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1.
Sci Rep ; 10(1): 4934, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188933

RESUMO

Prion diseases comprise a fatal neuropathy caused by the conversion of prion protein from a cellular (PrPC) to a pathological (PrPSc) isoform. Previously, we obtained an RNA aptamer, r(GGAGGAGGAGGA) (R12), that folds into a unique G-quadruplex. The R12 homodimer binds to a PrPC molecule, inhibiting PrPC-to-PrPSc conversion. Here, we developed a new RNA aptamer, r(GGAGGAGGAGGAGGAGGAGGAGGA) (R24), where two R12s are tandemly connected. The 50% inhibitory concentration for the formation of PrPSc (IC50) of R24 in scrapie-infected cell lines was ca. 100 nM, i.e., much lower than that of R12 by two orders. Except for some antibodies, R24 exhibited the lowest recorded IC50 and the highest anti-prion activity. We also developed a related aptamer, r(GGAGGAGGAGGA-A-GGAGGAGGAGGA) (R12-A-R12), IC50 being ca. 500 nM. The structure of a single R12-A-R12 molecule determined by NMR resembled that of the R12 homodimer. The quadruplex structure of either R24 or R12-A-R12 is unimolecular, and therefore the structure could be stably formed when they are administered to a prion-infected cell culture. This may be the reason they can exert high anti-prion activity.


Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas PrPSc/química , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/genética , Proteínas Priônicas , Relação Estrutura-Atividade
2.
Int J Rheumatol ; 2019: 5710340, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30906325

RESUMO

We examined whether the cathepsin K inhibitor, ONO-5334, administered alone or in combination with methotrexate (MTX), could ameliorate joint destruction evoked by collagen-induced arthritis (CIA) in female cynomolgus monkeys. CIA was induced by immunizing with bovine type II collagen. ONO-5334 (30 mg/kg/day) was orally administered once daily and MTX (10 mg/body/day) twice weekly for 9 weeks. X-ray (evaluation of joint destruction) and swelling (inflammatory) scores of proximal interphalangeal (PIP), distal interphalangeal (DIP), and metacarpophalangeal (MP) joints were evaluated. Urinary concentrations of C-terminal telopeptide of type I collagen (CTX-I) and type II collagen (CTX-II) were measured. Arthritis, accompanied by bone and cartilage destruction, was successfully induced in this collagen-induced arthritis monkey model. ONO-5334 showed no suppressive effect on joint swelling, while the joint swelling scores in the MTX and combination (ONO-5334 + MTX) groups were less than 50% compared with the control group. ONO-5334 decreased X-ray score by a mean of 64% (p<0.05 vs the control group), and MTX also decreased in X-ray score by a mean of 46% but with no statistical significance. Combination of ONO-5334 and MTX further decreased the X-ray score by 28% over MTX group (74% reduction vs the control group, p<0.01). Maximum increase in CTX-I (10-fold) and CTX-II (7-fold) compared to baseline was observed at 7 and 3 weeks after the first sensitization, respectively. After treatment with ONO-5334 alone or in combination with MTX, concentrations were maintained near baseline for both markers. In conclusion, ONO-5334 prevented joint destruction but not joint inflammation in this monkey CIA model. Concomitant use of ONO-5334 with MTX reduced architectural joint destruction compared to MTX alone; therefore, ONO-5334 may help to prevent joint destruction in combination with MTX for the treatment of rheumatoid arthritis.

