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1.
Anal Sci ; 40(8): 1529-1535, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38748393

RESUMO

This study introduces the α-rhamnose (Rham)-conjugated prodrug of SN-38 (Rham-SN-38) as a promising alternative to irinotecan. α-rhamnosidase, responsible for SN-38 release from Rham-SN-38, does not express in human cells, minimizing individual variability and side effects. The injection of the α-rhamnosidase into the tumor tissues makes it possible, for the first time, to activate the Rham-SN-38. Furthermore, α-rhamnosidase demonstrates significantly higher activity than carboxylesterase, the specific enzyme activating irinotecan. SN-38 release mediated by α-rhamnosidase completes within 2 h, with a kcat/Km value approximately 5.0 × 104-fold higher than that of irinotecan. The 50% inhibition concentration (IC50) of Rham-SN-38 against three types of cancer cells and one normal cell exceeds 4.5 × 103 nM. The addition of α-rhamnosidase significantly increases cytotoxicity, with IC50 comparable to free SN-38. The QIC50, an index reflecting the difference in cytotoxicity with and without α-rhamnosidase, exceeds approximately 1.0 × 102-fold. Rham-SN-38, synthesized in this study, demonstrates significant potential as a prodrug for cancer therapy.


Assuntos
Glicosídeo Hidrolases , Irinotecano , Pró-Fármacos , Irinotecano/farmacologia , Irinotecano/química , Humanos , Pró-Fármacos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/síntese química , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Relação Dose-Resposta a Droga , Linhagem Celular Tumoral
2.
Front Cell Neurosci ; 18: 1349878, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38433862

RESUMO

Protein kinase C γ (PKCγ), a neuronal isoform present exclusively in the central nervous system, is most abundantly expressed in cerebellar Purkinje cells (PCs). Targeted deletion of PKCγ causes a climbing fiber synapse elimination in developing PCs and motor deficit. However, physiological roles of PKCγ in adult mouse PCs are little understood. In this study, we aimed to unravel the roles of PKCγ in mature mouse PCs by deleting PKCγ from adult mouse PCs of PKCγfl/fl mice via cerebellar injection of adeno-associated virus (AAV) vectors expressing Cre recombinase under the control of the PC-specific L7-6 promoter. Whole cell patch-clamp recording of PCs showed higher intrinsic excitability in PCs virally lacking PKCγ [PKCγ-conditional knockout (PKCγ-cKO) PCs] than in wild-type (WT) mouse PCs in the zebrin-negative module, but not in the zebrin-positive module. AAV-mediated PKCγ re-expression in PKCγ-deficient mouse PCs in the zebrin-negative module restored the enhanced intrinsic excitability to a level comparable to that of wild-type mouse PCs. In parallel with higher intrinsic excitability, we found larger hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents in PKCγ-cKO PCs located in the zebrin-negative module, compared with those in WT mouse PCs in the same region. However, pharmacological inhibition of the HCN currents did not restore the enhanced intrinsic excitability in PKCγ-cKO PCs in the zebrin-negative module. These results suggested that PKCγ suppresses the intrinsic excitability in zebrin-negative PCs, which is likely independent of the HCN current inhibition.

4.
Surg Neurol Int ; 14: 278, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37680919

RESUMO

Background: There is no established treatment strategy for traumatic vertebral artery occlusion that does not require cervical spine repair surgery. Case Description: A 49-year-old man was brought to our hospital with traffic trauma. Fractures were observed in the left lateral mass and transverse process of Atlas and the left vertebral artery was occluded at the level of the foramen transversum of Atlas. No acute cerebral infarction was observed. Because the cervical spinal cord was not compressed by the fracture, no repair surgery was performed. Continuous intravenous heparin and oral aspirin were started for traumatic vertebral artery occlusion. Thereafter, the left vertebral artery spontaneously recanalized, but no cerebral infarction was observed. The patient was discharged home on day 16 of injury. Four days later, however, he was brought to our hospital with nausea and lightheadedness. Acute cerebral infarction was observed in the left posterior inferior cerebellar artery territory and a thrombus in the left vertebral artery V4 segment. Parent artery occlusion was performed to prevent further cerebral infarction due to distal embolization of the thrombus. No further cerebral infarction occurred after the operation and the patient was discharged home with a modified Rankin scale score of 1. Conclusion: In cases of traumatic vertebral artery occlusion without an occlusive mechanism, parent artery occlusion may be considered in terms of recanalization risk, regardless of the need for repair surgery.

