RESUMO
OBJECTIVE: To determine the microRNA (miR) signature in ankylosing spondylitis (AS) T helper (Th)17 cells. METHODS: Interleukin (IL)-17A-producing CD4+ T cells from patients with AS and healthy controls were FACS-sorted for miR sequencing and qPCR validation. miR-10b function was determined by miR mimic expression followed by cytokine measurement, transcriptome analysis, qPCR and luciferase assays. RESULTS: AS Th17 cells exhibited a miR signature characterised by upregulation of miR-155-5p, miR-210-3p and miR-10b. miR-10b has not been described previously in Th17 cells and was selected for further characterisation. miR-10b is transiently induced in in vitro differentiated Th17 cells. Transcriptome, qPCR and luciferase assays suggest that MAP3K7 is targeted by miR-10b. Both miR-10b overexpression and MAP3K7 silencing inhibited production of IL-17A by both total CD4 and differentiating Th17 cells. CONCLUSIONS: AS Th17 cells have a specific miR signature and upregulate miR-10b in vitro. Our data suggest that miR-10b is upregulated by proinflammatory cytokines and may act as a feedback loop to suppress IL-17A by targeting MAP3K7. miR-10b is a potential therapeutic candidate to suppress pathogenic Th17 cell function in patients with AS.
Assuntos
Interleucina-17/biossíntese , MAP Quinase Quinase Quinases/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Células Th17/metabolismo , Regulação para Cima , Adulto , Idoso , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Feminino , Inativação Gênica , Humanos , Interleucina-6/farmacologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Espondilite Anquilosante , Transcriptoma/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adulto JovemRESUMO
To assess the diagnostic potential of multi-slice single-photon emission computed tomography/computed tomography (SPECT/CT) for preoperative sentinel node (SN) mapping in early stage head and neck squamous cell carcinoma (HNSCC). Retrospective case-control study including data of consecutive HNSCC patients treated between November 2011 and December 2015. The diagnostic accuracy of multi-slice SPECT/CT was assessed with regard to the gold standard intraoperative gamma ray detection probe, using McNemar's test and calculating the area under the ROC curve. Additionally, the hot spot yield of SPECT/CT and planar lymphoscintigraphy (LS) was compared. Compared to the intraoperative gold standard, SPECT/CT showed an overall positive predictive value of 60.3% [confidence interval (CI) 46.6-73.0%)], a negative predictive value of 96.3% (CI 93.6-98.1%), and an accuracy of 90.8% (CI 89.1-92.4%). SPECT/CT detected more hot spots than LS and provided detailed anatomical information as well as relevant additional findings with potential impact on further patient management. Sentinel lymph node biopsy proved to be a reliable and safe procedure with an excellent SN excision rate (97%). Multi-slice SPECT/CT is a highly accurate diagnostic test and matches the gold standard intraoperative gamma ray detection probe.
Assuntos
Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/patologia , Linfocintigrafia , Biópsia de Linfonodo Sentinela/métodos , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Curva ROC , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)-E. As HLA-E is also over-expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA-E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV-seropositive donors than seronegative donors and was associated strongly with target cell HLA-E and NK cell NKG2C expression. NK cell cytotoxicity against HLA-E transfected lymphoma target cells (221.AEH) was â¼threefold higher with CMV, while NK cell cytotoxicity against non-transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a(+) ) and interferon (IFN)-γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)-15 were found to expand NKG2C(+) /NKG2A(-) NK cells preferentially from CMV-seronegative donors and increase NK cell cytotoxicity against HLA-E(+) tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C(+) NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA-E expression.
Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/análise , Latência Viral , Adolescente , Adulto , Antígenos CD57/imunologia , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade , Feminino , Voluntários Saudáveis , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células K562 , Ativação Linfocitária , Linfoma/imunologia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Adulto Jovem , Antígenos HLA-ERESUMO
A tailored-pulse-imploded core with a diameter of 70 µm is flashed by counterirradiating 110 fs, 7 TW laser pulses. Photon emission (>40 eV) from the core exceeds the emission from the imploded core by 6 times, even though the heating pulse energies are only one seventh of the implosion energy. The coupling efficiency from the heating laser to the core using counterirradiation is 14% from the enhancement of photon emission. Neutrons are also produced by counterpropagating fast deuterons accelerated by the photon pressure of the heating pulses. A collisional two-dimensional particle-in-cell simulation reveals that the collisionless two counterpropagating fast-electron currents induce mega-Gauss magnetic filaments in the center of the core due to the Weibel instability. The counterpropagating fast-electron currents are absolutely unstable and independent of the core density and resistivity. Fast electrons with energy below a few MeV are trapped by these filaments in the core region, inducing an additional coupling. This might lead to the observed bright photon emissions.
RESUMO
A novel direct core heating fusion process is introduced, in which a preimploded core is predominantly heated by energetic ions driven by LFEX, an extremely energetic ultrashort pulse laser. Consequently, we have observed the D(d,n)^{3}He-reacted neutrons (DD beam-fusion neutrons) with the yield of 5×10^{8} n/4π sr. Examination of the beam-fusion neutrons verified that the ions directly collide with the core plasma. While the hot electrons heat the whole core volume, the energetic ions deposit their energies locally in the core, forming hot spots for fuel ignition. As evidenced in the spectrum, the process simultaneously excited thermal neutrons with the yield of 6×10^{7} n/4π sr, raising the local core temperature from 0.8 to 1.8 keV. A one-dimensional hydrocode STAR 1D explains the shell implosion dynamics including the beam fusion and thermal fusion initiated by fast deuterons and carbon ions. A two-dimensional collisional particle-in-cell code predicts the core heating due to resistive processes driven by hot electrons, and also the generation of fast ions, which could be an additional heating source when they reach the core. Since the core density is limited to 2 g/cm^{3} in the current experiment, neither hot electrons nor fast ions can efficiently deposit their energy and the neutron yield remains low. In future work, we will achieve the higher core density (>10 g/cm^{3}); then hot electrons could contribute more to the core heating via drag heating. Together with hot electrons, the ion contribution to fast ignition is indispensable for realizing high-gain fusion. By virtue of its core heating and ignition, the proposed scheme can potentially achieve high gain fusion.
RESUMO
A compact fast core heating experiment is described. A 4-J 0.4-ns output of a laser-diode-pumped high-repetition laser HAMA is divided into four beams, two of which counterilluminate double-deuterated polystyrene foils separated by 100 µm for implosion. The remaining two beams, compressed to 110 fs for fast heating, illuminate the same paths. Hot electrons produced by the heating pulses heat the imploded core, emitting x-ray radiations >20 eV and yielding some 10(3) thermal neutrons.
RESUMO
BACKGROUND AND PURPOSE: The hemodynamics associated with cerebral AVMs have a significant impact on their clinical presentation. This study aimed to evaluate the hemodynamic features of AVMs using 3D phase-contrast MR imaging with dual velocity-encodings. MATERIALS AND METHODS: Thirty-two patients with supratentorial AVMs who had not received any previous treatment and had undergone 3D phase-contrast MR imaging were included in this study. The nidus diameter and volume were measured for classification of AVMs (small, medium, or large). Flow parameters measured included apparent AVM inflow, AVM inflow index, apparent AVM outflow, AVM outflow index, and the apparent AVM inflow-to-outflow ratio. Correlation coefficients between the nidus volume and each flow were calculated. The flow parameters between small and other AVMs as well as between nonhemorrhagic and hemorrhagic AVMs were compared. RESULTS: Patients were divided into hemorrhagic (n = 8) and nonhemorrhagic (n = 24) groups. The correlation coefficient between the nidus volume and the apparent AVM inflow and outflow was .83. The apparent AVM inflow and outflow in small AVMs were significantly smaller than in medium AVMs (P < .001 for both groups). The apparent AVM inflow-to-outflow ratio was significantly larger in the hemorrhagic AVMs than in the nonhemorrhagic AVMs (P = .02). CONCLUSIONS: The apparent AVM inflow-to-outflow ratio was the only significant parameter that differed between nonhemorrhagic and hemorrhagic AVMs, suggesting that a poor drainage system may increase AVM pressure, potentially causing cerebral hemorrhage.