3.
Annu Int Conf IEEE Eng Med Biol Soc ; 2019: 6872-6875, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31947419

RESUMO

For utilizing optogenetics in neuroscience research, a proper setup is necessary, which delivers sufficient light to target cells and minimizes unexpected side effects caused by light exposure. In this study, we were interested in the area of influence of optical surface stimulation on a spinal cord tissue. We built a 3D spinal cord structure of rat and utilized the Monte-Carlo methods to simulate the light transport in it. We first evaluated light propagation in homogeneous nervous tissue models. For a 10-mW, 470-nm light source, light intensity of 1 mW=mm2 was detected at depths of 1:14 and 1:77 mm in white and grey matter, respectively. This indicated a narrower spreading pattern of light in the white matter than in the grey matter. Since the grey matter, which contains the somatosensory pathways, is an important target of spinal cord stimulation, we focused on investigating how much light could reach this area in a multi-layered structure. The results showed that when an optical fiber was positioned in the center line of the spinal cord dorsal surface, most of the light energy was absorbed before reaching the grey matter. In contrast, when we put the fiber on a lateral position, 0:8 mm away from the central line, relatively sufficient light intensity could be detected deep into the lamina 5 area. The experimental results obtained herein suggest that tissue type and the position of stimulation could greatly affect the area of influence of light stimulation in a 3D spinal cord. It is important to consider the location of the interested neural pathways and plan a proper stimulation site before conducting optogenetic surface stimulation of the spinal cord.


Assuntos
Estimulação da Medula Espinal , Medula Espinal , Substância Branca , Animais , Substância Cinzenta , Optogenética , Ratos
4.
J Bone Miner Metab ; 37(4): 636-647, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30357565

RESUMO

This study evaluated the long-term effects of the cathepsin K inhibitor ONO-5334 on bone mass and strength in ovariectomised (OVX) cynomolgus monkeys. Animals were assigned to one of the following six groups: Sham (non-OVX), OVX control treated with vehicle, ONO-5334 1.2, 6 or 30 mg/kg/day, p.o., or alendronate (ALN) 0.05 mg/kg/2 weeks, i.v. for 16 months. Peripheral quantitative computed tomography (pQCT) analysis revealed that ONO-5334 increased not only trabecular bone mineral density (BMD) but also cortical BMD in the distal radius and the lumbar vertebra. ONO-5334 and ALN suppressed the deterioration of trabecular architecture by micro-CT analysis in the distal radius. Assessments of bone strength showed that ONO-5334 increased maximum load at the distal and midshaft radius. The linear regression lines between bone mass and strength in the lumbar vertebra were tended to be shifted towards increasing bone strength in the ONO-5334 6 and 30 mg/kg groups compared with the ALN groups. This indicated that bone strength was higher in the ONO-5334 groups than the ALN group, even though bone mineral content (BMC) and BMD were comparable. Subpopulation analysis revealed that, at similar integral BMC or BMD level, cortical bone mass for ONO-5334 was higher than for ALN; the opposite effects were observed for trabecular bone. In conclusion, ONO-5334 preferentially increased cortical bone, which may provide a greater contribution to bone strength. Since these results support a different mode of action for ONO-5334 compared with that of ALN, ONO-5334 may offer new therapeutic options to patients with osteoporosis.


Assuntos
Densidade Óssea/fisiologia , Catepsina K/antagonistas & inibidores , Osso Cortical/fisiologia , Ovariectomia , Tiazolidinas/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Osso Cortical/diagnóstico por imagem , Osso Cortical/efeitos dos fármacos , Feminino , Humanos , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/efeitos dos fármacos , Macaca fascicularis , Tamanho do Órgão , Rádio (Anatomia)/diagnóstico por imagem , Rádio (Anatomia)/efeitos dos fármacos , Tomografia Computadorizada por Raios X , Microtomografia por Raio-X
5.
Int J Rheumatol ; 2016: 8938916, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26949397