5.
J Control Release ; 2023 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-37080488

RESUMO

Herein, we report engineered macrophages, termed "MacTrigger," acting as a trigger to induce an inflammatory environment only in tumor tissues. This led to intensive anti-tumor effects based on the removal potential of foreign substances. The strength of this study is the utilization of two unique functions of macrophages: (1) their ability to migrate to tumor tissues and (2) polarization into the anti-inflammatory M2 phenotype in the presence of tumor tissues. The MacTrigger accelerated the release of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), when it was polarized to the M2 phenotype. When the MacTrigger was administered to tumor-bearing mice, tumor growth was significantly inhibited compared with the non-treatment group, the un-transfected macrophages group, and the group with engineered macrophages capable of randomly releasing TNF-α. Additionally, the ratio of the M1 phenotype to the M2 phenotype in tumor tissues was >1 only in the MacTrigger group. Moreover, the ratios of natural killer cells and CD8+T cells in tumor tissues were increased compared with other groups. These results indicate that MacTrigger can induce inflammation in tumor tissues, leading to effective anti-tumor effects. In normal tissues, especially the liver, notable side effects were not observed. This is because, in the liver, the MacTrigger was not polarized to the M2 phenotype and could not induce inflammation. These results suggest that the MacTrigger is a "trigger" that can induce inflammation only in tumor tissues, then allowing the body to attack tumor tissues through the innate immunity system.

6.
J Control Release ; 2023 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-37080897

RESUMO

Herein, we report engineered macrophages, termed "MacTrigger," acting as a trigger to induce an inflammatory environment only in tumor tissues. This led to intensive anti-tumor effects based on the removal potential of foreign substances. The strength of this study is the utilization of two unique functions of macrophages: (1) their ability to migrate to tumor tissues and (2) polarization into the anti-inflammatory M2 phenotype in the presence of tumor tissues. The MacTrigger accelerated the release of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), when it was polarized to the M2 phenotype. When the MacTrigger was administered to tumor-bearing mice, tumor growth was significantly inhibited compared with the non-treatment group, the un-transfected macrophages group, and the group with engineered macrophages capable of randomly releasing TNF-α. Additionally, the ratio of the M1 phenotype to the M2 phenotype in tumor tissues was >1 only in the MacTrigger group. Moreover, the ratios of natural killer cells and CD8+T cells in tumor tissues were increased compared with other groups. These results indicate that MacTrigger can induce inflammation in tumor tissues, leading to effective anti-tumor effects. In normal tissues, especially the liver, notable side effects were not observed. This is because, in the liver, the MacTrigger was not polarized to the M2 phenotype and could not induce inflammation. These results suggest that the MacTrigger is a "trigger" that can induce inflammation only in tumor tissues, then allowing the body to attack tumor tissues through the innate immunity system.

