RESUMO
PURPOSE: Sodium-glucose cotransporter 2 inhibitors (SGLT2is) are commonly prescribed anti-diabetic medications with various beneficial effects; however, they have also been associated with ketoacidosis. The aim of this study was to determine the incidence of SGLT2i-associated perioperative ketoacidosis (SAPKA) in surgical patients. METHODS: We conducted a multicenter, prospective cohort study across 16 centers in Japan, enrolling surgical patients with diabetes who were prescribed SGLT2is between January 2021 and August 2022. Patients were monitored until the third postoperative day to screen for SAPKA, defined as urine ketone positivity with a blood pH of < 7.30 and HCO3 level ≤ 18.0 mEq/L, excluding cases of respiratory acidosis. RESULTS: In total, 759 of the 762 evaluated patients were included in the final analysis. Among these, three patients (0.40%) had urine ketones with a blood pH of < 7.30; however, blood gas analysis revealed respiratory acidosis in all three, and none of them was considered to have SAPKA. The estimated incidence of SGLT2i-associated postoperative ketoacidosis was 0% (95% confidence interval, 0%-0.4%). CONCLUSIONS: The observed incidence of SAPKA in our general surgical population was lower than expected. However, given that the study was observational in nature, interpretation of study results warrants careful considerations for biases.
Assuntos
Complicações Pós-Operatórias , Inibidores do Transportador 2 de Sódio-Glicose , Humanos , Inibidores do Transportador 2 de Sódio-Glicose/efeitos adversos , Estudos Prospectivos , Masculino , Feminino , Pessoa de Meia-Idade , Incidência , Idoso , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/diagnóstico , Cetose/induzido quimicamente , Cetose/epidemiologia , Japão/epidemiologia , Estudos de Coortes , Adulto , Cetoacidose Diabética/epidemiologia , Cetoacidose Diabética/induzido quimicamenteRESUMO
BACKGROUND: Postoperative cognitive dysfunction may be associated with neuroinflammation, and sevoflurane suppresses surgery-induced inflammation. We hypothesized that low concentrations of sevoflurane would result in more impaired postoperative cognitive function compared to high concentrations. METHODS: Aged male Sprague-Dawley rats (n = 21, 17-22 months) were randomly assigned to 1 of 3 groups: control (C), sevoflurane 2% (S2), and sevoflurane 4% (S4). Rats in the S2 and S4 groups underwent open femoral fracture and intramedullary fixation of the left hind limb under 2 hours of sevoflurane anesthesia. Neurological outcomes were evaluated using the Morris water maze (MWM) test, and histopathological outcomes were assessed 28 days after surgery. RESULTS: The S2 group showed prolonged swimming latency compared to S4 on day 7 (difference of means, 34.4; 95% confidence interval [CI], 2.57-66.3; P = .031) and compared to the C group on day 9 (difference of means, -33.4; 95% CI, -65.3 to -1.55; P = .037). The intact CA1 cells in the S2 group were significantly less than those in the C and S4 groups (H statistic, 10.87; P = .006 versus C; P = .033 versus S4). CONCLUSIONS: We found that low concentrations of sevoflurane prolonged the swimming latency of the MWM compared to high concentrations and reduced intact CA1 hippocampal neurons in aged rats. These results suggest that low-concentration sevoflurane anesthesia may be more detrimental than high concentration for spatial cognitive function and postoperative impairment of hippocampal CA1 cells in aged rats.