Assuntos
Malformações Arteriovenosas Intracranianas , Hemorragia Cerebral , Hemodinâmica , Humanos , Imageamento Tridimensional , Malformações Arteriovenosas Intracranianas/diagnóstico por imagem , Imageamento por Ressonância MagnéticaRESUMO
Rotavirus (RV) is a common pathogen that causes acute gastroenteritis in childhood. Some cases with RV infection also have prerenal renal failure induced by dehydration associated with vomiting and diarrhea. Here, we report 4 patients with RV infection who developed postrenal renal failure induced by urinary tract obstruction with uroammoniac calculi or crystals. The patients did not have metabolic disorders or abnormalities of the urinary tract, and increased urinary excretion of uric acid was not recognized at discharge. In addition, no abnormalities in the uric acid transporter (URAT1) were found in any of the patients. Uric acid stone formation was considered to have originated from the low pH caused by dehydration and the increase of urinary uric acid excretion from damaged cells.
Assuntos
Injúria Renal Aguda/virologia , Gastroenterite/complicações , Cálculos Renais/etiologia , Infecções por Rotavirus/complicações , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/terapia , Feminino , Gastroenterite/terapia , Gastroenterite/virologia , Humanos , Lactente , Cálculos Renais/diagnóstico , Cálculos Renais/terapia , Masculino , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/terapiaRESUMO
Iron is one of the most studied chemical elements due to its sociotechnological and planetary importance; hence, understanding its structural transition dynamics is of vital interest. By combining a short pulse optical laser and an ultrashort free electron laser pulse, we have observed the subnanosecond structural dynamics of iron from high-quality x-ray diffraction data measured at 50-ps intervals up to 2500 ps. We unequivocally identify a three-wave structure during the initial compression and a two-wave structure during the decaying shock, involving all of the known structural types of iron (α-, γ-, and ε-phase). In the final stage, negative lattice pressures are generated by the propagation of rarefaction waves, leading to the formation of expanded phases and the recovery of γ-phase. Our observations demonstrate the unique capability of measuring the atomistic evolution during the entire lattice compression and release processes at unprecedented time and strain rate.
RESUMO
BACKGROUND: Bartter syndrome (BS) is a genetic disorder accompanied by hypokalaemic metabolic alkalosis. BS with sensorineural deafness (SND, OMIM602522) is a newly identified phenotype caused by mutations in the BSND gene that encodes barttin, a beta-subunit for chloride channel ClC-Ka and ClC-Kb and classified as type IV BS. Type IV BS features the most severe phenotype entailing life-threatening neonatal volume depletion and chronic renal failure developing during infancy. A recent report described a case of BS with SND from a consanguineous family who showed homozygous mutations in the CLCNKA and CLCNKB genes. This case indicated the possibility of the occurrence of digenic inheritance in BS with SND resulting from double mutations in the CLCNKA and CLCNKB genes. SUBJECT AND RESULTS: The current report concerns a 2-year-old girl from a non-consanguineous family with BS accompanied by SND. In our case, four loss-of-function mutations, consisting of mutations in both parental alleles in both CLCNKA and CLCNKB, were identified. The paternal allele had a nonsense mutation (Q260X) in CLCNKA and a splicing site mutation (IVS17+1 g>a) in CLCNKB. The maternal allele had a large deletion mutation (about 12 kbp) extending from CLCNKA to CLCNKB. Our case provides clear evidence that loss-of-function alleles in both alleles of both CLCNKA and CLCNKB results in a phenotype indistinguishable from that of mutations in BSND (type IV BS). CONCLUSIONS: Recent advances in genetics have resulted in a better understanding of many human inherited diseases, but most of them are monogenic disorders and more complex inheritance patterns remain unresolved. Our case provides clear evidence of digenic inheritance outside the scope of Mendelian inheritance disorders.