RESUMO

The role of cathepsin K in joint degradation in a model of collagen-induced arthritis (CIA) in cynomolgus monkey was examined using biochemical markers and histology. Joint swelling, urinary C-telopeptide of type II collagen (CTX-II), deoxypyridinoline (DPD), and N- and C-telopeptides of type I collagen (NTX and CTX-I, resp.) were analyzed. Immunohistochemistry of type II collagen, cathepsin K, and CTX-II were performed using joints. Joint swelling reached peak on day 42 and continued at this level. The CTX-II level peaked on day 28 and declined thereafter, while CTX-I, NTX, and DPD reached plateau on day 43. Joint swelling was positively correlated with CTX-II increases on days 20 and 42/43, with increases in CTX-I and NTX/Cr on days 42/43 and 84, and with DPD increases throughout the study period. Intense cathepsin K staining was observed in osteoclasts and in articular cartilage and synovial tissue in arthritic joints. CTX-II was present in the superficial layer of articular cartilage in CIA monkeys. Evidence from biochemical markers suggests that matrix degradation in the CIA model starts with degradation of cartilage, rather than bone resorption. Cathepsin K expressed in osteoclasts, articular cartilage, and synovial tissue may contribute to degradation of cartilage.

6.
Bone ; 86: 43-52, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26921823

RESUMO

We examined the effects of ONO-5334, a cathepsin K inhibitor, on bone markers, BMD, strength and histomorphometry in ovariectomized (OVX) cynomolgus monkeys. ONO-5334 (1.2, 6 and 30mg/kg/day, p.o.), alendronate (0.05mg/kg/2weeks, i.v.), or vehicle was administered to OVX monkeys (all groups N=20) for 16months. A concurrent Sham group (N=20) was also treated with vehicle for 16months. OVX significantly increased bone resorption and formation markers and decreased BMD in lumbar vertebra, femoral neck, proximal tibia and distal radius. Alendronate suppressed these parameters to a level similar to that in the Sham-operated monkeys. ONO-5334 at doses 6 and 30mg/kg decreased bone resorption markers to a level roughly half of that in the Sham group, while keeping bone formation markers level above that in the Sham monkeys. Changes in DXA BMD confirmed that ONO-5334 at doses 6 and 30mg/kg increased BMD to a level greater than that in the Sham group in all examined sites. In the proximal tibia, in vivo pQCT analysis showed that ONO-5334 at doses 6 and 30mg/kg suppressed trabecular BMD loss to the sham level. However, ONO-5334 increased cortical BMD, cortical area and cortical thickness to a level greater than that in the Sham group, suggesting that ONO-5334 improves both cortical BMD and cortical geometry. Histomorphometric analysis revealed that ONO-5334 suppressed bone formation rate (BFR) at osteonal site in the midshaft femur but did not influence OVX-induced increase in BFR at either the periosteal or endocortical surfaces. Unlike alendronate, ONO-5334 increased osteoclasts surface (Oc.S/BS) and serum tartrate-resistant acid phosphatise 5b (TRAP5b) activity, highlighting the difference in the mode of action between these two drugs. Our results suggest that ONO-5334 has therapeutic potential not only in vertebral bones, but also in non-vertebral bones.


Assuntos
Biomarcadores/sangue , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/anatomia & histologia , Osso e Ossos/fisiologia , Catepsina K/antagonistas & inibidores , Ovariectomia , Tiazolidinas/farmacologia , Absorciometria de Fóton , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/diagnóstico por imagem , Estradiol/sangue , Feminino , Macaca fascicularis , Tiazolidinas/administração & dosagem , Tiazolidinas/farmacocinética , Tíbia/anatomia & histologia , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacos , Tíbia/fisiologia , Tomografia Computadorizada por Raios X
7.
Nat Commun ; 6: 8018, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26292667

RESUMO

The Tohoku Medical Megabank Organization reports the whole-genome sequences of 1,070 healthy Japanese individuals and construction of a Japanese population reference panel (1KJPN). Here we identify through this high-coverage sequencing (32.4 × on average), 21.2 million, including 12 million novel, single-nucleotide variants (SNVs) at an estimated false discovery rate of <1.0%. This detailed analysis detected signatures for purifying selection on regulatory elements as well as coding regions. We also catalogue structural variants, including 3.4 million insertions and deletions, and 25,923 genic copy-number variants. The 1KJPN was effective for imputing genotypes of the Japanese population genome wide. These data demonstrate the value of high-coverage sequencing for constructing population-specific variant panels, which covers 99.0% SNVs of minor allele frequency ≥0.1%, and its value for identifying causal rare variants of complex human disease phenotypes in genetic association studies.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Variação Genética , Genoma Humano , Haplótipos , Humanos
8.
Annu Int Conf IEEE Eng Med Biol Soc ; 2015: 4578-81, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26737313