Assuntos
Anestésicos Inalatórios , Disfunção Cognitiva , Ratos , Animais , Masculino , Sevoflurano/efeitos adversos , Ratos Sprague-Dawley , Cognição , Hipocampo , Neurônios , Disfunção Cognitiva/induzido quimicamente , Aprendizagem em Labirinto , Anestésicos Inalatórios/efeitos adversosRESUMO
The toxic amyloid ß-peptide (Aß) is a key player in Alzheimer Disease (AD) pathogenesis and selective inhibition of the production of this peptide is sought for. Aß is produced by the sequential cleavage of the Aß precursor protein (APP) by ß-secretase (to yield APP-C-terminal fragment ß (APP-CTFß) and soluble APPß (sAPPß)) and γ-secretase (to yield Aß). We reasoned that proteins that associate with γ-secretase are likely to regulate Aß production and to be targets of pharmaceutical interventions and therefore performed a pull-down assay to screen for such proteins in rat brain. Interestingly, one of the purified proteins was potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), which has been shown to be involved in epilepsy. We found that silencing of HCN2 resulted in decreased secreted Aß levels. To further investigate the mechanism behind this reduction, we also determined the levels of full-length APP, sAPP and APP-CTF species after silencing of HCN2. A marked reduction in sAPP and APP-CTF, as well as glycosylated APP levels was detected. Decreased Aß, sAPP and APP-CTF levels were also detected after treatment with the HCN2 inhibitor ZD7288. These results indicate that the effect on Aß levels after HCN2 silencing or inhibition is due to altered APP maturation or processing by ß-secretase rather than a direct effect on γ-secretase. However, HCN2 and γ-secretase were found to be in close proximity, as evident by proximity ligation assay and immunoprecipitation. In summary, our results indicate that silencing or inhibition of HCN2 affects APP processing and thereby could serve as a potential treatment strategy.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais de Potássio/metabolismo , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Epilepsia/metabolismo , Feminino , Inativação Gênica , Glicosilação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Pirimidinas/química , Ratos , Ratos Sprague-DawleyRESUMO
Comprehensive experimental resources, such as ORFeome clone libraries and deletion mutant collections, are fundamental tools for elucidation of gene function. Data sets by omics analysis using these resources provide key information for functional analysis, modeling and simulation both in individual and systematic approaches. With the long-term goal of complete understanding of a cell, we have over the past decade created a variety of clone and mutant sets for functional genomics studies of Escherichia coli K-12. We have made these experimental resources freely available to the academic community worldwide. Accordingly, these resources have now been used in numerous investigations of a multitude of cell processes. Quality control is extremely important for evaluating results generated by these resources. Because the annotation has been changed since 2005, which we originally used for the construction, we have updated these genomic resources accordingly. Here, we describe GenoBase (http://ecoli.naist.jp/GB/), which contains key information about comprehensive experimental resources of E. coli K-12, their quality control and several omics data sets generated using these resources.
Assuntos
Bases de Dados Genéticas , Escherichia coli K12/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Genoma Bacteriano , Internet , Anotação de Sequência Molecular , MutaçãoRESUMO
In Alzheimer disease, oligomeric amyloid ß-peptide (Aß) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aß from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aß production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aß production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aß production (Aß40 and Aß42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aß production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Microdomínios da Membrana/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/isolamento & purificação , Peptídeos beta-Amiloides/biossíntese , Animais , Encéfalo/enzimologia , Moléculas de Adesão Celular Neuronais/genética , Cromatografia de Afinidade , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Presenilina-1/genética , Presenilina-1/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores Notch/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismo , Espectrometria de Massas em Tandem , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
BACKGROUND: It has long been recognized that analyzing the behaviour of the complex intracellular biological networks is important for breeding industrially useful microorganisms. However, because of the complexity of these biological networks, it is currently not possible to obtain all the desired microorganisms. In this study, we constructed a system for analyzing the effect of gene expression perturbations on the behavior of biological networks in Escherichia coli. Specifically, we utilized (13)C metabolic flux analysis ((13)C-MFA) to analyze the effect of perturbations to the expression levels of pgi and eno genes encoding phosphoglucose isomerase and enolase, respectively on metabolic fluxes. RESULTS: We constructed gene expression-controllable E. coli strains using a single-copy mini F plasmid. Using the pgi expression-controllable strain, we found that the specific growth rate correlated with the pgi expression level. (13)C-MFA of this strain revealed that the fluxes for the pentose phosphate pathway and Entner-Doudoroff pathway decreased, as the pgi expression lelvel increased. In addition, the glyoxylate shunt became active when the pgi expression level was almost zero. Moreover, the flux for the glyoxylate shunt increased when the pgi expression level decreased, but was significantly reduced in the pgi-knockout cells. Comparatively, eno expression could not be decreased compared to the parent strain, but we found that increased eno expression resulted in a decreased specific growth rate. (13)C-MFA revealed that the metabolic flux distribution was not altered by an increased eno expression level, but the overall metabolic activity of the central metabolism decreased. Furthermore, to evaluate the impact of perturbed expression of pgi and eno genes on changes in metabolic fluxes in E. coli quantitatively, metabolic sensitivity analysis was performed. As a result, the perturbed expression of pgi gene had a great impact to the metabolic flux changes in the branch point between the glycolysis and pentose phosphate pathway, isocitrate dehydrogenase reaction, anaplerotic pathways and Entner-Doudoroff pathway. In contrast, the impact of perturbed eno expression to the flux changes in E. coli metabolic network was small. CONCLUSIONS: Our results indicate that the response of metabolic fluxes to perturbation to pgi expression was different from that to eno expression; perturbations to pgi expression affect the reaction related to the Pgi protein function, the isocitrate dehydrogenase reaction, anaplerotic reactions and Entner-Doudoroff pathway. Meanwhile, eno expression seems to affect the overall metabolic activity, and the impact of perturbed eno expression on metabolic flux change is small. Using the gene expression control system reported here, it is expected that we can analyze the response and adaptation process of complex biological networks to gene expression perturbations.
