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1.
Int J Mol Sci ; 23(7)2022 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-35409407

RESUMEN

The Saccharomyces cerevisiae Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms N2-methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112. Furthermore, the CCA terminus is essential for methylation by Trm11-Trm112, and Trm11-Trm112 also only methylates tRNAs with a regular-size variable region. In addition, the G10-C25 base pair is required for methylation by Trm11-Trm112. The data also demonstrated that Trm11-Trm112 recognizes the anticodon-loop and that U38 in tRNAAla acts negatively in terms of methylation. Likewise, the U32-A38 base pair in tRNACys negatively affects methylation. The only exception in our in vitro study was tRNAValAAC1. Our experiments showed that the tRNAValAAC1 transcript was slowly methylated by Trm11-Trm112. However, position 10 in this tRNA was reported to be unmodified G. We purified tRNAValAAC1 from wild-type and trm11 gene deletion strains and confirmed that a portion of tRNAValAAC1 is methylated by Trm11-Trm112 in S. cerevisiae. Thus, our study explains the m2G10 modification pattern of all S. cerevisiae class I tRNAs and elucidates the Trm11-Trm112 binding sites.


Asunto(s)
Metiltransferasas , Proteínas de Saccharomyces cerevisiae , Guanina/metabolismo , Metilación , Metiltransferasas/metabolismo , Conformación de Ácido Nucleico , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN de Transferencia de Valina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismo
2.
Int J Parasitol Parasites Wildl ; 25: 100975, 2024 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-39297148

RESUMEN

We investigated intraerythrocytic Babesia parasites in 21 Japanese wild boars, Sus scrofa leucomystax, captured in Wakayama Prefecture on the mainland from 2008 to 2009 and in 31 Japanese wild boars from 2011 to 2013 in Kochi Prefecture on Shikoku Island, Japan. We detected small subunit ribosomal RNA (18S rRNA) gene (SSUrDNA) fragments of a Babesia species in 17 boars from Wakayama and 18 boars from Kochi. The nearly full SSUrDNA sequence (1669 bps) of this species was determined. A FASTA search revealed that the SSUrDNA sequence of the Babesia sp. in Japanese wild boars was the most homologous to those of several Babesia isolates reported as Babesia gibsoni. Phylogenetic analysis showed that the Babesia sp. found in Japanese wild boars was the closest relative to B. gibsoni but made a different clade from B. gibsoni. The Babesia sp. in Japanese wild boars was completely different from Babesia sp. Suis found in a European domestic pig, Sus scrofa domesticus. By microscopic examination, ring-shaped, oval and pear-shaped small sized intraerythrocytic parasites were observed on blood smears of 12 of 18 Japanese wild boars whose blood smears could be examined in Wakayama. We also detected SSUrDNA fragments of a Hepatozoon species in 6 of the 21 wild boars from Wakayama. The nearly full SSUrDNA sequence (1774 bps) of the Hepatozoon sp. was shown to be identical to that of Hepatozoon apri.

3.
Yakugaku Zasshi ; 140(2): 301-312, 2020.
Artículo en Japonés | MEDLINE | ID: mdl-32009049

RESUMEN

In Japanese pharmaceutical education, the Model Core Curriculum was revised in 2013 to train pharmacists who can appropriately evaluate literature and use evidence-based medicine (EBM). However, in the investigation of EBM education at pharmaceutical universities in 2015, it was found that literature evaluation was hardly performed in the education of undergraduate students. One of the reason is the lack of EBM lecturers at each universities. Therefore, we adopted team-based learning (TBL) to educate more than 50 undergraduate students on the practical evaluation of literatures and the understanding of EBM concepts. The learning outcomes of this strategy were evaluated using the scores of individual tests before and after the class. As a result, the mean scores on the post-test significantly improved from 4.34 to 6.42 out of 10 total points (p<0.001). We further administered a questionnaire survey regarding the understanding of EBM (the mean score was 4.12). In conclusion, it was suggested that TBL for a large number was effective in EBM education for providing knowledge of literature evaluation and the understanding of fundamental concepts.


Asunto(s)
Curriculum , Educación en Farmacia/métodos , Medicina Basada en la Evidencia/educación , Medicina Basada en la Evidencia/métodos , Aprendizaje , Estudiantes de Farmacia , Evaluación Educacional , Humanos
4.
J Neurosci ; 28(9): 2131-46, 2008 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-18305247

RESUMEN

Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRalpha1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRalpha1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.


Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Neuronas Motoras/clasificación , Neuronas Motoras/fisiología , Médula Espinal/citología , Animales , Animales Recién Nacidos , Caspasa 3/metabolismo , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/deficiencia , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/deficiencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Miembro Posterior/embriología , Miembro Posterior/crecimiento & desarrollo , Miembro Posterior/inervación , Ratones , Ratones Noqueados , Husos Musculares/embriología , Husos Musculares/crecimiento & desarrollo , Husos Musculares/metabolismo , Músculo Esquelético/metabolismo , Mutación Puntual , Proteínas Proto-Oncogénicas c-ret/deficiencia , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas de Transporte Vesicular de Acetilcolina/genética , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo
5.
J Histochem Cytochem ; 56(3): 253-65, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18040079

RESUMEN

We examined the precise intracellular translocation of gamma subtype of protein kinase C (gammaPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged gammaPKC (gammaPKC-GFP) on the plasma membrane. In contrast, treatment with Ca(2+) ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of gammaPKC-GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that gammaPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of gammaPKC-GFP on the plasma membrane, Ca(2+) ionophore and NMDA rapidly translocated gammaPKC-GFP to the plasma membrane and then restricted gammaPKC-GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca(2+) influx alone induced the association of gammaPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of gammaPKC in response to various stimuli suggests a novel mechanism for PKC activation.


Asunto(s)
Activadores de Enzimas/farmacología , Proteína Quinasa C/metabolismo , Animales , Células CHO , Calcimicina/farmacología , Calcio/metabolismo , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Activación Enzimática , Ionóforos/farmacología , Isoenzimas/metabolismo , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , N-Metilaspartato/farmacología , Transporte de Proteínas , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Fracciones Subcelulares/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Uridina Trifosfato/farmacología
6.
Yakugaku Zasshi ; 138(5): 655-666, 2018.
Artículo en Japonés | MEDLINE | ID: mdl-29710010

RESUMEN

 Practicing evidence-based medicine (EBM) is likely to gain importance for clinical pharmacists in the relatively near future in Japan. An educational program including research and the critical appraisal of literature was required for pharmacy students as of 2015. We organized a six-month practical EBM course for pharmacy students at Hyogo University of Health Sciences. To evaluate its effectiveness, students took a 10-question test after completing the course. The mean score of six students was 8.33±1.79 points. We also conducted a 1-day practical EBM workshop for pharmacists. Changes in knowledge and skills related to EBM were evaluated based on the responses to 10 questions. Knowledge and skills related to several variables improved significantly after the workshop (6.36 points before versus 9.09 points after the workshop; p=0.023). The results suggested that our EBM educational course is effective in improving EBM-related knowledge and skills of pharmacists and pharmacy students.


Asunto(s)
Curriculum , Educación en Farmacia/métodos , Medicina Basada en la Evidencia/educación , Farmacéuticos , Estudiantes de Farmacia , Competencia Clínica , Educación , Humanos , Japón , Conocimiento , Farmacéuticos/psicología , Estudiantes de Farmacia/psicología
7.
Yakugaku Zasshi ; 137(8): 987-998, 2017.
Artículo en Japonés | MEDLINE | ID: mdl-28768952

RESUMEN

This study evaluated the effect of an evidence-based medicine (EBM) educational program on EBM-related knowledge and skills of pharmacists and pharmacy students. Our preliminary educational program included the following four sessions: 1) ice breaker, 2) formulation of answerable clinical questions from virtual clinical scenario using the PICO criteria, 3) critical appraisal of the literature using a checklist, and 4) critical appraisal of the results and integrating the evidence with experience and patients values. Change in knowledge and skills related to EBM were evaluated using pre- and post-seminar 4-point scale questionnaires comprising of 14 questions. A total of 23 pharmacists, 1 care manager, and 5 pharmacy students participated in our EBM educational seminar. Knowledge and skills related to several variables improved significantly post-seminar (pre-seminar 2.80 versus 3.26 post-seminar; p<0.001). Specifically, the skills of formulating answerable clinical questions from virtual clinical scenario and critical appraisal of the literature using a checklist improved. Our findings suggested that EBM educational program using problem-based learning was effective in improving EBM-related knowledge and skills of pharmacists and pharmacy students.


