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Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing selenium instead of sulfur). SeP functions for the transport of selenium to specific tissues in a receptor-dependent manner. Apolipoprotein E receptor 2 (ApoER2) has been identified as a SeP receptor. However, diverse variants of ApoER2 have been reported, and the details of its tissue specificity and the molecular mechanism of its efficiency remain unclear. In the present study, we found that human T lymphoma Jurkat cells have a high ability to utilize selenium via SeP, while this ability was low in human rhabdomyosarcoma cells. We identified an ApoER2 variant with a high affinity for SeP in Jurkat cells. This variant had a dissociation constant value of 0.67 nM and a highly glycosylated O-linked sugar domain. Moreover, the acidification of intracellular vesicles was necessary for selenium transport via SeP in both cell types. In rhabdomyosarcoma cells, SeP underwent proteolytic degradation in lysosomes and transported selenium in a Sec lyase-dependent manner. However, in Jurkat cells, SeP transported selenium in Sec lyase-independent manner. These findings indicate a preferential selenium transport pathway involving SeP and high-affinity ApoER2 in a Sec lyase-independent manner. Herein, we provide a novel dynamic transport pathway for selenium via SeP.
Assuntos
Liases , Selênio , Humanos , Liases/metabolismo , Selênio/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteína P/genética , Selenoproteína P/metabolismo , Selenoproteínas , Células JurkatRESUMO
An analytical method based on LC-MS/MS was developed for the determination of chlorothalonil metabolite I in livestock products. Chlorothalonil metabolite I in livestock products was extracted with acetone. The crude extracts were defatted by acetonitrile and n-hexane partitioning. Cleanup was carried out using a combination of ethylene diamine-N-propyl silylation silica gel (PSA) and silica gel (SI) mini columns with acidic condition. The sample solution was subjected to LC-MS/MS using an external solvent calibration curve. The average recovery (n=5) of chlorothalonil metabolite I from five types of livestock products (cattle muscle, cattle fat, cattle liver, milk and egg) spike at the maximum residue limits (MRLs) or at a uniform limit of 0.01 mg/kg was 97.1-102.9%, with a relative standard deviation of 1.4-6.8%. The limit of quantitation of the developed method was calculated to be 0.01 mg/kg.
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Gado , Espectrometria de Massas em Tandem , Animais , Bovinos , Cromatografia Líquida , Sílica GelRESUMO
Tachykinin-like peptides, such as substance P, neurokinin A, and neurokinin B, are among the earliest discovered and best-studied neuropeptide families, and research on them has contributed greatly to our understanding of the endocrine control of many physiological processes. However, there are still many orphan tachykinin receptor homologs for which cognate ligands have not yet been identified, especially in small invertebrates, such as the nematode Caenorhabditis elegans (C. elegans). We here show that the C. elegans nlp-58 gene encodes putative ligands for the orphan G protein-coupled receptor (GPCR) TKR-1, which is a worm ortholog of tachykinin receptors. We first determine, through an unbiased biochemical screen, that a peptide derived from the NLP-58 preprotein stimulates TKR-1. Three mature peptides that are predicted to be generated from NLP-58 show potent agonist activity against TKR-1. We designate these peptides as C. elegans tachykinin (CeTK)-1, -2, and -3. The CeTK peptides contain the C-terminal sequence GLR-amide, which is shared by tachykinin-like peptides in other invertebrate species. nlp-58 exhibits a strongly restricted expression pattern in several neurons, implying that CeTKs behave as neuropeptides. The discovery of CeTKs provides important information to aid our understanding of tachykinin-like peptides and their functional interaction with GPCRs.
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Caenorhabditis elegans/metabolismo , Taquicininas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cricetulus , Taquicininas/química , Taquicininas/genética , Taquicininas/isolamento & purificaçãoRESUMO
An analytical method based on LC-MS/MS was developed for the determination of asulam in livestock products. Asulam in livestock products was extracted with acetone. The crude extracts were defatted by acetonitrile and n-hexane partitioning. Cleanup was carried out using a combination of ethylene diamine-N-propyl silylation silica gel (PSA) and octadecyl silylated silica gel (C18) mini columns with acidic condition. The sample solution was subjected to LC-MS/MS using an external solvent calibration curve. The average recovery (n=5) of Asulam from four types of livestock products (bovine muscle, bovine fat, bovine liver and milk) spike at the maximum residue limits (MRLs) or at a uniform limit of 0.01 mg/kg was 92.7-98.7%, with a relative standard deviation of 3.1-11.6%. The limit of quantitation of the developed method was calculated to be 0.01 mg/kg.