Assuntos
Isótopos de Carbono/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Glucose-6-Fosfato Isomerase/genética , Fosfopiruvato Hidratase/genética , Carbono/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Glucose-6-Fosfato Isomerase/metabolismo , Glioxilatos/metabolismo , Via de Pentose Fosfato , Fosfopiruvato Hidratase/metabolismoRESUMO
BACKGROUND: Systems biology and functional genomics require genome-wide datasets and resources. Complete sets of cloned open reading frames (ORFs) have been made for about a dozen bacterial species and allow researchers to express and study complete proteomes in a high-throughput fashion. RESULTS: We have constructed an open reading frame (ORFeome) collection of 3974 or 94% of the known Escherichia coli K-12 ORFs in Gateway entry vector pENTR/Zeo. The collection has been used for protein expression and protein interaction studies. For example, we have compared interactions among YgjD, YjeE and YeaZ proteins in E. coli, Streptococcus pneumoniae, and Staphylococcus aureus. We also compare this ORFeome with other Gateway-compatible bacterial ORFeomes and show its utility for comparative functional genomics. CONCLUSIONS: The E. coli ORFeome provides a useful resource for functional genomics and other areas of protein research in a highly flexible format. Our comparison with other ORFeomes makes comparative analyses straighforward and facilitates direct comparisons of many proteins across many genomes.
Assuntos
Escherichia coli K12/genética , Fases de Leitura Aberta , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Análise de Sequência de DNA , Staphylococcus aureus/genética , Streptococcus pneumoniae/genéticaRESUMO
While direct to consumer health-related genetic testing (DTCGT) has potential to provide accessible genetic information and empower individuals to make informed healthcare decisions, it attracts concern associated with regulatory gaps, clinical utility and potential for harm. Understanding public reactions to DTCGT is vital to facilitate considered regulatory, health care and consumer protection strategies. Yet little is known, particularly outside the dominant US market, about how the general public view and might engage with DTCGT outside traditional health care systems. This paper addresses this knowledge gap with the first empirical study to investigate general public views across four countries, each at different stages of market development. US (n = 1000), UK (n = 1014), Japanese (n = 1018) and Australian (n = 1000) respondents completed an online experimental survey assessing comprehension, risk perceptions, and potential psychological and behavioural outcomes by type of test (disease pre-disposition and drug sensitivity), severity, lifestyle factors, and family history. Results showed generally low awareness and intention to purchase across countries, highest in the US and lowest in Japan. Results also showed clear preference for within-country purchases (less in Japan), with reports returned via doctors far more important in Japan. All respondents were more likely to act on test results, where there was higher genetic or lifestyle risk of developing a disease. Statistical comparisons of demographic and health-related variables across countries point to the need for further analyses designed to explain much needed cross-cultural, cross-health care system and developed versus developing market differences.
Assuntos
Triagem e Testes Direto ao Consumidor/psicologia , Testes Genéticos , Conhecimentos, Atitudes e Prática em Saúde , Relações Públicas , Adulto , Idoso , Austrália , Comportamento do Consumidor , Comparação Transcultural , Triagem e Testes Direto ao Consumidor/organização & administração , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Classe Social , Reino Unido , Estados UnidosRESUMO
Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.
