinas/farmacocinética , Tiazolidinas/toxicidade , Acetaldeído/farmacocinética , Células CACO-2 , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , PermeabilidadeRESUMO
A method for the analysis of intact glucuronides and sulfates of common neurotransmitters serotonin (5-HT) and dopamine (DA) as well as of 5-hydroxy-3-indoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat brain microdialysates by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Enzyme-assisted synthesis using rat liver microsomes as a biocatalyst was employed for the production of 5-HT-, 5-HIAA-, DOPAC-, and HVA-glucuronides for reference compounds. The sulfate conjugates were synthesized either chemically or enzymatically using a rat liver S9 fraction. The LC-MS/MS method was validated by determining the limits of detection and quantitation, linearity, and repeatability for the quantitative analysis of 5-HT and DA and their glucuronides, as well as of 5-HIAA, DOPAC, and HVA and their sulfate-conjugates. In this study, 5-HT-glucuronide was for the first time detected in rat brain. The concentration of 5-HT-glucuronide (1.0-1.7 nM) was up to 2.5 times higher than that of free 5-HT (0.4-2.1 nM) in rat brain microdialysates, whereas the concentration of DA-glucuronide (1.0-1.4 nM) was at the same level or lower than the free DA (1.2-2.4 nM). The acidic metabolites of neurotransmitters, 5-HIAA, HVA, and DOPAC, were found in free and sulfated form, whereas their glucuronidation was not observed.
Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Glucuronídeos/análise , Serotonina/metabolismo , Sulfatos/análise , Animais , Cromatografia Líquida , Dopamina/análise , Dopamina/química , Glucuronídeos/biossíntese , Glucuronídeos/química , Humanos , Hidrólise , Masculino , Microdiálise , Microssomos Hepáticos , Ratos , Ratos Sprague-Dawley , Padrões de Referência , Serotonina/análise , Serotonina/química , Estereoisomerismo , Sulfatos/síntese química , Sulfatos/química , Sulfatos/metabolismo , Espectrometria de Massas em TandemRESUMO
Immature hippocampal synapses express presynaptic kainate receptors (KARs), which tonically inhibit glutamate release. Presynaptic maturation involves activity-dependent downregulation of the tonic KAR activity and consequent increase in release probability; however, the molecular mechanisms underlying this developmental process are unknown. Here, we have investigated whether brain derived neurotrophic factor (BDNF), a secreted protein implicated in developmental plasticity in several areas of the brain, controls presynaptic maturation by regulating KARs. Application of BDNF in neonate hippocampal slices resulted in increase in synaptic transmission that fully occluded the immature-type KAR activity in area CA1. Conversely, genetic ablation of BDNF was associated with delayed synaptic maturation and persistent presynaptic KAR activity, suggesting a role for endogenous BDNF in the developmental regulation of KAR function. In addition, our data suggests a critical role for BDNF TrkB signaling in fast activity-dependent regulation of KARs. Selective acute inhibition of TrkB receptors using a chemical-genetic approach prevented rapid change in synapse dynamics and loss of tonic KAR activity that is typically seen in response to induction of LTP at immature synapses. Together, these data show that BDNF-TrkB-dependent maturation of glutamatergic synapses is tightly associated with a loss of endogenous KAR activity. The coordinated action of these two receptor mechanisms has immediate physiological relevance in controlling presynaptic efficacy and transmission dynamics at CA3-CA1 synapses at a stage of development when functional contact already exists but transmission is weak.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Regulação para Baixo/fisiologia , Receptores de Ácido Caínico/metabolismo , Receptores Pré-Sinápticos/antagonistas & inibidores , Receptores Pré-Sinápticos/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Fator Neurotrófico Derivado do Encéfalo/deficiência , Regulação para Baixo/genética , Potenciais Pós-Sinápticos Excitadores/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Técnicas de Introdução de Genes , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor trkB/genética , Transdução de Sinais/genética , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
A new method to synthesize N-unsubstituted pyrazoles on a solid support is described. The solid support acts as a protecting group for the amino acid. N-Protected amino acid is N-nitrosated, and the subsequent treatment with acetic anhydride in a microwave reactor yields mesoionic sydnones that react in situ in 1,3-dipolar cycloaddition reactions with alkynes. Traceless cleavage of the products gives N-unsubstituted pyrazoles in high overall yields.
Assuntos
Aminoácidos/química , Pirazóis/síntese química , Sidnonas/química , Alcinos/química , Catálise , Estrutura Molecular , Pirazóis/químicaRESUMO
Study was made of the 1,3-dipolar cycloaddition of polymer-bound alkynes to azomethine imines generated in situ from N-aminopyridine iodides. Aromatization of the cycloadducts gives polymer-bound pyrazolopyridines that can be released from the resin as carboxylic acids with trifluoroacetic acid or as methyl esters with sodium methoxide.