Assuntos
Gado , Espectrometria de Massas em Tandem , Animais , Carbamatos , Bovinos , Cromatografia LíquidaRESUMO
For proper functioning of the nervous system, it is crucial that neurons find their appropriate partners and build the correct neural connection patterns. Although cell adhesion molecules (CAMs) have been studied for many years as essential players in neural connections, we have yet to unravel the code by which CAMs encode synaptic specificity. We analyzed the effects of mutations in CAM genes on the morphology and synapses of a set of sensory neurons in the Caenorhabditis elegans male tail. B-type ray sensory neurons express 10 genes encoding CAMs. We examined the effect on axon trajectory and localization of pre-synaptic components in viable mutants of nine of these. We found axon trajectory defects in mutants of UNC-40/DCC, SAX-3/ROBO, and FMI-1/Flamingo/Celsr1. None of the mutations caused loss of pre-synaptic components in axons, and in several the level even appeared to increase, suggesting possible accumulation of pre-synapses. B-type sensory neurons fasciculate with a second type of ray sensory neuron, the A-type, in axon commissures. We found a CAM expressed in A-type functions additively with a CAM expressed in B-type in axon guidance, and lack of a CAM expressed in B-type affected A-type axon guidance. Overall, single and multiple mutants of CAM genes had limited effects on ray neuron trajectories and accumulation of synaptic components.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Masculino , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Receptoras Sensoriais/metabolismo , Diferenciação CelularRESUMO
OBJECTIVE: As information required to rehabilitate, we aimed to clarify the use of switch devices, language development, upper extremity function, and quality of life (QOL) in patients with spinal muscular atrophy (SMA) type 1 in Japan. METHODS: A questionnaire survey was conducted on the parents of these patients. RESULTS: A total of 48 completed questionnaires were collected. The median age of patients with SMA type 1 was 8 years (range, 13 months to 27 years). The patients used a switch device were 46.5%. Their language development was delayed, and their voluntary movement in the distal upper extremities was relatively retained. In QOL, parents' labor and burden for the care of patients were high. CONCLUSIONS: It is necessary to provide specialist support that contributes to the spread of communication devices, which improve their motor function, language development and overall QOL.
Assuntos
Desenvolvimento da Linguagem , Qualidade de Vida , Tecnologia Assistiva , Atrofias Musculares Espinais da Infância/fisiopatologia , Extremidade Superior/fisiopatologia , Adolescente , Criança , Pré-Escolar , Humanos , Lactente , Japão , Pais , Inquéritos e Questionários , Adulto JovemRESUMO
[This corrects the article DOI: 10.3389/fneur.2019.00693.].
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Optical limiting effects of silicon phthalocyanine (SiPc) oligomers have been examined in the Q band region in order to demonstrate a novel concept for controlling the wavelength of optical limiting effects.
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Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is clinically characterized by early-onset dementia, stroke, spondylosis deformans, and alopecia. In CARASIL cases, brain magnetic resonance imaging reveals severe white matter hyperintensities (WMHs), lacunar infarctions, and microbleeds. CARASIL is caused by a homozygous mutation in high-temperature requirement A serine peptidase 1 (HTRA1). Recently, it was reported that several heterozygous mutations in HTRA1 also cause cerebral small vessel disease (CSVD). Although patients with heterozygous HTRA1-related CSVD (symptomatic carriers) are reported to have a milder form of CARASIL, little is known about the clinical and genetic differences between the two diseases. Given this gap in the literature, we collected clinical information on HTRA1-related CSVD from a review of the literature to help clarify the differences between symptomatic carriers and CARASIL and the features of both diseases. Forty-six symptomatic carriers and 28 patients with CARASIL were investigated. Twenty-eight mutations in symptomatic carriers and 22 mutations in CARASIL were identified. Missense mutations in symptomatic carriers are more frequently identified in the linker or loop 3 (L3)/loop D (LD) domains, which are critical sites in activating protease activity. The ages at onset of neurological symptoms/signs were significantly higher in symptomatic carriers than in CARASIL, and the frequency of characteristic extraneurological findings and confluent WMHs were significantly higher in CARASIL than in symptomatic carriers. As previously reported, heterozygous HTRA1-related CSVD has a milder clinical presentation of CARASIL. It seems that haploinsufficiency can cause CSVD among symptomatic carriers according to the several patients with heterozygous nonsense/frameshift mutations. However, the differing locations of mutations found in the two diseases indicate that distinct molecular mechanisms influence the development of CSVD in patients with HTRA1-related CSVD. These findings further support continued careful examination of the pathogenicity of mutations located outside the linker or LD/L3 domain in symptomatic carriers.
