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1.
Phys Rev Lett ; 126(4): 041301, 2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33576660

RESUMO

We constrain the coupling between axionlike particles (ALPs) and photons, measured with the superconducting resonant detection circuit of a cryogenic Penning trap. By searching the noise spectrum of our fixed-frequency resonant circuit for peaks caused by dark matter ALPs converting into photons in the strong magnetic field of the Penning-trap magnet, we are able to constrain the coupling of ALPs with masses around 2.7906-2.7914 neV/c^{2} to g_{aγ}<1×10^{-11} GeV^{-1}. This is more than one order of magnitude lower than the best laboratory haloscope and approximately 5 times lower than the CERN axion solar telescope (CAST), setting limits in a mass and coupling range which is not constrained by astrophysical observations. Our approach can be extended to many other Penning-trap experiments and has the potential to provide broad limits in the low ALP mass range.

2.
Science ; 358(6366): 1081-1084, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29170238

RESUMO

Precise knowledge of the fundamental properties of the proton is essential for our understanding of atomic structure as well as for precise tests of fundamental symmetries. We report on a direct high-precision measurement of the magnetic moment µp of the proton in units of the nuclear magneton µN The result, µp = 2.79284734462 (±0.00000000082) µN, has a fractional precision of 0.3 parts per billion, improves the previous best measurement by a factor of 11, and is consistent with the currently accepted value. This was achieved with the use of an optimized double-Penning trap technique. Provided a similar measurement of the antiproton magnetic moment can be performed, this result will enable a test of the fundamental symmetry between matter and antimatter in the baryonic sector at the 10-10 level.

3.
PLoS One ; 12(7): e0180981, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28686706

RESUMO

Plasminogen (Plg) is a precursor of plasmin that degrades fibrin. A race-specific A620T mutation in Plg, also known as Plg-Tochigi, originally identified in a patient with recurrent venous thromboembolism, causes dysplasminogenemia with reduced plasmin activity. The Plg-A620T mutation is present in 3-4% of individuals in East Asian populations, and as many as 50,000 Japanese are estimated to be homozygous for the mutant 620T allele. In the present study, to understand the changes of thrombotic phenotypes in individuals with the mutant 620T allele, we generated knock-in mice carrying the homozygous Plg-A622T mutation (PlgT/T), an equivalent to the A620T mutation in human Plg. PlgT/T mice grew normally but showed severely reduced plasmin activity activated by urokinase, equivalent to ~8% of that in wild-type mice. In vitro fibrin clot lysis in plasma was significantly slower in PlgT/T mice than in wild-type mice. However, all experimental models of electrolytic deep vein thrombosis, tissue factor-induced pulmonary embolism, transient focal brain ischaemic stroke, or skin-wound healing showed largely similar phenotypes between PlgT/T mice and wild-type mice. Protein S-K196E mutation (Pros1E/E) is a race-specific genetic risk factor for venous thromboembolism. Coexistence in mice of PlgT/T and Pros1E/E did not affect pulmonary embolism symptoms, compared with those in Pros1E/E mice. Hence, the present study showed that the Plg-A622T mutation, which confers ~8% plasmin activity, does not increase the risk of thrombotic diseases in mice under experimental thrombotic conditions and does not modify the thrombotic phenotype observed in Pros1E/E mice. PlgT/T mice can be used to investigate the potential pathophysiological impact of the Plg-A620T mutation.


Assuntos
Conjuntivite/genética , Técnicas de Introdução de Genes , Mutação , Fenótipo , Plasminogênio/deficiência , Plasminogênio/genética , Dermatopatias Genéticas/genética , Tromboembolia Venosa/genética , Substituição de Aminoácidos , Animais , Isquemia Encefálica/sangue , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Conjuntivite/sangue , Conjuntivite/patologia , Modelos Animais de Doenças , Feminino , Fibrina/genética , Fibrina/metabolismo , Fibrinolisina/genética , Fibrinolisina/metabolismo , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Plasminogênio/metabolismo , Proteína S/genética , Proteína S/metabolismo , Embolia Pulmonar/sangue , Embolia Pulmonar/genética , Embolia Pulmonar/patologia , Dermatopatias Genéticas/sangue , Dermatopatias Genéticas/patologia , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Tromboembolia Venosa/sangue , Tromboembolia Venosa/patologia , Trombose Venosa/sangue , Trombose Venosa/genética , Trombose Venosa/patologia , Cicatrização/fisiologia
4.
J Innate Immun ; 9(2): 217-228, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28068663