7.
Neuroradiol J ; 36(4): 453-459, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36607169

RESUMO

OBJECTIVES: Thrombi in cerebral large vessel occlusion associated with active cancer are often fibrin and platelet-rich white thrombi. However, evaluating the thrombus composition in a short time before thrombectomy is often ineffective. We sought to determine factors related to white thrombi in acute ischemic stroke due to large vessel occlusion in cancer patients. METHODS: Consecutive cancer patients undergoing thrombectomy for acute ischemic stroke due to large vessel occlusion between January 2018 and May 2022 were retrospectively reviewed. The patients were classified into white thrombus and red thrombus groups on the basis of the pathological findings of retrieved thrombi. Patient characteristics and laboratory findings were compared between the two groups. RESULTS: There were 12 patients in the white thrombus group and 11 patients in the red thrombus group. Active cancer was significantly more in the white thrombus group than in the red thrombus group (91.7% vs. 36.3%, p = 0.0094). Internal carotid artery occlusion was significantly less in the white thrombus group than in the red thrombus group (0% vs. 36.4%, p = 0.037). Among laboratory findings, D-dimer levels were an independent factor associated with white thrombi (odds ratio 8.97 [95% confidence interval 1.71-368.99], p < 0.0001). The cutoff value of D-dimer levels for predicting white thrombi was 3.5 µg/mL (83.3% sensitivity and 100% specificity). CONCLUSIONS: In acute ischemic stroke in cancer patients, active cancer, no internal carotid artery occlusion, and higher D-dimer levels (≥3.5 µg/mL) may be associated with occlusion with fibrin and platelet-rich white thrombi.


Assuntos
Arteriopatias Oclusivas , Isquemia Encefálica , AVC Isquêmico , Neoplasias , Acidente Vascular Cerebral , Trombose , Humanos , AVC Isquêmico/diagnóstico por imagem , AVC Isquêmico/complicações , Estudos Retrospectivos , Trombectomia , Fibrina , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/complicações , Isquemia Encefálica/complicações , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/patologia
8.
Commun Biol ; 5(1): 979, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114373

RESUMO

Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. By using a split Cre recombinase fused with mCherry-binding nanobodies or designed ankyrin repeat proteins, we created Cre recombinase dependent on red fluorescent protein (RFP) (Cre-DOR). Functional binding units for monomeric RFPs are different from those for polymeric RFPs. We confirmed selective target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors. In estrogen receptor-beta (Esr2)-mRFP1 mice and gastrin-releasing peptide receptor (Grpr)-mRFP1 rats, we confirmed that Cre-DOR can be used for selective tracing of the neural projection from RFP-expressing specific neurons. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can modulate Cre-DOR efficiency. Our results provide a method for manipulating gene expression in specific cells expressing RFPs and expand the repertory of nanobody-based genetic tools.


Assuntos
Receptores da Bombesina , Anticorpos de Domínio Único , Animais , Integrases , Proteínas Luminescentes , Camundongos , Camundongos Transgênicos , Ratos , Receptores de Estrogênio , Anticorpos de Domínio Único/genética , Proteína Vermelha Fluorescente
9.
Mol Brain ; 14(1): 33, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33588899

RESUMO

GABAergic interneurons play a critical role in tuning neural networks in the central nervous system, and their defects are associated with neuropsychiatric disorders. Currently, the mDlx enhancer is solely used for adeno-associated virus (AAV) vector-mediated transgene delivery into cortical interneurons. Here, we developed a new inhibitory neuron-specific promoter (designated as the mGAD65 promoter), with a length of 2.5 kb, from a mouse genome upstream of exon 1 of the Gad2 gene encoding glutamic acid decarboxylase (GAD) 65. Intravenous infusion of blood-brain barrier-penetrating AAV-PHP.B expressing an enhanced green fluorescent protein under the control of the mGAD65 promoter transduced the whole brain in an inhibitory neuron-specific manner. The specificity and efficiency of the mGAD65 promoter for GABAergic interneurons, which was assessed at the motor cortex, were almost identical to or slightly higher than those of the mDlx enhancer. Immunohistochemical analysis revealed that the mGAD65 promoter preferentially transduced parvalbumin (PV)-expressing interneurons. Notably, the mGAD65 promoter transduced chandelier cells more efficiently than the mDlx enhancer and robustly labeled their synaptic boutons, called the cartridge, targeting the axon initial segments of excitatory pyramidal neurons. To test the ability of the mGAD65 promoter to express a functional molecule, we virally expressed G-CaMP, a fluorescent Ca2+ indicator, in the motor cortex, and this enabled us to monitor spontaneous and drug-induced Ca2+ activity in GABAergic inhibitory neurons. These results suggest that the mGAD65 promoter is useful for AAV-mediated targeting and manipulation of GABAergic neurons with the dominance of cortical PV-expressing neurons, including chandelier cells.