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We succeeded in cloning the rhesus monkey nociceptin/orphanin FQ peptide (NOP) receptor. The nucleotide sequence and amino acid sequence of the rhesus monkey NOP receptor were 95.9% and 97.8%, respectively, identical to the human NOP receptor. There was no significant difference between the rhesus monkey NOP receptor and the human NOP receptor in the binding affinity of [(125)I] [Thy(14)]nociceptin and the binding of [(35)S]guanosine 5'-O-(gamma thio)triphospate ([(35)S]GTPgammaS) stimulated by nociceptin/orphanin FQ (N/OFQ). A selective NOP receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one ((+)-J-113397) inhibited the [(35)S]GTPgammaS binding activated by N/OFQ using the membrane of the rhesus monkey NOP receptor. The antagonistic activity of (+)-J-113397 to the rhesus monkey NOP receptor was comparable to that to the human NOP receptor. Thus, N/OFQ acts via activation of the NOP receptor in both human and rhesus monkeys without significant species differences.
Assuntos
Benzimidazóis/farmacologia , Peptídeos Opioides/metabolismo , Piperidinas/farmacologia , Receptores Opioides/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Macaca mulatta , Dados de Sequência Molecular , Antagonistas de Entorpecentes , Receptores Opioides/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Receptor de Nociceptina , NociceptinaRESUMO
Background: Mutations in the high-temperature requirement A serine peptidase 1 (HTRA1) cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Most carriers for HTRA1 mutations are asymptomatic, but more than 10 mutations have been reported in symptomatic carriers. The molecular differences between the mutations identified in symptomatic carriers and mutations identified only in CARASIL patients are unclear. HTRA1 is a serine protease that forms homotrimers, with each HTRA1 subunit activating the adjacent HTRA1 via the sensor domain of loop 3 (L3) and the activation domain of loop D (LD). Previously, we analyzed four HTRA1 mutant proteins identified in symptomatic carriers and found that they were unable to form trimers or had mutations in the LD or L3 domain. The mutant HTRA1s with these properties are presumed to inhibit trimer-dependent activation cascade. Indeed, these mutant HTRA1s inhibited wild-type (WT) protease activity. In this study, we further analyzed 15 missense HTRA1s to clarify the molecular character of mutant HTRA1s identified in symptomatic carriers. Methods: We analyzed 12 missense HTRA1s identified in symptomatic carriers (hetero-HTRA1) and three missense HTRA1s found only in CARASIL (CARASIL-HTRA1). The protease activity of the purified recombinant mutant HTRA1s was measured using fluorescein isothiocyanate-labeled casein as substrate. Oligomeric structure was evaluated by size-exclusion chromatography. The protease activities of mixtures of WT with each mutant HTRA1 were also measured. Results: Five hetero-HTRA1s had normal protease activity and were excluded from further analysis. Four of the seven hetero-HTRA1s and one of the three CARASIL-HTRA1s were unable to form trimers. The other three hetero-HTRA1s had mutations in the LD domain. Together with our previous work, 10 of 11 hetero-HTRA1s and two of six CARASIL-HTRA1s were either defective in trimerization or had mutations in the LD or L3 domain (P = 0.006). By contrast, eight of 11 hetero-HTRA1s and two of six CARASIL-HTRA1 inhibited WT protease activity (P = 0.162). Conclusions: HTRA1 mutations identified in symptomatic carriers have the property of interfering the trimer-dependent activation cascade of HTRA1.
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Electron paramagnetic resonance spectroscopy has unmasked for the first time the spin-delocalised doublet- and triplet-ground states of azacalix[4]arene cation radicals.