RESUMO

Recent studies have demonstrated that active gait training can recover voluntary locomotive ability of paralyzed rats. Rehabilitation devices used for studying spinal cord injury to date are usually fixed on a treadmill, but they have been used only slightly for active training. To process active rehabilitation, a wearable, lightweight device with adequate output is needed. Pouch motors, soft pneumatic actuators, are extremely light and have other benefits such as low cost, easy fabrication, and highly customizable design. They can be used to develop active gait rehabilitation devices. However, performance details of different motor designs have not been examined. As described herein, to build a wearable gait assistive device for rat study, we specifically examine how to design small pouch motors with a good contraction ratio and force output. Results show that pouch performance decreases dramatically with size, but better output is obtainable by separation into small 0.8 length-to-width ratio rooms. We used this knowledge to produce an assistive robot suit for gait rehabilitation and to test it with paralyzed rats. Results show that these small pouches can produce sufficient power to control hip joint movements during gait training. They can reveal the potential for new pouch motor applications for spinal cord injury studies.


Assuntos
Marcha , Animais , Teste de Esforço , Terapia por Exercício , Ratos , Equipamentos de Autoajuda , Traumatismos da Medula Espinal
9.
Blood ; 124(24): 3587-96, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25298035

RESUMO

Ecotropic viral integration site 1 (Evi1) is a transcription factor that is highly expressed in hematopoietic stem cells and is crucial for their self-renewal capacity. Aberrant expression of Evi1 is observed in 5% to 10% of de novo acute myeloid leukemia (AML) patients and predicts poor prognosis, reflecting multiple leukemogenic properties of Evi1. Here, we show that thrombopoietin (THPO) signaling is implicated in growth and survival of Evi1-expressing cells using a mouse model of Evi1 leukemia. We first identified that the expression of megakaryocytic surface molecules such as ITGA2B (CD41) and the THPO receptor, MPL, positively correlates with EVI1 expression in AML patients. In agreement with this finding, a subpopulation of bone marrow and spleen cells derived from Evi1 leukemia mice expressed both CD41 and Mpl. CD41(+) Evi1 leukemia cells induced secondary leukemia more efficiently than CD41(-) cells in a serial bone marrow transplantation assay. Importantly, the CD41(+) cells predominantly expressing Mpl effectively proliferated and survived on OP9 stromal cells in the presence of THPO via upregulating BCL-xL expression, suggesting an essential role of the THPO/MPL/BCL-xL cascade in enhancing the progression of Evi1 leukemia. These observations provide a novel aspect of the diverse functions of Evi1 in leukemogenesis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Leucemia/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Oncogênicas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Receptores de Trombopoetina/metabolismo , Transdução de Sinais , Trombopoetina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica/genética , Leucemia/genética , Leucemia/patologia , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Oncogênicas/genética , Glicoproteína IIb da Membrana de Plaquetas/genética , Proto-Oncogenes/genética , Receptores de Trombopoetina/genética , Trombopoetina/genética , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Proteína bcl-X/biossíntese , Proteína bcl-X/genética
10.
BMC Genomics ; 15: 673, 2014 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-25109789

RESUMO

BACKGROUND: Validation of single nucleotide variations in whole-genome sequencing is critical for studying disease-related variations in large populations. A combination of different types of next-generation sequencers for analyzing individual genomes may be an efficient means of validating multiple single nucleotide variations calls simultaneously. RESULTS: Here, we analyzed 12 independent Japanese genomes using two next-generation sequencing platforms: the Illumina HiSeq 2500 platform for whole-genome sequencing (average depth 32.4×), and the Ion Proton semiconductor sequencer for whole exome sequencing (average depth 109×). Single nucleotide polymorphism (SNP) calls based on the Illumina Human Omni 2.5-8 SNP chip data were used as the reference. We compared the variant calls for the 12 samples, and found that the concordance between the two next-generation sequencing platforms varied between 83% and 97%. CONCLUSIONS: Our results show the versatility and usefulness of the combination of exome sequencing with whole-genome sequencing in studies of human population genetics and demonstrate that combining data from multiple sequencing platforms is an efficient approach to validate and supplement SNP calls.