Assuntos
Proteínas de Escherichia coli/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Modelos Moleculares , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Espectrometria de Massas em Tandem , Difração de Raios XRESUMO
We report a case of suspected acute pancreatitis after bilateral total knee arthroplasty. Airway management including jaw thrust maneuvers was performed. Postoperatively, the patient complained of nausea with vomiting. Further examination revealed high serum amylase levels (1703 IU x l(-1)), and an abdominal CT scan revealed confined inflammation near the head of the pancreas. The amylase isozyme patterns identified the salivary-type amylase. A gastroduodenoscopy procedure performed on the 21st postoperative day revealed a duodenal ulcer. We speculated that the primary cause of hyperamylasemia was accumulation of amylase in the parotid glands and the CT findings showed the inflammation of the pancreas itself or extrapancreatic exdates resulting from duodenal ulcer.
Assuntos
Anestesia Geral , Pancreatite/etiologia , Doença Aguda , Idoso , Úlcera Duodenal/complicações , Feminino , Humanos , Complicações Pós-OperatóriasRESUMO
We experienced anesthetic management of a patient undergoing surgeries for bilateral femoral trochanter fractures before the onset and during the healing stages of takotsubo cardiomyopathy (TCM). While performing the surgery before the onset of TCM, we administered 0.5% intrathecal isobaric bupivacaine 15 mg and the procedure was completed uneventfully. On the other hand, while performing the surgery during the healing stages of TCM, vasopressor infusion was required for treating hypotension with extrasystoles despite administrating only 14.5 mg of 0.05% intrathecal isobaric bupivacaine. It is therefore suggested that cardiac function during the healing stages of TCM was more compromised than before the onset of the disease.
Assuntos
Raquianestesia , Fraturas do Fêmur/complicações , Fraturas do Fêmur/cirurgia , Cardiomiopatia de Takotsubo/complicações , Idoso de 80 Anos ou mais , Bupivacaína , Feminino , Humanos , Hipotensão/tratamento farmacológico , Complicações Intraoperatórias/tratamento farmacológico , Assistência Perioperatória , Fenilefrina/administração & dosagem , Vasoconstritores/administração & dosagemRESUMO
Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qß RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qß replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of ß-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qß replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qß replicase while maintaining the encoded gene function.
Assuntos
Q beta Replicase/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , RNA/genética , Sequência de Aminoácidos/genética , Aminoácidos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fenótipo , Domínios Proteicos/genética , beta-Galactosidase/genéticaRESUMO
BACKGROUND: Increased levels of the pathogenic amyloid ß-peptide (Aß), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aß in neurons. METHODS: MAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aß production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aß42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis. RESULTS: Immunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aß42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aß42. Furthermore, overexpression of MAO-B enhanced Aß production. CONCLUSIONS: This study shows that MAO-B levels are increased not only in astrocytes but also in pyramidal neurons in AD brain. The study also suggests that MAO-B regulates Aß production in neurons via γ-secretase and thereby provides a key to understanding the relationship between MAO-B and AD pathogenesis. Potentially, the γ-secretase/MAO-B association may be a target for reducing Aß levels using protein-protein interaction breakers.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Monoaminoxidase/metabolismo , Neurônios/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Animais , Axônios/metabolismo , Encéfalo/patologia , Linhagem Celular Transformada , Dendritos/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Monoaminoxidase/genética , Neurônios/ultraestrutura , Presenilina-1/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , TransfecçãoRESUMO
The transmembrane protease complex γ-secretase is a key enzyme in Alzheimer disease pathogenesis as it liberates the neurotoxic amyloid ß-peptide (Aß); however, the mechanism of regulation of its activity in various cell types and subcellular compartments is largely unknown. Several γ-secretase inhibitors have been developed, but none have been released due to side-effects that appear to arise from reduced processing of Notch, one of many γ-secretase substrates. Hence, it is desirable to specifically inhibit Aß production. In our previous studies, we have identified several γ-secretase-associated proteins (GSAPs) from brain, which affect Aß production without having any major effects on Notch processing. In the present study using detergent-resistant membranes prepared from brain, we have identified four GSAPs that affect Aß production to a greater extent than Notch processing. We evaluated the interaction between GSAPs and γ-secretase in various cell types and their mRNA expression in various human organs. Using an in situ proximity ligation assay, we demonstrated that many GSAPs showed considerably greater interaction with γ-secretase in neurons than in human embryonic kidney cells stably over-expressing APP, and showed that several GSAPs are highly expressed in human brain. This study underscores the importance of studying protein-protein interactions in relevant cell types, and suggests that reducing Aß production by interfering with brain- or neuron-specific γ-secretase/GSAP interactions may reduce the risk of unwanted side-effects associated with treatment of Alzheimer disease.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Células Cultivadas , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas/genética , RNA Interferente Pequeno , Receptores Notch/metabolismoRESUMO