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We assessed achromatic luminance and isoluminant chromatic contrast sensitivity functions of a 20-year-old male. He showed severe motor and intellectual disabilities after advanced adrenoleukodystrophy, which started at the age of 7. Optokinetic nystagmus (OKN) to drifting gratings was used to assess his contrast sensitivities. Although the achromatic luminance contrast sensitivities were lower over the entire range of spatial frequencies tested than those of the healthy adults, they were preserved to the level comparable to healthy 7-year-old children, except for the frequency of 1 and 4 cycles/degree. In contrast, both of the red-green and blue chromatic contrast sensitivities were remarkably lower in all frequency range compare to healthy children and adults. These results indicate that it is possible for even an advanced case of ALD to show residual visual capacity that was preserved to a remarkable extent.
Assuntos
Adrenoleucodistrofia/complicações , Adrenoleucodistrofia/fisiopatologia , Transtornos da Visão/etiologia , Transtornos da Visão/fisiopatologia , Córtex Visual/fisiopatologia , Vias Visuais/fisiopatologia , Adulto , Fatores Etários , Criança , Percepção de Cores/fisiologia , Sensibilidades de Contraste/fisiologia , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/etiologia , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Humanos , Luminescência , Imageamento por Ressonância Magnética , Masculino , Exame Neurológico , Estimulação Luminosa , Valor Preditivo dos Testes , Transtornos da Visão/diagnóstico , Testes Visuais , Córtex Visual/patologia , Vias Visuais/patologiaRESUMO
A pulse control and data acquisition system with high time resolution of 300 ps and data-averaging rate of 100 kHz for a pulsed-ESR spectrometer was constructed. The system consists of two sets of 3.34 GHz data timing generator and a dual-channel analog-to-digital converter with 1 GHz samples, both of which are commercially available. We developed a scheme and software to control these equipments with high efficiency in pulsing design, data acquisition, and accurate synchronization of the devices. The system was developed at Ku band (17.5 GHz) and demonstrated some examples of electron spin echo and double quantum measurements.
Assuntos
Conversão Análogo-Digital , Espectroscopia de Ressonância de Spin Eletrônica/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , RetroalimentaçãoRESUMO
A method for the determination of hymexazol in agricultural produdcts by gas chromatography with a highly sensitive nitrogen-phosphorus detector (GC-NPD) was investigated. Hymexazol was extracted with acetonitrile, and the acetonitrile layer was separated by salting-out. The water layer was loaded onto a Chem-Elut column. Hymexazol in the water layer was adsorbed on the column, and eluted with ethyl acetate. The acetonitrile layer and the eluate were mixed and evaporated. The residue was dissolved in ethyl acetate, and the sample solution was cleaned up on a C18 column. Hymexazol in the eluate was analyzed by GC-NPD with a high-polarity capillary column (DB-FFAP) and highly deactivated inlet liner. Recoveries of hymexazol spiked in agricultural products (tomato, lemon, soybean and other samples) at the level of 0.1 mug/g ranged from 65.0 to 84.7%. The limit of detection was 0.02microg/g.
Assuntos
Agroquímicos/análise , Cromatografia Gasosa/instrumentação , Produtos Agrícolas/química , Oxazóis/análiseRESUMO
A survey of pesticide residues in 490 imported cereal products on the Tokyo market from April 1994 to March 2006 was carried out. Eight kinds of organophosphorus pesticides (chlorpyrifos, chlorpyrifos-methyl, DDVP, diazinon, etrimfos, malathion, MEP and pirimiphos-methyl) were detected at levels between Tr (below 0.01 ppm) and 0.82 ppm from 91 samples. In our investigations, chlorpyrifos-methyl and malathion tended to be detected in samples from America, pirimiphos-methyl in those from Europe, and MEP in those from Oceania. Thus, pesticide residues seemed to be different in produce from different areas. Residue levels of these pesticides were calculated as between 0.08 and 13.2% of their ADI values according to the daily intake of cereal products. Therefore, these cereal products should be safe for normal usage.