RESUMO

Collectins are C-type lectins that are involved in innate immunity as pattern recognition molecules. Recently, collectin kidney 1 (CL-K1) has been discovered, and in vitro studies have shown that CL-K1 binds to microbes and activates the lectin complement pathway. However, in vivo functions of CL-K1 against microbes have not been elucidated. To investigate the biological functions of CL-K1, we generated CL-K1 knockout (CL-K1-/-) mice and then performed a Streptococcus pneumoniae infection analysis. First, we found that recombinant human CL-K1 bound to S. pneumoniae in a calcium-dependent manner, and induced complement activation. CL-K1-/- mice sera formed less C3 deposition on S. pneumoniae. Furthermore, immunofluorescence analysis in the wild-type (WT) mice demonstrated that CL-K1 and C3 were localized on S. pneumoniae in infected lungs. CL-K1-/- mice revealed decreased phagocytosis of S. pneumoniae. Consequently, less S. pneumoniae clearance was observed in their lungs. CL-K1-/- mice showed severe pulmonary inflammation and weight loss in comparison with WT mice. Finally, the decreased clearance and severe pulmonary inflammation caused by S. pneumoniae infection might cause higher CL-K1-/- mice lethality. Our results suggest that CL-K1 might play an important role in host protection against S. pneumoniae infection through the activation of the lectin complement pathway.


Assuntos
Colectinas/metabolismo , Complemento C3/metabolismo , Pulmão/imunologia , Infecções Pneumocócicas/imunologia , Pneumonia/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Streptococcus pneumoniae/fisiologia , Animais , Carga Bacteriana , Colectinas/genética , Lectina de Ligação a Manose da Via do Complemento/genética , Humanos , Imunidade Inata , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Receptores de Reconhecimento de Padrão/genética , Transgenes/genética
5.
Biochim Biophys Acta Gen Subj ; 1861(2): 1-14, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27864148

RESUMO

BACKGROUND: Pentraxins (PTXs) are a superfamily of multifunctional conserved proteins involved in acute-phase responses. Recently, we have shown that collectin placenta 1 (CL-P1) and C-reactive protein (CRP) mediated complement activation and failed to form terminal complement complex (TCC) in normal serum conditions because of complement factor H inhibition. METHODS: We used CL-P1 expressing CHO/ldlA7 cells to study the interaction with PTXs. Soluble type CL-P1 was used in an ELISA assay for the binding, C3 and TCC deposition experiments. Furthermore, we used our previously established CL-P1 expressing HEK293 cells for the C3 fragment and TCC deposition assay. RESULTS: We demonstrated that CL-P1 also bound serum amyloid p component (SAP) and pentraxin 3 (PTX3) to activate the classical pathway and the alternative pathway using factor B. CRP and PTX3 further amplified complement deposition by properdin. We found that CRP and PTX3 recruit CFH, whereas SAP recruits C4 binding protein on CL-P1 expressing cell surfaces to prevent the formation of TCC in normal serum conditions. In addition, depletion of CFH, C4BP and complement factor I (CFI) failed to prevent TCC formation both in ELISA and cell experiments. Furthermore, soluble complement receptor 1, an inhibitor of all complement pathways prevents PTX induced TCC formation. CONCLUSION: Our current study hypothesizes that the interaction of pentraxins with CL-P1 is involved in complement activation. GENERAL SIGNIFICANCE: CL-P1 might generally inhibit PTX induced complement activation and host damage to protect self-tissues.


Assuntos
Proteína C-Reativa/metabolismo , Colectinas/metabolismo , Ativação do Complemento/fisiologia , Componente Amiloide P Sérico/metabolismo , Reação de Fase Aguda/metabolismo , Animais , Células CHO , Linhagem Celular , Fator H do Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/fisiologia , Cricetulus , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia
6.
Biochim Biophys Acta ; 1860(6): 1118-28, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26922829

RESUMO

BACKGROUND: C-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown. METHODS: We prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1. RESULTS: Here, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions. GENERAL SIGNIFICANCE: The interaction of collectin CL-P1 with CFH might be key for preventing attack on "self" as a result of complement activation induced by the CL-P1 and CRP interaction.