Assuntos
Encéfalo/metabolismo , Dependovirus/metabolismo , Neurônios GABAérgicos/metabolismo , Plasmídeos/metabolismo , Transdução Genética , Animais , Cálcio/metabolismo , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Injeções Intravenosas , Interneurônios/metabolismo , Camundongos Endogâmicos C57BL , Córtex Motor/metabolismo , Neurônios/metabolismo , Parvalbuminas/metabolismo , Regiões Promotoras Genéticas
10.
Anal Sci ; 37(3): 529-532, 2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33390414

RESUMO

We previously developed a hydrolase-based fluorescence amplification method for antigen-specific cell labelling, in which fluorescent substrates stained cells by a non-covalent hydrophobic interaction. To improve the substrates retention in cells, we examined the effect of a chloroacetyl group modification on the substrate retention. We found that the chloroacetyl group suppressed the dissociation of the substrate after forming a covalent bond with intracellular proteins. However, the slow reaction speed of the chloroacetyl group allowed dissociation for cells in the early stage of the staining reaction.


Assuntos
Acetatos/química , Corantes Fluorescentes/química , Coloração e Rotulagem , Humanos , Células K562 , Microscopia de Fluorescência , Células Tumorais Cultivadas
11.
Nat Neurosci ; 22(8): 1289-1305, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285612

RESUMO

The effects of autonomic innervation of tumors on tumor growth remain unclear. Here we developed a series of genetic techniques to manipulate autonomic innervation in a tumor- and fiber-type-specific manner in mice with human breast cancer xenografts and in rats with chemically induced breast tumors. Breast cancer growth and progression were accelerated following stimulation of sympathetic nerves in tumors, but were reduced following stimulation of parasympathetic nerves. Tumor-specific sympathetic denervation suppressed tumor growth and downregulated the expression of immune checkpoint molecules (programed death-1 (PD-1), programed death ligand-1 (PD-L1), and FOXP3) to a greater extent than with pharmacological α- or ß-adrenergic receptor blockers. Genetically induced simulation of parasympathetic innervation of tumors decreased PD-1 and PD-L1 expression. In humans, a retrospective analysis of breast cancer specimens from 29 patients revealed that increased sympathetic and decreased parasympathetic nerve density in tumors were associated with poor clinical outcomes and correlated with higher expression of immune checkpoint molecules. These findings suggest that autonomic innervation of tumors regulates breast cancer progression.


Assuntos
Fibras Autônomas Pré-Ganglionares/patologia , Neoplasias da Mama/patologia , Antagonistas Adrenérgicos/farmacologia , Animais , Antígeno B7-H1/genética , Denervação , Progressão da Doença , Feminino , Fatores de Transcrição Forkhead/genética , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Sistema Nervoso Parassimpático/patologia , Receptor de Morte Celular Programada 1/genética , Ratos , Estudos Retrospectivos , Estresse Psicológico/psicologia , Sistema Nervoso Simpático/patologia
12.
Sci Rep ; 8(1): 5749, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29636528

RESUMO

Homoserine dehydrogenase (EC 1.1.1.3, HSD) is an important regulatory enzyme in the aspartate pathway, which mediates synthesis of methionine, threonine and isoleucine from aspartate. Here, HSD from the hyperthermophilic archaeon Sulfolobus tokodaii (StHSD) was found to be inhibited by cysteine, which acted as a competitive inhibitor of homoserine with a Ki of 11 µM and uncompetitive an inhibitor of NAD and NADP with Ki's of 0.55 and 1.2 mM, respectively. Initial velocity and product (NADH) inhibition analyses of homoserine oxidation indicated that StHSD first binds NAD and then homoserine through a sequentially ordered mechanism. This suggests that feedback inhibition of StHSD by cysteine occurs through the formation of an enzyme-NAD-cysteine complex. Structural analysis of StHSD complexed with cysteine and NAD revealed that cysteine situates within the homoserine binding site. The distance between the sulfur atom of cysteine and the C4 atom of the nicotinamide ring was approximately 1.9 Å, close enough to form a covalent bond. The UV absorption-difference spectrum of StHSD with and without cysteine in the presence of NAD, exhibited a peak at 325 nm, which also suggests formation of a covalent bond between cysteine and the nicotinamide ring.