Assuntos
Grão Comestível/química , Resíduos de Praguicidas/análiseRESUMO
A survey of pesticide residues in 316 baby foods on the Tokyo market from April 1999 to June 2005 was carried out. Fifteen kinds of pesticides, including organophosphorus, organochlorine, carbamate, pyrethroid and others, were detected between at levels Tr (below 0.01 ppm) and 0.55 ppm from 28 samples. The baby foods in which residual pesticides were detected were produced from flour, leaf vegetable and fruits. The pesticides were detected in baby foods produced with minimal processing, such as straining and squeezing. For the baby foods containing pesticides, the amounts of intake of the pesticides were calculated from the recommended servings, and compared with the ADI values. Residual pesticide levels in baby food were between 0.06 and 16.6% of the ADI. Therefore, these foods should be safe in normal usage.
Assuntos
Alimentos Infantis/análise , Resíduos de Praguicidas/análise , Carbamatos/análise , Frutas/química , Hidrocarbonetos Clorados/análise , Carne/análise , Compostos Organofosforados/análise , Piretrinas/análise , Verduras/químicaRESUMO
A simplified method for the determination of forchlorfenuron in agricultural products by HPLC with UV detection was investigated. A chopped sample homogenate from agricultural products was extracted with acetone. The extract was filtrated and concentrated. The residues was loaded onto a Chem Elut column and extracted with ethyl acetate. The crude extract was purified on Oasis HLB and Bond Elut PSA mini-columns using a mixture of methanol and ethyl acetate. Forchlorfenuron was analyzed by HPLC with UV detection (263 nm). HPLC separation was performed on an ODS column with methanol-water as the mobile phase. Recoveries of forchlorfenuron from several agricultural products fortified at the level of 0.1 microg/g were in the range of 87.6-99.5%. The limit of detection (S/N=3) was 0.005 microg/g in the sample.
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Cromatografia Líquida de Alta Pressão , Produtos Agrícolas/química , Compostos de Fenilureia/análise , Reguladores de Crescimento de Plantas/análise , Piridinas/análise , Técnicas de Química Analítica/métodosRESUMO
Pesictide residues in 343 samples of domestic rice and 32 samples of imported rice purchased on the Tokyo market from April 1995 to March 2005 were investigated. Residues of eleven kinds of pesticides (including organophosphorus, organochlorine, carbamate, pyrethroid and organonitrogen pesticides and bromide) were detected at levels between trace and 1 ppm in 47 domestic rice samples. DDVP and bromide residues were detected at levels between 0.01 ppm and 5 ppm in 18 imported rice samples. For rice samples that were found to containing pesticides, the amounts of pesticide intake were calculated according to the daily intake of rice, and they were compared with the ADI values. Since residual pesticide levels were 17/10,000-2/5 of ADI, all the rice samples were considered to be safe for human consumption.
Assuntos
Contaminação de Alimentos/análise , Oryza/química , Resíduos de Praguicidas/análise , Concentração Máxima Permitida , TóquioRESUMO
Several types of associative learning are dependent upon the presence or absence of food, and are crucial for the survival of most animals. Target of rapamycin (TOR), a kinase which exists as a component of two complexes, TOR complex 1 (TORC1) and TOR complex 2 (TORC2), is known to act as a nutrient sensor in numerous organisms. However, the in vivo roles of TOR signaling in the nervous system remain largely unclear, partly because its multifunctionality and requirement for survival make it difficult to investigate. Here, using pharmacological inhibitors and genetic analyses, we show that TORC1 and TORC2 contribute to associative learning between salt and food availability in the nematode Caenorhabditis elegans in a process called taste associative learning. Worms migrate to salt concentrations experienced previously during feeding, but they avoid salt concentrations experienced under starvation conditions. Administration of the TOR inhibitor rapamycin causes a behavioral defect after starvation conditioning. Worms lacking either RICT-1 or SINH-1, two TORC2 components, show defects in migration to high salt levels after learning under both fed and starved conditions. We also analyzed the behavioral phenotypes of mutants of the putative TORC1 substrate RSKS-1 (the C. elegans homolog of the mammalian S6 kinase S6K) and the putative TORC2 substrates SGK-1 and PKC-2 (homologs of the serum and glucocorticoid-induced kinase 1, SGK1, and protein kinase C-α, PKC-α, respectively) and found that neuronal RSKS-1 and PKC-2, as well as intestinal SGK-1, are involved in taste associative learning. Our findings shed light on the functions of TOR signaling in behavioral plasticity and provide insight into the mechanisms by which information sensed in the intestine affects the nervous system to modulate food-searching behaviors.