Assuntos
Proteína C-Reativa/química , Colectinas/química , Ativação do Complemento , Receptores Depuradores/química , Animais , Proteína C-Reativa/fisiologia , Células CHO , Colectinas/fisiologia , Fator H do Complemento/química , Cricetulus , Células HEK293 , Humanos , Receptores Depuradores/fisiologia
7.
Biochim Biophys Acta ; 1840(12): 3345-56, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25199873

RESUMO

BACKGROUND: Collectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD. METHODS: We constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively. RESULTS: CL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL. CONCLUSIONS: CL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL. GENERAL SIGNIFICANCE: We considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body.

8.
Dev Comp Immunol ; 35(1): 51-61, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20713082

RESUMO

Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30kDa protein from hemocytes of the freshwater crayfish, Pacifastacus leniusculus, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type peptidoglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the Pl-SPH1 and lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two Pl-SPHs are involved in this activation possibly by forming a complex with LGBP and without a PGRP.


Assuntos
Proteínas de Transporte/imunologia , Catecol Oxidase/imunologia , Precursores Enzimáticos/imunologia , Imunidade Inata , Peptidoglicano/metabolismo , Serina Proteases/genética , Serina Proteases/imunologia , Sequência de Aminoácidos , Animais , Astacoidea/classificação , Astacoidea/genética , Astacoidea/imunologia , Baculoviridae/genética , Sequência de Bases , Células Cultivadas , Regulação da Expressão Gênica , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/genética , Serina Proteases/química , Regulação para Cima
9.
Plant Mol Biol ; 72(1-2): 137-52, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19851874

RESUMO

The control of seed germination under environmental conditions, where plants will be grown, is important for the adaptability of plants. Low-temperature is one of the most common environmental stress factors that affect plant growth and development and places a major limit on crop productivity in cultivated areas. Previously, qLTG3-1, a major quantitative trait locus controlling low-temperature tolerance at the germination stage in rice (called low-temperature germinability) was identified, which encodes a protein of unknown function. To identify genes targeted by qLTG3-1, a genome-wide expression profiling analysis using the 44 K Rice Oligo microarray was performed. Because the expression of qLTG3-1 was dramatically increased at 1 day after incubation, the expression profiles at this time were compared between Hayamasari, which has a loss-of-function qLTG3-1 allele, and a near isogenic line with a functional allele. A total of 4,587 genes showed significant differences between their expression levels in the two lines. Most of these genes might be involved in the process of seed germination itself, and then a focus was made on qLTG3-1 dependently induced or suppressed genes, defined as 'qLTG3-1 dependent' genes. Twenty-nine 'qLTG3-1 dependent' genes with diverse functions were categorized, implying that disruption of cellular homeostasis leads to a wide range of metabolic alterations and diverse cross-talk between various signaling pathways. In particular, genes involved in defense responses were up-regulated by qLTG3-1, indicating that qLTG3-1 expression is required for the expression of defense response genes in low-temperature germinability in rice.


Assuntos
Temperatura Baixa , Genoma de Planta/fisiologia , Germinação/fisiologia , Oryza/genética , Oryza/fisiologia , Genoma de Planta/genética , Germinação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Locos de Características Quantitativas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/genética , Sementes/fisiologia
10.
J Plant Physiol ; 166(14): 1537-43, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19410335

RESUMO

The rice Dart/nDart transposon system belongs to the hAT superfamily of class II transposons. The nonautonomous element nDart is active in intact rice plants. The autonomous element Dart was identified based on sequence similarity to nDart. Because the rice genome sequence of Nipponbare contains at least 51 Dart elements, it is not clear whether Dart elements are expressed or whether they are transposable. This study characterized for the expression of the predicted ORF of Dart. RNA blotting analysis revealed three transcripts of different lengths. Only the longest transcript (2.5kb) corresponding to the predicted ORF of the Dart element produced a functional TPase. The other transcripts had a frame-shift generating a premature stop codon through alternative splicing. These transcripts were expressed from either of two potentially autonomous Dart elements, Dart01/28 and Dart02. The frequency of alternative splicing differed between the transcripts of the derivative elements. More than 90% of the transcripts from Dart02 were alternatively spliced, compared with only 3% from Dart01/28. The element-specific expression and alternative splicing may control the transposition of nDart.