Assuntos
Síndrome de Bartter/complicações , Síndrome de Bartter/genética , Canais de Cloreto/genética , Perda Auditiva Neurossensorial/complicações , Perda Auditiva Neurossensorial/genética , Mutação , Alelos , Sequência de Bases , Pré-Escolar , Códon sem Sentido , DNA/genética , Análise Mutacional de DNA , Primers do DNA/genética , Feminino , Heterozigoto , Humanos , Masculino , Fenótipo , Sítios de Splice de RNA , Deleção de SequênciaAssuntos
Acidose Tubular Renal/genética , Proteínas de Transporte de Ânions , Antiporters , Proteínas de Transporte/genética , Anormalidades do Olho/genética , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutação , Acidose Tubular Renal/patologia , Acidose Tubular Renal/fisiopatologia , Adolescente , Adulto , Substituição de Aminoácidos , Cegueira , Proteínas de Transporte/fisiologia , Pré-Escolar , Cromossomos Humanos Par 4/genética , Consanguinidade , Análise Mutacional de DNA , Anormalidades do Olho/fisiopatologia , Feminino , Homozigoto , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Proteínas SLC4A , Simportadores de Sódio-BicarbonatoRESUMO
To understand the effect of trisomic chromosome 21 on the cause of Down syndrome (DS), DNA methylation in the CpG island, which regulates the expression of adjacent genes, was investigated with the DNAs of chromosome 21 isolated from DS patients and their parents. A methylation-sensitive enzyme, BssHII, was used to digest DNAs of chromosome 21, and the resulting DNA fragments were subjected to RLGS (restriction landmark genomic scanning). Surprisingly, the CpG island of the h2-calponin gene was shown to be specifically methylated by comparative studies with RLGS and Southern blot analysis. In association with this methylation, h2-calponin gene expression was attenuated to the normal level, although other genes in the DS region of chromosome 21 were expressed dose dependently at 1.5 times the normal level. These results and the high miscarriage rate associated with trisomy 21 embryos imply that the altered in vivo methylation that attenuates downstream gene expression, which is otherwise lethal, permits the generation of DS neonates. The h2-calponin gene detected by the RLGS procedure may be one such gene that is attenuated.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Ilhas de CpG/genética , Metilação de DNA , Síndrome de Down/genética , Regulação da Expressão Gênica/genética , Sequência de Bases , Cromossomos Humanos Par 21/genética , Clonagem Molecular , DNA-Citosina Metilases , Humanos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Mapeamento por Restrição/métodos , Transcrição Gênica , CalponinasRESUMO
A 54-year-old man was pointed out a mass shadow during treatment of suspected acute pyothorax. He was diagnosed as large cell carcinoma by transbronchial biopsy. Preoperative chemotherapy and right lower lobectomy were done. In pathological examination, he was diagnosed as pulmonary pleomorpic carcinoma. Postoperatively, he had rapid metastases of the pancreas, skin, muscle, brain and oral cavity. We reported a case of pulmonary pleomorphic carcinoma with rapid and peculiar metastases.
Assuntos
Carcinoma de Células Grandes/secundário , Neoplasias Pulmonares/patologia , Linfonodos/patologia , Pneumonectomia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/secundário , Carcinoma de Células Grandes/tratamento farmacológico , Carcinoma de Células Grandes/cirurgia , Cisplatino/administração & dosagem , Terapia Combinada , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/cirurgia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Musculares/secundário , Paclitaxel/administração & dosagem , Neoplasias Pancreáticas/secundário , Neoplasias Cutâneas/secundárioRESUMO
Natural killer (NK) cells are key components of the innate immune system, providing potent antitumor immunity. Here, we show that the tumor growth factor-ß (TGF-ß)/SMAD signaling pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia (ALL). We characterized NK cells in 50 consecutive children with B-ALL at diagnosis, end induction and during maintenance therapy compared with age-matched controls. ALL-NK cells at diagnosis had an inhibitory phenotype associated with impaired function, most notably interferon-γ production and cytotoxicity. By maintenance therapy, these phenotypic and functional abnormalities partially normalized; however, cytotoxicity against autologous blasts remained impaired. We identified ALL-derived TGF-ß1 to be an important mediator of leukemia-induced NK cell dysfunction. The TGF-ß/SMAD signaling pathway was constitutively activated in ALL-NK cells at diagnosis and end induction when compared with healthy controls and patients during maintenance therapy. Culture of ALL blasts with healthy NK cells induced NK dysfunction and an inhibitory phenotype, mediated by activation of the TGF-ß/SMAD signaling pathway, and abrogated by blocking TGF-ß. These data indicate that by regulating the TGF-ß/SMAD pathway, ALL blasts induce changes in NK cells to evade innate immune surveillance, thus highlighting the importance of developing novel therapies to target this inhibitory pathway and restore antileukemic cytotoxicity.