Assuntos
Exoma/genética , Genômica/instrumentação , Polimorfismo de Nucleotídeo Único , Semicondutores , Análise de Sequência de DNA/instrumentação , Composição de Bases , Feminino , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Reprodutibilidade dos Testes
11.
Bone ; 65: 1-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24784023

RESUMO

This study examined the effect of ONO-5334, a cathepsin K inhibitor, on bone turnover, mineral density (BMD), mechanical strength and microstructure in ovariectomized (OVX) cynomolgus monkeys. Vehicle, ONO-5334 (3, 10 or 30 mg/kg) or alendronate (0.5 mg/kg) was orally administered for eight months to sham- and OVX-operated monkeys. ONO-5334 dose-dependently suppressed OVX-induced increase in bone turnover markers (urinary C-terminal cross-linking telopeptide of type I collagen (CTX) and serum osteocalcin). At the dose of 30 mg/kg, ONO-5334 maintained urinary CTX at nearly zero level and kept serum osteocalcin around the level of the sham animals. Marker levels in the alendronate-treated animals were similar to those in the sham animals throughout the study. ONO-5334 dose-dependently reversed the effect of OVX on vertebral BMD as measured by dual-energy X-ray absorptiometry (DXA) with improvement of bone mechanical strength. Both ONO-5334 and alendronate suppressed OVX-induced changes in vertebral microstructure and turnover state. In the femoral neck, peripheral quantitative computed tomography (pQCT) analysis showed that ONO-5334 increased total and cortical BMD. In particular, ONO-5334 significantly increased cortical BMD with improvement of bone mechanical strength. In microstructural analysis, alendronate suppressed OVX-induced increase in femoral mid-shaft osteonal bone formation rate (BFR) to a level below that recorded in the sham group, whereas ONO-5334 at 30 mg/kg did not suppress periosteal, osteonal and endocortical BFR. This finding supports the significant effect of ONO-5334 on cortical BMD and mechanical strength in the femoral neck. The results of this study suggest that ONO-5334 has good therapeutic potential for the treatment of osteoporosis.


Assuntos
Osso e Ossos/efeitos dos fármacos , Catepsina K/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Ovariectomia , Tiazolidinas/farmacologia , Absorciometria de Fóton , Animais , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Osso e Ossos/ultraestrutura , Feminino , Macaca fascicularis
12.
Opt Express ; 22(7): 8533-40, 2014 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-24718225

RESUMO

A novel wavelength combiner using non-uniform refractive index distribution within a multimode interference device is proposed and simulated. The refractive index step creates separate localized modes with different effective refractive indices and two modes are strongly excited which form the basis of an interferometer. We applied the concept to 1.30/1.31 µm and 1.31/1.55 µm wavelength combiners on an InP substrate. The lengths of the devices are 1272 µm and 484 µm with simulated insertion losses of 0.6 dB and 0.67 dB respectively.