Assuntos
Elementos de DNA Transponíveis/genética , Oryza/genética , Transcrição Genética/genética , Processamento Alternativo/genética , Northern Blotting , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Proc Natl Acad Sci U S A ; 105(34): 12623-8, 2008 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-18719107

RESUMO

Tolerance to abiotic stress is an important agronomic trait in crops and is controlled by many genes, which are called quantitative trait loci (QTLs). Identification of these QTLs will contribute not only to the understanding of plant biology but also for plant breeding, to achieve stable crop production around the world. Previously, we mapped three QTLs controlling low-temperature tolerance at the germination stage (called low-temperature germinability). To understand the molecular basis of one of these QTLs, qLTG3-1 (quantitative trait locus for low-temperature germinability on chromosome 3), map-based cloning was performed, and this QTL was shown to be encoded by a protein of unknown function. The QTL qLTG3-1 is strongly expressed in the embryo during seed germination. Before and during seed germination, specific localization of beta-glucuronidase staining in the tissues covering the embryo, which involved the epiblast covering the coleoptile and the aleurone layer of the seed coat, was observed. Expression of qLTG3-1 was tightly associated with vacuolation of the tissues covering the embryo. This may cause tissue weakening, resulting in reduction of the mechanical resistance to the growth potential of the coleoptile. These phenomena are quite similar to the model system of seed germination presented by dicot plants, suggesting that this model may be conserved in both dicot and monocot plants.


Assuntos
Aclimatação/genética , Temperatura Baixa , Germinação/genética , Oryza/genética , Locos de Características Quantitativas/genética , Mapeamento Cromossômico , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Dados de Sequência Molecular , Oryza/fisiologia , Locos de Características Quantitativas/fisiologia
12.
Mol Genet Genomics ; 279(5): 499-507, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18293011

RESUMO

Elucidation of the genetic basis of the control of leaf shape could be of use in the manipulation of crop traits, leading to more stable and increased crop production. To improve our understanding of the process controlling leaf shape, we identified a mutant gene in rice that causes a significant decrease in the width of the leaf blade, termed narrow leaf 7 (nal7). This spontaneous mutation of nal7 occurred during the process of developing advanced back-crossed progeny derived from crosses of rice varieties with wild type leaf phenotype. While the mutation resulted in reduced leaf width, no significant morphological changes at the cellular level in leaves were observed, except in bulli-form cells. The NAL7 locus encodes a flavin-containing monooxygenase, which displays sequence homology with YUCCA. Inspection of a structural model of NAL7 suggests that the mutation results in an inactive enzyme. The IAA content in the nal7 mutant was altered compared with that of wild type. The nal7 mutant overexpressing NAL7 cDNA exhibited overgrowth and abnormal morphology of the root, which was likely to be due to auxin overproduction. These results indicate that NAL7 is involved in auxin biosynthesis.


Assuntos
Genes de Plantas/fisiologia , Ácidos Indolacéticos/farmacologia , Oryza/genética , Folhas de Planta/anatomia & histologia , Folhas de Planta/efeitos dos fármacos , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Tamanho do Órgão/efeitos dos fármacos , Oryza/anatomia & histologia , Oryza/efeitos dos fármacos , Oryza/metabolismo
13.
J Biol Chem ; 282(52): 37316-24, 2007 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-17967901

RESUMO

In mammals, the cornified cell envelope forms beneath the plasma membrane in epithelia and provides a vital physical barrier consisting of insoluble proteins cross-linked by transglutaminase (TGase). In the horseshoe crab Tachypleus tridentatus, TGase is stored in hemocytes and secreted in response to the simulation of bacterial lipopolysaccharides. Here we characterized a TGase substrate designated as caraxin that was identified in horseshoe crab cuticle. One of the homologs, caraxin-1, possessed a unique domain structure consisting of N-and C-terminal heptad repeats and a central domain with a tandem-repeated structure of a pentapeptide. Western blotting showed the specific localization of caraxin-1 in sub-cuticular epidermis. Moreover, we identified the pentapeptide motif to be a chitin-binding unit. Analytical ultracentrifugation revealed that caraxin-1 exists as an oligomer with 310-350 kDa, which is approximately 20-mer based on the molecular mass of the monomer. The oligomers were cross-linked by TGase to form an elaborate mesh with honeycomb structures, which was electron-microscopically found to be different from the clotting mesh triggered by lipopolysaccharide-induced hemocyte exocytosis. We determined several cross-linking sites in the N-and C-terminal domains of caraxin-1. The replacements of Leu to Pro at positions 36 and 118 in caraxin-1 reduced the alpha-helix content, which destroyed the TGase-dependent mesh, thus indicating the importance of the N-and C-terminal domains for the proper mesh formation. In arthropods, TGase-dependent protein cross-linking may be involved in the initial stage of host defense at the sub-cuticular epidermis, as in the case of mammalian skin.