Assuntos
Citotoxicidade Imunológica/imunologia , Evasão da Resposta Imune/imunologia , Células Matadoras Naturais/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Lactente , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Masculino , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Transdução de Sinais , Células Tumorais Cultivadas , Microambiente Tumoral/imunologiaRESUMO
Conformational changes associated with the functional states of the molecule of troponin were studied using SH-direct fluorogenic reagents, N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM) and N-(1-anilinonaphthyl-4) maleimide (ANM). 1. The fluorescence parameters of ANM-troponin, intensity, and polarization, did not change on combining it with tropomyosin alone, but markedly changed when F-actin was further added to the system. 2. The conformation around the dye-labeled sulfhydryl group(s) was shown to be susceptible to Ca2+ in terms of fluorescence intensity of the label, thermal transition of the conformation, and the microenvironment near the label. 3. On addition of Ca2+, the fluorescence characteristics of the two systems, ANM-troponin . tropomyosin and ANM-troponin . tropomyosin . F-actin complexes, were altered in opposite directions. When BIPM was used in place of ANM, similar changes were observed: a simple decrease in the intensity when pCa was decreased from 7.4 to 5.5 in the system without F-actin and a sigmoidal increase in the range from pCa 7 to 6 in the system with F-actin. Heavy meromyosin, when added to the latter complex (the reconstituted thin filaments), made the profile of its Ca2+ concentration dependence of fluorescence similar to that of the former complex. When tropomyosin was labeled in place of troponin, similar results were obtained. The data obtained imply that the Ca2+-induced conformational changes of troponin are markedly modified when detached from actin, and that heavy meromyosin weakens the interaction of the troponin . tropomyosin complex with F-actin.
Assuntos
Actinas , Cálcio , Proteínas Musculares , Tropomiosina , Troponina , Animais , Ácido Egtázico , Cinética , Substâncias Macromoleculares , Músculos , Conformação Proteica , Coelhos , Espectrometria de Fluorescência , Compostos de Sulfidrila , Temperatura , ViscosidadeRESUMO
The local conformational changes in the tropomyosin molecule under various conditions were studied by means of fluorimetry using SH-directed fluorescent dyes, N-(1-anilinonaphthyl-4)maleimide (ANM) and N-(3-pyrene)maleimide (PRM). 1. The fluorescence intensity, polarization and the emmission maximum of ANM-tropomyosin were found to be susceptible to ionic strength, but in different ways. The changes in these parameters suggest that the fluorescence-labeled sulfhydryl group or groups become more buried in a hydrophobic internal region by salt-induced depolymerization of aggregate and by adding F-actin to tropomyosin. 2. Titration of the labeled tropomyosin with F-actin revealed a cooperative nature in ANM labeling and a simple saturation kinetics in PRM labeling. The dissociation constant of F-actin to PRM-tropomyosin was calculated to be 5.8-10(-6) M. 3. Temperature dependence of the fluorescence polarization showed a thermal transition in the conformation of ANM- or PRM-tropomyosin at around 30 degrees C. Flexibility or segmental motion of the region containing the fluorophore was suppressed significantly on adding troponin and markedly on adding F-actin. 4. Measurements of the quantum yield and polarization of the ANM-tropomyosin-F-actin complex suggested that troponin strengthened the binding between the two proteins and that Ca2+ reversed this effect.