Assuntos
Cisteína/química , Cisteína/metabolismo , Homosserina Desidrogenase/química , Homosserina Desidrogenase/metabolismo , Substâncias Macromoleculares/química , NAD/química , NAD/metabolismo , Ligantes , Substâncias Macromoleculares/metabolismo , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Análise Espectral
13.
J Comp Neurol ; 526(3): 373-396, 2018 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-29063602

RESUMO

Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.


Assuntos
Integrases/metabolismo , Proteínas Luminescentes/metabolismo , Inibição Neural/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Animais , Encéfalo/citologia , Antagonistas de Estrogênios/farmacologia , Regulação da Expressão Gênica/genética , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Integrases/genética , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição PAX2/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Somatostatina/metabolismo , Tamoxifeno/farmacologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Polipeptídeo N-Acetilgalactosaminiltransferase
14.
Proc Natl Acad Sci U S A ; 114(49): 13042-13047, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29158391

RESUMO

Although maternal nurturing behavior is extremely important for the preservation of a species, our knowledge of the biological underpinnings of these behaviors is insufficient. Here we show that the degree of a mother's nurturing behavior is regulated by factors present during her own fetal development. We found that Cin85-deficient (Cin85-/-) mother mice had reduced pituitary hormone prolactin (PRL) secretion as a result of excessive dopamine signaling in the brain. Their offspring matured normally and produced their own pups; however, nurturing behaviors such as pup retrieval and nursing were strongly inhibited. Surprisingly, when WT embryos were transplanted into the fallopian tubes of Cin85-/- mice, they also exhibited inhibited nurturing behavior as adults. Conversely, when Cin85-/- embryos were transplanted into the fallopian tubes of WT mice, the resultant pups exhibited normal nurturing behaviors as adults. When PRL was administered to Cin85-/- mice during late pregnancy, a higher proportion of the resultant pups exhibited nurturing behaviors as adults. This correlates with our findings that neural circuitry associated with nurturing behaviors was less active in pups born to Cin85-/- mothers, but PRL administration to mothers restored neural activity to normal levels. These results suggest that the prenatal period is extremely important in determining the expression of nurturing behaviors in the subsequent generation, and that maternal PRL is one of the critical factors for expression. In conclusion, perinatally secreted maternal PRL affects the expression of nurturing behaviors not only in a mother, but also in her pups when they have reached adulthood.


Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Comportamento Materno , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Efeitos Tardios da Exposição Pré-Natal/genética , Prolactina/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Animais Recém-Nascidos , Comportamento Animal , Encéfalo/fisiopatologia , Transferência Embrionária , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Mães , Proteínas de Neoplasias/deficiência , Proteínas do Tecido Nervoso/deficiência , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Prolactina/metabolismo , Maturidade Sexual/fisiologia , Transdução de Sinais
15.
PLoS One ; 11(5): e0155687, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27195805

RESUMO

Despite the strength of the Cre/loxP recombination system in animal models, its application in rats trails that in mice because of the lack of relevant reporter strains. Here, we generated a floxed STOP tdTomato rat that conditionally expresses a red fluorescent protein variant (tdTomato) in the presence of exogenous Cre recombinase. The tdTomato signal vividly visualizes neurons including their projection fibers and spines without any histological enhancement. In addition, a transgenic rat line (FLAME) that ubiquitously expresses tdTomato was successfully established by injecting intracytoplasmic Cre mRNA into fertilized ova. Our rat reporter system will facilitate connectome studies as well as the visualization of the fine structures of genetically identified cells for long periods both in vivo and ex vivo. Furthermore, FLAME is an ideal model for organ transplantation research owing to improved traceability of cells/tissues.