Assuntos
Quitina/química , Transglutaminases/química , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Dicroísmo Circular , Exocitose , Hemócitos/metabolismo , Caranguejos Ferradura/metabolismo , Cinética , Microscopia Eletrônica de Varredura , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Ultracentrifugação
14.
J Biol Chem ; 282(46): 33545-52, 2007 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-17855345

RESUMO

Hemolymph coagulation in arthropods plays key roles in host defense, including sealing wounds to staunch bleeding and immobilizing invading microorganisms. We have previously reported that horseshoe crab transglutaminase (TGase) promotes cross-linking of a clotting protein (coagulin) with hemocyte-derived proteins (proxins), resulting in the formation of stable coagulin fibrils. Here we show that TGase also cross-links proxins to another hemocyte-derived protein named stablin. Stablin is a cysteine-rich protein of 131 residues. Surface plasmon resonance analysis revealed the specific interaction of stablin with proxin-1 at K(d) = 4.0 x 10(-9) m. Stablin was predominantly localized in the large granules of hemocytes and secreted by lipopolysaccharide-induced exocytosis. Interestingly, stablin bound to chitin at K(d) = 1.5 x 10(-8) m, as determined by using a quartz-crystal microbalance. Stablin also interacted with lipopolysaccharides and lipoteichoic acids and exhibited bacterial agglutinating activity against Gram-positive and -negative bacteria. Immunostaining showed that stablin is co-localized with coagulin in the clotting fibrils that effectively trap bacteria. Moreover, an anti-stablin antibody strongly inhibited the proper formation of the clotting fibrils. These data suggest that stablin promotes the formation of the clotting mesh and the immobilization of invading microbes at injury sites. In arthropods, the TGase-mediated cross-linking may play an important role in the initial stage of host defense, wound closure, and healing, as in the case of mammals.


Assuntos
Cisteína/química , Cicatrização , Sequência de Aminoácidos , Animais , Sequência de Bases , Reagentes para Ligações Cruzadas/farmacologia , Hemócitos/metabolismo , Hemolinfa/metabolismo , Caranguejos Ferradura , Cinética , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Ácidos Teicoicos/química , Transglutaminases/metabolismo
15.
FEBS J ; 272(18): 4774-86, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16156796

RESUMO

Arthropod cuticles play an important role as the first barrier against invading pathogens. We extensively determined the sequences of horseshoe crab cuticular proteins. Proteins extracted from a part of the ventral side of the cuticle were purified by chitin-affinity chromatography, and separated by two-dimensional SDS/PAGE. Proteins appearing on the gel were designated high molecular mass chitin-binding proteins, and these proteins were then grouped into classes based on their approximate isoelectric points and predominant amino acid compositions. Members of groups designated basic G, basic Y, and acidic S groups contained a so-called Rebers and Riddiford consensus found in arthropod cuticular proteins. Proteins designated acidic DE25 and DE29 each contained a Cys-rich domain with sequences similar to those of insect peritrophic matrix proteins and chitinases. In contrast, basic QH4 and QH10 contained no consensus sequences found in known chitin-binding proteins. Alternatively, a low molecular mass chitin-binding fraction was prepared by size exclusion chromatography, and 15 low molecular mass chitin-binding proteins, named P1 through P15, were isolated. With the exception of P9 and P15, all were found to be identical to known antimicrobial peptides. P9 consisted of a Kunitz-type chymotrypsin inhibitor sequence, and P15 contained a Cys-rich motif found in insulin-like growth factor-binding proteins. Interestingly, we observed transglutaminase-dependent polymerization of nearly all high molecular mass chitin-binding proteins, a finding suggests that transglutaminase-dependent cross-linking plays an important role in host defense in the arthropod cuticle, analogous to that observed in the epidermal cornified cell envelope in mammals.