γ-Secretase is a transmembrane protease complex that is responsible for the processing of a multitude of type 1 transmembrane proteins, including the amyloid precursor protein and Notch. γ-Secretase processing of amyloid precursor protein results in the release of the amyloid ß-peptide (Aß), which is involved in the pathogenesis in Alzheimer's disease. Processing of Notch leads to the release of its intracellular domain, which is important for cell development. γ-Secretase associated proteins (GSAPs) could be of importance for substrate selection, and we have previously shown that affinity purification of γ-secretase in combination with mass spectrometry can be used for finding such proteins. In the present study, we used this methodology to screen for novel GSAPs from human brain, and studied their effect on Aß production in a comprehensive gene knockdown approach. Silencing of probable phospholipid-transporting ATPase IIA, brain-derived neurotrophic factor/neurotrophin-3 growth factor receptor precursor and proton myo-inositol cotransporter (SLC2A13) showed a clear reduction of Aß and these proteins were selected for further studies on Aß production and Notch cleavage using small interfering RNA-mediated gene silencing, as well as an overexpression approach. Silencing of these reduced Aß secretion in a small interfering RNA dose-dependent manner. Interestingly, SLC2A13 had a lower effect on Notch processing. Furthermore, overexpression of SLC2A13 increased Aß40 generation. Finally, the interaction between γ-secretase and SLC2A13 was confirmed using immunoprecipitation and a proximity ligation assay. In summary, SLC2A13 was identified as a novel GSAP that regulates Aß production without affecting Notch cleavage. We suggest that SLC2A13 could be a target for Aß lowering therapy aimed at treating Alzheimer's disease.
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Fragmentos de Peptídeos/genética , Prótons , Receptores Notch/genética , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/biossíntese , Animais , Química Encefálica , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Carbamatos/farmacologia , Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Inositol/metabolismo , Camundongos , Microssomos/química , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Anotação de Sequência Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/biossíntese , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Estabilidade Proteica , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
To date, many researchers in Japan have been working on the assumption that the cause of autistic spectrum disorders is attributable to some disorder in the ability of the child. We have been working on the premise that the autistic spectrum disorders are brought about by some form of relationship disturbances in early infancy, and have been attempting to validate this hypothesis through early intervention. We have been examining the developmental process of affective communication in infants with autistic spectrum disorders. We postulated approach-avoidance motivational conflict (Richer, 1993) as the primary factor impeding development of affective communication, focusing therapeutic intervention on this perspective. As a result, attachment behavior was remarkably improved in the children. Through a clinical course of an example, the problem pertaining to the process of acquiring the function of language cognition is discussed. Starting with the concept that the meaning of any given object is not stipulated as a unique existence, but by context, it is pointed out that in order to relate the meaning of some object to a child, sharing with the child "which" attributes of the object the child should focus upon and "how" it should be dealt with are indispensable. This is then followed by discussion of how this cannot be accomplished without deepening of affective communication upon the basis of attachment formation.
Assuntos
Transtorno Autístico/psicologia , Comunicação , Conflito Psicológico , Emoções , Relações Mãe-Filho , Motivação , Apego ao Objeto , Adulto , Cognição , Humanos , Lactente , Desenvolvimento da Linguagem , MasculinoRESUMO
Genetic interaction networks are especially useful for functional assignment of genes and gaining new insights into the systems-level organization of the cell. While studying interactions of nonessential genes can be relatively straight-forward via use of deletion mutants, different approaches must be used to reveal interactions of essential genes due to their indispensability. One method shown to be useful for revealing interactions of essential genes requires tagging the query protein. However, this approach can be complicated by mutational effects of potential hypomorphic alleles. Here, we describe a pilot study for a new scheme of systematically studying the interactions of essential genes. Our method uses a low-copy, F-based, complementing plasmid, pFE604T, from which the essential gene is conditionally expressed. The essential gene is expressed at lower levels, producing a moderate growth defect in a query host. Secondary mutations are introduced into the query host by conjugation and the resultant exconjugants are scored for growth by imaging them over time. We report results from studying five essential query genes: dnaN, ftsW, trmD, yrfF and yjgP, showing (on average) interactions with nearly 80 nonessential genes. This system should prove useful for genome-wide analyses of other essential genes in E. coli K-12.