Assuntos
Alcinos/química , Compostos Azo/química , Iminas/química , Pirazóis/síntese química , Piridinas/síntese química , Metanol/química , Modelos Químicos , Polímeros/química , Pirazóis/farmacologia , Piridinas/farmacologia , Tecnologia FarmacêuticaRESUMO
Membrane components of bacteria and fungi are recognized by Toll-like receptors (TLRs) which, when activated, induce several inflammatory mediators important in the host defense. As the liver is constantly exposed to ingested bacteria, hepatic TLRs must be broadly responsive and highly regulated to prevent uncontrolled inflammatory activation. Although several hepatic cells express microbe recognition molecules and inflammatory mediators in vitro, the regulation and cellular localization of these proteins in vivo remain uncertain. The expression and regulation of TLR-2 and TLR-4, and the cytokine expression patterns were evaluated in mouse tissues using a model of acute inflammation induced by intraperitoneal injection of LPS. Five hours after intraperitoneal LPS, induction of TLR-4 was evident in lung, while the low hepatic TLR-4 expression was non-inducible. TLR-2 mRNA and protein were induced both in lung and liver TLR-4 dependently. However, IL-1alpha also contributed to this induction, and IL-1R1 antibody attenuated the TLR-2 increase. Immunoelectron microscopy showed accumulation of cytoplasmic TLR-2 to vesicles near the hepatocyte plasma membrane in the space of Disse, to the sinusoidal endothelium and to the Kupffer cells. NF-kappaB activation was clear in Kupffer cells and hepatocytes during LPS-challenge, suggesting these cells to be the main source of in vivo cytokine production. Hepatic cytokine response to LPS was remarkably rapid in liver, whereas lung responded less acutely. Secondary inflammatory challenge attenuated the TLR-2 response. The innate immune system of the liver is rapidly and transiently activated during endotoxemia by mechanism involving both TLR-4 and TLR-2.
Assuntos
Endotoxemia/imunologia , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Pulmão/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Animais , Membrana Celular/metabolismo , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Inflamação/imunologia , Células de Kupffer/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Monocinas/fisiologia , NF-kappa B/metabolismo , Regulação para CimaRESUMO
The feasibility of ultra thin-layer chromatography atmospheric pressure matrix-assisted laser desorption ionization mass spectrometry (UTLC-AP-MALDI-MS) has been studied in the analysis of small molecules. Because of a thinner adsorbent layer, the monolithic UTLC plates provide 10-100 times better sensitivity in MALDI analysis than conventional high performance thin-layer chromatography (HPTLC) plates. The limits of detection down to a low picomole range are demonstrated by UTLC-AP-MALDI-MS. Other advantages of UTLC over HPTLC include faster separations and lower solvent consumption. The performances of AP-MALDI-MS and vacuum MALDI-MS have been compared in the analysis of small drug molecules directly from the UTLC plates. The desorption from the irregular surface of UTLC plates with an external AP-MALDI ion source combined with an ion trap instrument provides clearly less variation in measurements of m/z values when compared with a vacuum MALDI-time-of-flight (TOF) instrument. The performance of the UTLC-AP-MALDI-MS method has been applied successfully to the purity analysis of synthesis products produced by solid-phase parallel synthesis method.
Assuntos
Cromatografia em Camada Fina/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Pressão Atmosférica , Metoprolol/análise , Metoprolol/química , Midazolam/análise , Midazolam/química , Modelos Moleculares , Peso Molecular , Sensibilidade e Especificidade , Verapamil/análise , Verapamil/químicaRESUMO
Solid-phase methods are of a great significance in organic synthesis. Recent developments of these methods are providing new ways to construct libraries of small organic molecules. Five-membered heterocyclic compounds, which can be utilized in a variety of applications, are formed in the 1,3-dipolar cycloaddition reaction between dipolarophiles and dipoles. This review deals with the solid-phase synthesis of heterocycles via [3+2] cycloaddition reaction. Cycloaddition reactions of polymer-bound dipoles and polymer-bound dipolarophiles and intramolecular solid-phase cycloadditions are discussed in separate sections. Reactions of dipolarophiles such as alkenes, alkynes, and imines with dipoles such as azomethine ylides, azomethine imines, nitrile imines, azides, nitrones, and nitrile oxides are described. The recent literature up to December 2003 is covered.
Assuntos
Química Orgânica/métodos , Hidrocarbonetos Cíclicos/síntese química , Compostos Orgânicos/química , Compostos Orgânicos/síntese química , Hidrocarbonetos Cíclicos/química , Indicadores e Reagentes , Conformação MolecularRESUMO
Endotoxin [lipopolysaccharide (LPS)] from Gram-negative bacteria is found in amniotic fluid in intrauterine infections that associate with the risk for spontaneous premature birth, bronchopulmonary dysplasia (BPD), and respiratory distress syndrome. Toll-like receptor 4 (TLR4) is the signaling receptor for LPS. The aim was to investigate the primary inflammatory response in mice shortly after administration of LPS to the dam (14 and 17 d of pregnancy), to the newborn, or into the amniotic fluid. The expression levels of TLR4, IL-1, tumor necrosis factor-alpha, IL-6, IL-10, macrophage inflammatory protein-2, and IL-1 receptor 1 were studied with ribonuclease protection assay. In addition, TLR4 protein was analyzed with Western blotting. The fetal membranes expressed TLR4 mRNA and protein and showed an acute cytokine response to LPS when LPS was administrated into the amniotic fluid. There was distinct ontogeny in the responsiveness of fetal lung to LPS: on fetal day 14 (term 20 d), both the expression of TLR4 and the acute cytokine response were undetectable 5 h after LPS; they became detectable by fetal day 17. TLR4 and the cytokine response further increased after birth. In maternal lung, the TLR4 expression was strongest and up-regulated in parallel with the induction of the cytokines. We propose that TLR4 controls the magnitude of the LPS-induced cytokine response during the perinatal period.