13.
Cancer Sci ; 105(5): 512-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24612037

RESUMO

Leukemia stem cells (LSC) are resistant to conventional chemotherapy and persistent LSC after chemotherapy are supposed to be a major cause of relapse. However, information on genetic or epigenetic regulation of stem cell properties is still limited and LSC-targeted drugs have scarcely been identified. Epigenetic regulators are associated with many cellular processes including maintenance of stem cells. Of note are polycomb group proteins, because they potentially control stemness, and can be pharmacologically targeted by a selective inhibitor (DZNep). Therefore, we investigated the therapeutic potential of EZH2 inhibition in mixed lineage leukemia (MLL) fusion leukemia. Intriguingly, EZH2 inhibition by DZNep or shRNA not only suppressed MLL fusion leukemia proliferation but also reduced leukemia initiating cells (LIC) frequency. Expression analysis suggested that p16 upregulation was responsible for LICs reduction. Knockdown of p16 canceled the survival advantage of mice treated with DZNep. Chromatin immunoprecipitation assays demonstrated that EZH2 was highly enriched around the transcription-start-site of p16, together with H3K27 methylation marks in MLL/ENL and Hoxa9/Meis1 transduced cells but not in E2A/HLF transduced cells. Although high expression of Hoxa9 in MLL fusion leukemia is supposed to be responsible for the recruitment of EZH2, our data also suggest that there may be some other mechanisms independent of Hoxa9 activation to suppress p16 expression, because expression levels of Hoxa9 and p16 were not inversely related between MLL/ENL and Hoxa9/Meis1 transduced cells. In summary, our findings show that EZH2 is a potential therapeutic target of MLL fusion leukemia stem cells.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Leucemia/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Complexo Repressor Polycomb 2/antagonistas & inibidores , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Leucemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Meis1 , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Ativação Transcricional , Transplante Heterólogo , Regulação para Cima
14.
Cell Struct Funct ; 39(1): 23-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24334484

RESUMO

Microfold (M) cells are intestinal epithelial cells specialized for sampling and transport of luminal antigens to gut-associated lymphoid tissue for initiation of both mucosal and systemic immune responses. Therefore, M-cell targeted vaccination has the potential to be a better immunization strategy. Glycoprotein 2 (GP2), an antigen uptake receptor for FimH(+) bacteria on M cells, can be a good target for this purpose. Aptamers are oligonucleotides that bind to a variety of target molecules with high specificity and affinity. Together with its low toxic feature, aptamers serves as a tool of molecular-targeted delivery. In this study, we used Systematic Evolution of Ligands by EXponential enrichment (SELEX) to isolate aptamers specific to murine GP2 (mGP2). After ten rounds of SELEX, eleven different aptamer sequences were selected. Among them, the most frequently appeared sequence (~60%) were aptamer NO. 1 (Apt1), and the second most (~7%) were aptamer NO. 5 (Apt5). In vitro binding experiment confirmed that only Apt1 and Apt5 specifically bound to mGP2 among eleven aptamers initially selected. Apt1 showed the strongest affinity with mGP2, with the Kd value of 110±2.6 nM evaluated by BIACORE. Binding assays with mutants of Apt1 suggest that, in addition to the loop structure, the nucleotide sequence, AAAUA, in the loop is important for binding to mGP2. Furthermore, this aptamer was able to bind to mGP2 expressed on the cell surface. These results suggest that this mGP2-specific aptamer could serve as a valuable tool for testing M-cell-targeted vaccine delivery in the murine model system.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Proteínas Ligadas por GPI/metabolismo , Técnica de Seleção de Aptâmeros , Animais , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Especificidade por Substrato
15.
PLoS One ; 8(4): e60205, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23560077

RESUMO

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.


Assuntos
Blastocisto/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas/genética , Zigoto/metabolismo , 5-Metilcitosina/metabolismo , Animais , Blastocisto/citologia , Cromatina/genética , Cromatina/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores Sexuais , Zigoto/citologia
16.
J Toxicol Sci ; 38(2): 291-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23535408