Assuntos
Proteínas Luminescentes/genética , Ratos Transgênicos , Animais , Linhagem da Célula , Cromossomos Artificiais Bacterianos , Dependovirus , Eletroporação , Eritrócitos/citologia , Feminino , Fertilização , Fibroblastos/metabolismo , Genes Reporter , Hipocampo/metabolismo , Imageamento Tridimensional , Integrases , Macrófagos/metabolismo , Masculino , RNA Mensageiro/metabolismo , Ratos , Transgenes , Proteína Vermelha Fluorescente
16.
J Mol Endocrinol ; 56(2): 113-22, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26643909

RESUMO

Increasing thermogenesis in white adipose tissues can be used to treat individuals at high risk for obesity and cardiovascular disease. The objective of this study was to determine the function of EP300-interacting inhibitor of differentiation (EID1), an inhibitor of muscle differentiation, in the induction of beige adipocytes from adipose mesenchymal stem cells (ADMSCs). Subcutaneous adipose tissue was obtained from healthy women undergoing abdominoplasty. ADMSCs were isolated in vitro, grown, and transfected with EID1 or EID1 siRNA, and differentiation was induced after 48 h by administering rosiglitazone. The effects of EID1 expression under the control of the aP2 promoter (aP2-EID1) were also evaluated in mature adipocytes that were differentiated from ADMSCs. Transfection of EID1 into ADMSCs reduced triglyceride accumulation while increasing levels of thermogenic proteins, such as PGC1α, TFAM, and mitochondrial uncoupling protein 1 (UCP1), all of which are markers of energy expenditure and mitochondrial activity. Furthermore, increased expression of the beige phenotype markers CITED1 and CD137 was observed. Transfection of aP2-EID1 transfection induced the conversion of mature white adipocytes to beige adipocytes, as evidenced by increased expression of PGC1α, UCP1, TFAM, and CITED1. These results indicate that EID1 can modulate ADMSCs, inducing a brown/beige lineage. EID1 may also activate beiging in white adipocytes obtained from subcutaneous human adipose tissue.


Assuntos
Adipócitos Brancos/fisiologia , Adipogenia , Células-Tronco Mesenquimais/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Adulto , Proteínas de Ciclo Celular , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Proteínas Nucleares/genética , PPAR gama/fisiologia , Proteínas Repressoras/genética , Gordura Subcutânea/citologia , Adulto Jovem
17.
Biochem Biophys Rep ; 3: 14-17, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29124164

RESUMO

Homoserine dehydrogenase (HSD; 305 amino acid residues) catalyzes an NAD(P)-dependent reversible reaction between l-homoserine and aspartate 4-semialdehyde and is involved in the aspartate pathway. HSD from the hyperthermophilic archaeon Sulfolobus tokodaii was markedly activated (2.5-fold) by the addition of 0.8 mM dithiothreitol. The crystal structure of the homodimer indicated that the activation was caused by cleavage of the disulfide bond formed between two cysteine residues (C303) in the C-terminal regions of the two subunits.

18.
J Biol Chem ; 286(36): 31885-95, 2011 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-21771796

RESUMO

The clustered protocadherins (Pcdhs), Pcdh-α, -ß, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-ß cluster control region (CCR) containing six HS sites (HS16, 17, 17', 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-ß gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-ß genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-ß expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.


Assuntos
Caderinas/genética , Família Multigênica , Neuropeptídeos/genética , Animais , Proteínas Relacionadas a Caderinas , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Camundongos , Neurônios , Protocaderinas , Sequências Reguladoras de Ácido Nucleico
19.
J Bone Miner Metab ; 28(2): 139-48, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19795094