Asunto(s)
Educación en Farmacia/métodos , Medicina Basada en la Evidencia , Farmacéuticos , Evaluación de Programas y Proyectos de Salud/estadística & datos numéricos , Estudiantes de Farmacia , Competencia Clínica , Femenino , Estructura de Grupo , Humanos , Conocimiento , Masculino , Farmacéuticos/psicología , Estudiantes de Farmacia/psicología
8.
Parasitol Int ; 65(5 Pt B): 567-571, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26972181

RESUMEN

Babesia microti, a rodent babesia, is known as a pathogen of zoonosis, human babesiosis, is composed of several genotypes of small subunit ribosomal RNA gene (SSUrDNA) and different genotypes have been suggested to have different infectivity and pathogenicity to humans. We established a real-time PCR assay using SYBR Green I, which allows specific detection and absolute quantification for each SSUrDNA-type-B. microti of four SSUrDNA-types found in Japanese rodents even in mixed infection. In this assay, four genotype-specific primer pairs targeted on internal transcribed spacer 1 or 2 sequences were used. Primer pairs have the characteristics for a high specificity for homologous genotype DNA. The calibration curves of cycle threshold (Ct) values versus log concentrations of DNA for all four genotypes were linear over 107 fold range of DNA concentrations with correlation coefficient from 0.95 to 1 and sufficient amplification efficiency from 90% to 110%. The standard curves for all four genotypes were not changed even in the presence of heterologous DNA. In this paper, we introduce how to establish and perform the genotype-specific real-time PCR and our practical experimental tips to be recommended.

9.
Parasitol Int ; 65(5 Pt B): 526-531, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27033029

RESUMEN

Babesiosis has attracted attention as a zoonotic disease. The disease is caused in immunocompetent individuals almost solely by Babesia microti, a rodent babesia. Most cases of human babesiosis by B. microti have been reported in the endemic foci of the Northeastern coastal areas and upper Midwest regions of the United States, while some sporadic cases have recently been reported in several Asian countries including Japan. Our previous surveys identified that four small subunit ribosomal RNA gene (SSUrDNA) types of B. microti parasitize Japanese rodents. Indirect fluorescent antibody test (IFAT) is often performed for the diagnosis of babesiosis together with microscopical examination of thin blood smears and PCR. We established IFAT against four SSUrDNA-types of B. microti using erythrocytes of SCID mice or Syrian hamsters infected with each SSUrDNA-type B. microti. The results of IFAT for sera of ICR mice or Syrian hamsters infected with each SSUrDNA-type B. microti demonstrated that the four SSUrDNA-types of B. microti have different serotypes. Here, we report technical or practical procedures of IFAT, which gains sufficiently stable results, including procedures of continuous in vivo culture of B. microti.

10.
Parasitol Int ; 60(4): 403-9, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21723958

RESUMEN

We have developed a real-time PCR assay that can rapidly and differentially detect and quantify four genotypes of small subunit ribosomal RNA gene (SSUrDNA) of Babesia microti (Kobe-, Otsu-, Nagano- and US-types). In this assay, four genotype-specific pairs of primers targeted on internal transcribed spacer (ITS) 1 or 2 sequences were used and amplicons by each pair of primers were quantitatively detected by fluorescent SYBR Green I. The four genotype-specific pairs of primers displayed the high specificity for homologous genotype DNA. The standard curves of cycle threshold (Ct) values versus amount of target DNA per reaction (log) for all four genotypes were linear and the correlation coefficient (Rsq) values for the curves were from 0.970 to 0.997. The standard curves were almost identical even in the presence of heterologous genotype DNA. This assay could detect 10-30 fg purified DNA (equivalent to the amount of 1-5 parasite DNA) of each genotype B. microti. This assay could also detect each genotype B. microti infection in blood with 3×10(-6)%-1×10(-5)% parasitemia. This assay was applicable to field rodent and tick samples to reveal mixed infection in several samples, for which a single genotype of B. microti had been detected by direct sequencing analyses in our previous studies. This assay also seemed to be applicable to clinical human samples, showing Kobe-type positive results for the first Japanese babesiosis patient and the asymptomatic donor, both infected with Kobe-type B. microti.


Asunto(s)
Babesia microti/genética , Babesiosis/diagnóstico , Coinfección/diagnóstico , Dermatoglifia del ADN/métodos , ADN Protozoario/genética , ADN Ribosómico/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades de los Roedores/diagnóstico , Animales , Babesia microti/clasificación , Babesia microti/aislamiento & purificación , Babesiosis/sangre , Babesiosis/parasitología , Benzotiazoles , Coinfección/sangre , Coinfección/parasitología , Cartilla de ADN/genética , ADN Protozoario/análisis , ADN Ribosómico/análisis , Diaminas , Colorantes Fluorescentes/análisis , Genotipo , Humanos , Límite de Detección , Compuestos Orgánicos/análisis , Quinolinas , Enfermedades de los Roedores/sangre , Enfermedades de los Roedores/parasitología , Roedores , Análisis de Secuencia de ADN , Garrapatas
11.
Acta Histochem Cytochem ; 39(6): 173-81, 2006 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-17327904