Assuntos
Quitina/metabolismo , Proteínas/isolamento & purificação , Transglutaminases/metabolismo , Animais , Sequência de Bases , Quitinases , Cromatografia de Afinidade , Proteínas da Matriz Extracelular , Caranguejos Ferradura , Sistema Imunitário/química , Imunidade Inata , Dados de Sequência Molecular , Peso Molecular , Proteínas/classificação , Análise de Sequência de Proteína
16.
FEBS Lett ; 579(11): 2499-506, 2005 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-15848195

RESUMO

We describe the regulation mechanisms of the Na(+)-dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco-2. Exposure of Caco-2 cells to S-nitrosothiol, such as S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione, and the NO-donor, NOC12, concentration- and time-dependently increased Na(+)-dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (V(max)) of Na(+)-dependent alanine uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na(+)-dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) and N-(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT-PCR revealed that Caco-2 cells expressed the Na(+)-dependent neutral amino acid transporter ASCT2, but not the other Na(+)-dependent neutral amino acid transporters ATB(0,+) and B(0)AT1. These results suggested that functional up-regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis.


Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , S-Nitrosotióis/farmacologia , Sódio/farmacologia , Alanina/metabolismo , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Humanos , Cinética , Antígenos de Histocompatibilidade Menor , Doadores de Óxido Nítrico/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacologia , S-Nitrosotióis/metabolismo , Regulação para Cima/efeitos dos fármacos
17.
J Pharm Sci ; 93(4): 877-85, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14999725

RESUMO

Effects of various surfactants on the transport of rhodamine123, a P-glycoprotein (P-gp) substrate, across the isolated rat intestinal membranes were examined by an in vitro diffusion chamber system. The jejunal serosal-to-mucosal transport (Jsm) of rhodamine123 was more than threefold greater than its mucosal-to-serosal transport (Jms), suggesting that the net movement of rhodamine123 across the rat jejunum was preferentially secretory direction. There exists a regional difference in the intestinal transport of rhodamine123 and the secretory directed transport was remarkably observed in the jejunum. The Jsm/Jms ratio of rhodamine123 decreased in the presence of 0.3 mM verapamil and 10 mM sodium azide (NaN3) + 1 mM sodium fluoride (NaF), confirming that rhodamine123 might be secreted from the intestinal tissue into the lumen by a P-gp-mediated efflux system. Nonionic surfactants [0.1% Cremophor EL, Tween 80 and n-dodecyl-beta-D-maltopyranoside (LM)] reduced the Jsm/Jms ratio of rhodamine123, whereas its ratio was not influenced in the presence of 0.1% cationic surfactant (hexadecyltrimethylammonium bromide, C16TAB) and anionic surfactant (sodium dodecyl sulfate, SDS). Therefore, these findings suggested that charge of surfactants was possibly related to the action of these surfactants on the intestinal absorption of P-gp substrates. On the other hand, the transfer of rhodamine123 was not affected by the addition of Cremophor EL to the serosal side. Because the c.m.c. of Cremophor EL is 0.0095 w/v%, interactions between rhodamine123 and the micellar form of Cremophor EL may decrease the P-gp-mediated efflux of rhodamine123 at higher concentrations. In the kinetic analysis, the Vmax value (nmol/min/g wet tissue) of rhodamine123 decreased, although the Km value (mM) was constant in the presence of Cremophor EL. Therefore, Cremophor EL inhibited the efflux transport of rhodamine123 in a noncompetitive manner. Cremophor EL did not affect the transport of [14C]Gly-Sar and [3H]3-O-methyl-D-glucose, suggesting that the action of Cremophor EL might be P-gp specific. These findings indicated that nonionic surfactants including Cremophor EL and Tween 80 may be useful pharmaceutical excipients for inhibiting the function of P-gp, thereby increasing the intestinal absorption of various drugs, which are secreted by a P-gp-mediated efflux system in the intestine.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Glicerol/análogos & derivados , Glicerol/farmacologia , Mucosa Intestinal/metabolismo , Tensoativos/farmacologia , 3-O-Metilglucose/farmacocinética , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Diálise , Cultura em Câmaras de Difusão , Dipeptídeos/farmacocinética , Corantes Fluorescentes , Técnicas In Vitro , Absorção Intestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Membranas/metabolismo , Micelas , Ratos , Ratos Wistar , Rodamina 123/farmacocinética
18.
Phys Rev Lett ; 90(24): 243401, 2003 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-12857191

RESUMO

For the first time, we observed the dependence of the ddmu formation rate and the dmu hyperfine-transition rate on the ortho-para state in muon-catalyzed fusion in the solid D2 state, and found that the effect is even opposite to a recent theoretical prediction. We also determined the back-decay rate and the hyperfine-transition rate via scattering in solid state by using the ortho-para dependence. A theory to describe properly our experimental result is called for to understand the nature of muon-catalyzed fusion in the solid state.

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