RESUMO

The type III inorganic phosphate (Pi) transporter Pit-1 was previously found to be preferentially expressed in developing long bones. Several studies also described a regulation of its expression in cultured bone cells by osteotropic factors, suggesting a role of this transporter in bone metabolism. In the present study, we investigated the effects of the transgenic overexpression of Pit-1 in Wistar male rats on calcium phosphate and bone metabolism. A threefold increase and doubling of Pi transport activity were recorded in primary cultured osteoblastic cells derived from calvaria of two transgenic (Tg) lines compared with wild-type littermates (WT), respectively. Skeletal development was not affected by the transgene, and bone mass, analyzed by DXA, was slightly decreased in Tg compared with WT. Enhanced Pi uptake in calvaria-derived osteoblasts from Pit-1 Tg was associated with a significantly decreased expression of alkaline phosphatase activity and a normal deposition and calcification of the collagenous matrix. In 4-month-old adult Tg rats, serum Pi and renal Pi transport were increased compared with WT. The decrease of serum Ca concentration was associated with increased serum parathyroid hormone levels. Variations in serum Pi in Pit-1 Tg rats were negatively correlated with serum fibroblast growth factor-23, whereas 1,25-dihydroxyvitamin D(3) was not affected by Pit-1 overexpression. In conclusion, transgenic Pit-1 overexpression in rats affected bone and calcium phosphate metabolism. It also decreased alkaline phosphatase activity in osteoblasts without influencing bone matrix mineralization as well as skeletal development.


Assuntos
Densidade Óssea/genética , Osso e Ossos/metabolismo , Cálcio/metabolismo , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/biossíntese , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/fisiologia , Alanina/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Osso e Ossos/química , Osso e Ossos/diagnóstico por imagem , Calcitriol/sangue , Cálcio/sangue , Diferenciação Celular/genética , Fatores de Crescimento de Fibroblastos/sangue , Hidroxiapatitas/metabolismo , Masculino , Camundongos , Osteoblastos/metabolismo , Hormônio Paratireóideo/sangue , Fosfatos/sangue , Radiografia , Ratos , Ratos Transgênicos , Ratos Wistar , Crânio/citologia , Crânio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Tíbia/citologia , Tíbia/diagnóstico por imagem
20.
Gene ; 430(1-2): 86-94, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19038318

RESUMO

The mouse protocadherin (Pcdh) clusters, Pcdh-alpha, -beta, and -gamma, are located on chromosome 18. Many polymorphic variations are found in the Pcdh-alpha genes in wild-derived and laboratory mouse strains. In comparing the expression levels of Pcdh-alpha isoforms among several strains, we observed lower expression levels of Pcdh-alpha9 in BLG2 and BFM/2, and of Pcdh-alpha8 in C57BL/6 (B6) than in the other strains. For Pcdh-alpha8, high DNA methylation (72.7%) in the promoter region was found only in B6, whereas 36.4-44.3% methylation was seen in the other strains. On the other hand, the Pcdh-alpha9 DNA-methylation levels were similar (23.6-36.3%) among the strains regardless of the difference in expression levels. Interestingly, however, the Pcdh-alpha9 variable exon in both BLG2 and BFM/2 included a premature termination codon (PTC) generated by a nucleotide deletion or insertion. Treatment with emetine, a potent inhibitor of nonsense-mediated mRNA decay (NMD), increased the expression level of Pcdh-alpha9 from the BLG2-Pcdh-alpha locus. These data indicate that the transcription levels of mature Pcdh-alpha mRNAs are decreased by the DNA-methylation state of the Pcdh-alpha promoter regions and by the NMD pathway during RNA maturation. And we correct some previous data on Sugino, H., Toyama, T., Taguchi, Y., Esumi, S., Miyazaki, M., Yagi, T., (2004) Negative and positive effects of an IAP-LTR on nearby Pcdaalpha gene expression in the central nervous system and neuroblastoma cell lines, Gene 337 91-103.


Assuntos
Caderinas/genética , Códon sem Sentido/metabolismo , Metilação de DNA , Mutação da Fase de Leitura/genética , Regiões Promotoras Genéticas/genética , Estabilidade de RNA , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Caderinas/química , Caderinas/metabolismo , Metilação de DNA/efeitos dos fármacos , Emetina/farmacologia , Éxons/genética , Perfilação da Expressão Gênica , Genoma/genética , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Splicing de RNA/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie
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