RESUMEN

PICK1 is a protein which was initially identified as a protein kinase Calpha (alphaPKC) binding protein using the yeast two-hybrid system. In addition to alphaPKC, the PICK1 complex binds to and regulates various transmembrane proteins including receptors and transporters. However, it has not been clarified when and where PICK1 binds to alphaPKC. We examined the spatio-temporal interaction of PICK1 and PKC using live imaging techniques and showed that the activated alphaPKC binds to PICK1 and transports it to the plasma membrane. Although the membrane translocation of PICK1 requires the activation of alphaPKC, PICK1 is retained on the membrane even after PKC moves back to the cytosol. These results suggest that the interaction between alphaPKC and PICK1 is transient and may not be necessary for the regulation of receptors/transporters by PICK1 or by alphaPKC on the membrane.

12.
Genes Cells ; 11(9): 1051-70, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16923125

RESUMEN

To elucidate the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in cellular signaling, we constructed and expressed a pseudosubstrate of PDK1, designated as deltaAL-PIF, and characterized its properties in cultured cells. deltaAL-PIF consists of two fused proteins of the protein kinase Cdelta (deltaPKC) activation loop (deltaAL) and PDK1-interacting fragment (PIF). The phosphorylation of deltaAL-PIF was detected with anti-deltaPKC phospho-Thr505-specific antibody and was increased in proportion to the expression level of co-expressed GST-PDK1, indicating that it acts as a pseudosubstrate of PDK1. In cells expressing deltaAL-PIF, basal phosphorylation level at the activation loop of PKBalpha, deltaPKC and gammaPKC was reduced, compared with that in control cells, suggesting that deltaAL-PIF functions as an inhibitory molecule for PDK1. deltaAL-PIF affected the stability, translocation and endogenous activity of PKCs. These effects of deltaAL-PIF on gammaPKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing deltaAL-PIF for 3 days. These findings revealed that deltaAL-PIF served as an effective pseudosubstrate and an inhibitory molecule for PDK1, suggesting that this molecule can be used as a tool for investigating PDK-mediated cellular functions as well as being applicable for anti-cancer therapy.


Asunto(s)
Expresión Génica , Proteína Quinasa C-delta/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Adenosina Trifosfato/farmacología , Animales , Apoptosis/efectos de los fármacos , Células CHO , Células COS , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Cricetulus , Activación Enzimática/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Ácido Glutámico/farmacología , Ratones , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosfotreonina/metabolismo , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Proteínas Recombinantes de Fusión/química
13.
Genes Cells ; 9(10): 945-57, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15461665

RESUMEN

To elucidate spatial and temporal profiles of the protein kinase C (PKC) activation in relation to neuronal functions including synaptic plasticity, we tried to detect PKC translocation in living brain slices. We first developed brain region-specific and inducible gammaPKC-GFP transgenic mice using a tetracycline (tet)-regulated system. In the transgenic mice, the expression of gammaPKC-GFP was region-specifically regulated by the promoter and abolished by the administration of doxycycline. Cerebellar slices from the mice were utilized for intracellular recording and fluorescence imaging of gammaPKC-GFP in Purkinje cells. GFP fluorescence was uniformly distributed from soma to dendritic arbor. When mGluR agonists were applied, the intensity was transiently increased at the edge of the dendrite and concomitantly decreased in the cytoplasm, indicating that gammaPKC translocated to the plasma membrane. This transient change in the pattern of GFP fluorescence simultaneously occurred throughout the Purkinje cell dendrites by agonist stimulation. Translocation of gammaPKC-GFP was also induced by electrical stimulation of parallel fibres. However, the event was not restricted at the distal dendrites, propagated forwardly along the dendritic tree and reached to the proximal trunk close to the soma. Time course of the propagation was slower than the electrical signal and Ca(2+) waves and faster than conveying molecules through microtubules. The present results indicate that PKC signals activated locally by parallel fibre input could propagate to the soma through dendrites in living Purkinje neurones. The findings may provide us with a new insight for understanding molecular mechanisms of the synaptic plasticity including cerebellar long-term depression.


Asunto(s)
Dendritas/metabolismo , Proteína Quinasa C/metabolismo , Células de Purkinje/metabolismo , Animales , Antibacterianos/farmacología , Encéfalo/metabolismo , Doxiciclina/farmacología , Electrofisiología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Ratones , Ratones Transgénicos , Proteína Quinasa C/genética , Transporte de Proteínas/fisiología
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