Assuntos
Endotoxinas/metabolismo , Glicoproteínas de Membrana/biossíntese , Receptores de Superfície Celular/biossíntese , Alelos , Animais , Animais Recém-Nascidos , Western Blotting , Quimiocina CXCL2 , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação , Interleucina-1/biossíntese , Interleucina-10/biossíntese , Interleucina-6/biossíntese , Lipopolissacarídeos/metabolismo , Pneumopatias/microbiologia , Camundongos , Camundongos Endogâmicos DBA , Monocinas/biossíntese , Placenta/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Receptores de Interleucina-1/biossíntese , Receptores Tipo I de Interleucina-1 , Ribonucleases/metabolismo , Transdução de Sinais , Fatores de Tempo , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/biossíntese , Regulação para CimaRESUMO
The luteinizing hormone receptor (LHR) is a G protein-coupled receptor involved in regulation of ovarian and testicular functions. Here we show that the receptor is present also in specific areas of the peripheral and central nervous system and may thus have a broader functional role than has been anticipated. Full-length LHR mRNA and two receptor protein species of M(r) 90,000 and 73,000, representing mature and precursor forms, respectively, were expressed in adult and developing rat nervous tissue, starting at fetal day 14.5. The receptor was capable of ligand binding because it was purified by ligand affinity chromatography, and human chorionic gonadotropin and LH were able to displace (125)I-labeled human chorionic gonadotropin binding to fetal head membranes in a dose-dependent manner. Finally, two 5'-flanking sequences ( approximately 2 and 4 kb) of the rat LHR gene were shown to direct expression of the lacZ reporter to specific areas of the peripheral and central nervous system in fetal and adult transgenic mice, especially to structures associated with sensory, memory, reproductive behavior, and autonomic functions. Importantly, the transgene activity was confined to neurons and colocalized with the cytochrome P450 side chain cleavage enzyme. Taken together, these results indicate that the neuronal LHR is a functional protein, implicating a role in neuronal development and function, possibly by means of regulating synthesis of neurosteroids.
Assuntos
Neurônios/química , Receptores do LH/análise , Animais , Encéfalo/metabolismo , Gonadotropina Coriônica/metabolismo , Feminino , Feto/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores do LH/química , Receptores do LH/genética , TransgenesRESUMO
The solid-phase synthesis of 1,2,3-triazoles via 1,3-dipolar cycloaddition of polymer-bound azides to various alkynes is reported. Polymer-bound azides were synthesized from polymer-bound halides and sodium azide and reacted with alkynes to produce polymer-bound 1,2,3-triazoles. Cleavage of the triazoles was performed with trifluoroacetic acid. A traceless synthesis of 1,2,3-triazoles was developed using 2-methoxy-substituted resin (polymer-bound 4-hydroxy-2-methoxybenzyl alcohol). In addition, a synthesis of 4-hydroxybenzyl-substituted 1,2,3-triazoles from the bromo-Wang resin (4-(bromomethyl)phenoxymethyl polystyrene) was achieved.
Assuntos
Tecnologia Farmacêutica/métodos , Triazóis/síntese química , Triazóis/químicaRESUMO
Recent evidence suggests a role for phosphatidylinositol (PI) 3-kinase in various inflammatory responses. In this study, the consequences of LPS-induced PI 3-kinase activation on cytokine and chemokine expression and the intracellular mechanisms of inflammatory activation were examined in mouse macrophages. LPS stimulation induced a complex formation between PI 3-kinase and myeloid differentiation factor 88 (MyD88), which was followed by an induction of IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein (MIP)-2. The induction of IL-1beta, but not of MIP-2 or TNF-alpha, was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin. The nuclear factor-kappaB (NF-kappaB) inhibitor pyrrolidinedithiocarbamate (PDTC) blocked the induction of IL-1beta and TNF-alpha, but had no effect on MIP-2 expression. Inhibition of PI 3-kinase decreased the LPS-induced transcriptional activity of NF-kappaB, but it had no effect on the nuclear DNA binding activity of NF-kappaB. These findings suggest that, while NF-kappaB nuclear localization and DNA binding are necessary, they are not sufficient for transcriptional activation of the IL-1beta gene in the absence of PI 3-kinase activity. Taken together, our results demonstrate that activation of Toll-like receptor (TLR)-4 results in PI 3-kinase-MyD88 complex formation, and that PI 3-kinase activity selectively leads to cytokine induction downstream of TLR4.