Assuntos
Actinas , Cálcio , Proteínas Musculares , Tropomiosina , Troponina , Sítios de Ligação , Substâncias Macromoleculares , Maleimidas , Naftalenos , Ligação Proteica , Conformação Proteica , Teoria Quântica , Espectrometria de FluorescênciaRESUMO
Myosin A (Ascaris myosin A) was prepared from the body wall musculature of Ascaris lumbricoides var. suis showing a single peak in analytical ultracentrifuge pattern, and its sedimentation coefficient was about 6 S which was close to the values reported for other myosins. The ATPase activities determined in various media were 3-4 times lower than those of rabbit skeletal myosin A. Nevertheless, the maximum level of the superprecipitation of actomyosin was 4 times higher for Ascaris myosin A than for rabbit skeletal myosin A in the presence of 0.01 mM ATP. It appears that Ascaris myosin A is capable of contracting the actomyosin system in a lower concentration of ATP than that for the usual actomyosin system.
Assuntos
Actomiosina/química , Ascaris lumbricoides/metabolismo , Músculo Esquelético/metabolismo , Músculos/metabolismo , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/metabolismo , Ultracentrifugação/métodos , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Animais , Cálcio/química , Coelhos , Temperatura , Fatores de TempoRESUMO
N-(1-Anilinonaphthyl-4)maleimide (ANM) (m.p. 207-208.5 degrees C), nonfluorescent, reacts selectively with thiols to give addition products which are strongly fluorescent (excitation maximum 355 nm, emission maximum 448 nm, in ethanol). 0.7 mole of ANM was introduced into thiol groups of egg albumin without modifying any other amino acid residues. In the fluorescence spectra of the reaction products of ANM with thiols, the quantum yields increase and the emission maxima shift towards the blue as the solvent polarity decreases. This remarkable solvent dependence was described in terms ofZ value of the solvents. ANM is thus expected to be a useful fluorescent hydrophobic probe directed to thiol groups in protein.
Assuntos
Corantes Fluorescentes/química , Maleimidas/química , Proteínas/química , Compostos de Sulfidrila/química , Albuminas/química , Etanol/química , Modelos Químicos , Óvulo/metabolismo , Ligação Proteica , Solventes/química , Espectrometria de Fluorescência/métodos , Espectrofotometria Infravermelho/métodos , Água/químicaRESUMO
The mechanism of calmodulin-stimulated alkaline phosphatase activity was studied in the rat. In calmodulin-treated rats (2.5 micrograms/animal, intraperitoneally) alkaline phosphatase (ALP) activity was elevated 11-fold in the ileum, 1.5-fold in the duodenum and calvarium, 3-fold in serum, and not at all in liver. The elevated ALP activity was prevented by prior treatment with flunarizine, a calcium channel blocker, and by W-7, a calmodulin antagonist. cAMP content in ileum paralleled the timing and changes in ALP activity, but was not elevated in the duodenum or calvarium. Calcium ionophore A23187 and calcitonin treatment also increased ileal, duodenal, and calvarial ALP activity, but by less than the response to calmodulin. All of these treatments caused a 2-fold elevation in serum 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3) levels. Pretreatment of the animals with parathyroid hormone prevented the rise of both ALP activity and of 1,25(OH)2D3. Administration of 1,25(OH)2D3 alone stimulated a different pattern of increased ALP activity, greater in duodenum than ileum. The uptake of 45Ca by calmodulin was also elevated in ileum and calvarium. These data suggest that shifts in calcium movement, perhaps mediated by vitamin D, can alter ALP activity, and may provide a mechanism for rapid control of the secretion of this enzyme.
Assuntos
Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Íleo/enzimologia , Animais , Calcitonina/farmacologia , Calcitriol/sangue , Calmodulina/farmacologia , AMP Cíclico/metabolismo , Flunarizina/farmacologia , Isoenzimas/metabolismo , Masculino , Hormônio Paratireóideo/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Caspase-11 is a key regulator of proinflammatory cytokine IL-1beta maturation and pathological apoptosis. Caspase-11 is not expressed in most tissues under normal condition, but highly inducible upon pathological stimulation such as in the presence of lipopolysaccharide (LPS). Here, we describe the identification and characterization of wedelolactone, a natural compound that inhibits LPS-induced caspase-11 expression in cultured cells by inhibiting NF-kappaB-mediated transcription. We demonstrate that wedelolactone is an inhibitor of IKK, a kinase critical for activation of NF-kappaB by mediating phosphorylation and degradation of IkappaBalpha.