RESUMO

Hypothyroidism induced by xenobiotic treatment was analyzed for possible underlying mechanism(s) on the basis of different responses of the thyroid gland and the liver, using a newly-created database of repeated-dose toxicity of 500 chemicals. Two mechanisms are proposed: direct inhibition of thyroid hormone biosynthesis in the thyroid gland, and stimulated degradation of thyroid hormone by induction of hepatic drug-metabolizing enzymes. In the database there were 10 chemicals inducing hypertrophy/hyperplasia of follicular cells in the thyroid gland and having data on thyroid glands. On the basis of the chemical structure and information available in the literature, we judged three chemicals to be typical thioamide derivatives that act directly on the thyroid gland, and the others as non-thioamide derivatives that were unlikely to have any direct action on the thyroid gland. All these chemicals were classified into two groups using the ratios of relative weight increase rate of thyroid gland versus that of the liver. These values were at least 1.7, but 3.2 or more in the most of the cases for thioamide derivatives, and 1.2 or less for non-thioamide derivatives. This background analysis suggests the feasibility of parameter-supported speculation on the possible underlying mechanism when new repeated-dose toxicity data on hypothyroidism becomes available.


Assuntos
Hipotireoidismo/induzido quimicamente , Tioamidas/toxicidade , Animais , Relação Dose-Resposta a Droga , Humanos , Hiperplasia , Fígado/efeitos dos fármacos , Fígado/patologia , Tamanho do Órgão/efeitos dos fármacos , Tioamidas/administração & dosagem , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/patologia
17.
J Reprod Dev ; 59(3): 258-65, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23449310

RESUMO

Degradation of maternally stored mRNAs after fertilization is an essential process for mammalian embryogenesis. Maternal mRNA degradation depending on deadenylases in mammalian early embryos has been mostly speculated, rather than directly demonstrated. Previously, we found that gene expression of nocturnin, which functions as a circadian clock-controlled deadenylase in mammalian cells, was clearly changed during the maternal-to-zygotic transition (MZT). Here, we investigated the possible role of nocturnin during mouse MZT. First, we examined the expression profile and localization of nocturnin in mouse oocytes and early embryos. The abundance of Nocturnin mRNA level was significantly decreased from the MII to 4-cell stages and slightly increased from the 8-cell to blastocyst stages, whereas the Nocturnin protein level was almost stable in all examined cells including GV and MII oocytes and early embryos. Nocturnin was localized in both the cytoplasm and the nucleus of all examined cells. We then examined the effect of loss or gain of Nocturnin function on early embryonic development. Knockdown of Nocturnin by injection of Nocturnin antisense expression vector into 1-cell embryos resulted in the delay of early embryonic development to the early blastocyst stage. Moreover, Nocturnin-overexpressed embryos by injection of Nocturnin expression vector impaired their development from the 1-cell to 2-cell or 4-cell stages. These results suggest that precise expression of nocturnin is critical to proper development of early mouse embryos. Functional analysis of nocturnin may contribute to the understanding of the possible role of the deadenylase at mouse MZT.


Assuntos
Blastocisto/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Zigoto/fisiologia , Animais , Blastocisto/citologia , Núcleo Celular/metabolismo , Ritmo Circadiano , Citoplasma/metabolismo , Técnicas de Cultura Embrionária , Feminino , Fertilização , Fertilização In Vitro , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos ICR , Oócitos/citologia , Gravidez , Prenhez , Fatores de Tempo
18.
Biol Open ; 2(2): 170-82, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23429752

RESUMO

During the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin-proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT.

19.
Nucleic Acids Res ; 41(2): 1355-62, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23180780

RESUMO

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP(C)) of PrP into an abnormal form (PrP(Sc)) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP(C) is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP(Sc) level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP(C), resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.


Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas PrPC/química , Animais , Aptâmeros de Nucleotídeos/farmacologia , Bovinos , Linhagem Celular , Camundongos , Modelos Moleculares , Peptídeos/química , Proteínas PrPSc/análise
20.
Opt Express ; 20(26): B371-6, 2012 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-23262875

RESUMO

A novel polarization splitter on an InP substrate utilizing an MMI coupler loaded with a dielectric and gold layer pad is proposed and simulated. A tilted joint is used for adjusting the phases of TE and TM modes. The MMI section is less than 540 µm. Simulations show that the device has a polarization extinction ratio over 23 dB and an insertion loss below 0.7 dB over the entire C-band for both TE and TM polarizations. The device design was optimized to maximize the wavelength range and tolerance for manufacturing variations.

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