Amida has no homology with known proteins, but showed significant homology to a clone AA from mouse expressed sequence tag EST library. Transcripts of approximately 1. Maximal electroconvulsive seizure with pretreatment with cycloheximide, which superinduce immediate early genes in the hippocampus, did not increase the mRNA Fig.
Amida mRNA was mainly detected in the hippocampus and dentate gyrus by in situ hybridization data not shown. To confirm that Amida is associated to Arc, a reconstruction experiment in vitro was performed. Amida protein was translated in vitro and [ 35 S]methionine-labeled using the reticulocyte lysate system. This discrepancy may be due to the acidity. The mixture was incubated with the lysate of PC12 cells which abundantly express Arc protein. Then, immunoprecipitation with anti-Arc-specific antiserum was performed. Labeled Amida protein was specifically co-precipitated, while the in vitro translated products of other clones were not Fig.
Amida is associated with Arc in vitro and in vivo. A , in vitro association of Amida and Arc. In vitro translated and 35 S-labeled Amida was incubated with lysate from PC12 cells which express Arc protein abundantly. Lane 1 represents Amida. Lanes 2 and 3 show the other two-hybrid positive clones which could not bind Arc protein under these conditions.
B , in vivo association of Amida and Arc. After 48 h transfection, the lysate was immunoblotted with anti-Arc antibody lane 1. The lysate lane 2 was also immunoprecipitated with anti-Myc antibody, and the resultant supernatant lane 3 and precipitate lane 4 were immunoblotted with anti-EGFP antibody. After 48 h, cells were lysed and immunoprecipitated with anti-Myc monoclonal antibody.
EGFP-Arc fusion protein of 80 kDa was specifically precipitated with Myc-tagged Amida protein, while no signal was seen when control Myc-tag peptide was co-expressed. Furthermore, we tried to examine whether Amida interacts with the other proteins nonspecifically or not. An intense fluorescent signal was exclusively detected inside the nucleus Fig. Three days after transfection, most of the COS-7 cells expressing the EGFP-Amida fusion protein showed a small and round morphology which is characteristic of cells undergoing apoptosis.
Similar changes were seen in other cell lines such as NG data not shown.
Amida protein localizes in the nucleus and induces apoptosis. Arrows indicate the same positions in B and C. Condensation and fragmentation of the nuclei of Amida-transfected cells were seen. NG cells were also transfected with the same expression vector containing the Myc-tagged Amida F-H. F shows immunocytochemistry using anti-Myc monoclonal antibody with rhodamine. To confirm that the Amida expressing cells were dying by apoptosis, a double staining study using the TUNEL reaction and immunocytochemistry against Myc-tag was performed. It was thought that Amida has two putative nuclear localization signals which abundantly contain positively charged residues such as lysine or arginine.
Deletion mutants D1, D2, and D3 as shown in Fig. Constructs of Amida deletion mutants. The abilities of each mutant for the association with Arc, nuclear localization and cell death are summarized. Morphological analyses to determine the effects of the nuclear localization signals. Intracellular distribution of EGFP fusion proteins of each deletion construct was examined. Analysis of nuclear localization and cell death induction by D3. Arrows in D show healthy morphology of the nuclei and in H induced cell death. We investigated the ability of each deletion mutant to promote cell death.
These results indicate that the D3 region is sufficient but nuclear localization of this region is necessary for promotion of cell death as summarized in Fig. To investigate the domain of Amida which interacts with Arc, each deletion mutant was co-transfected with Arc and co-precipitated depicted in Fig. The immunoprecipitate was immunoblotted with anti-GFP antibody Fig.
A control experiment was carried out using a vector which encodes only EGFP peptide, which had no ability to interact with Myc-tagged Arc Data not shown. Co-precipitation of the proteins from Amida deletion mutants and Arc. Note that only a faint band was visible in D3 mutant expressing cell lysate. We investigated the co-operative function of Arc and Amida. Amida changes the subcellular distribution of Arc. After 72 h, cells were fixed and stained with DAPI.
The Promise of Amida Buddha
The proportion of cells with pyknotic nuclei from the EGFP-positive cells were calculated and analyzed with the statistical t test Fig. Overexpression of Amida significantly increased the proportion of pyknotic nuclei, while Arc had no influence on cell death compared with vector alone Fig. These results indicate that Arc has no influence on cell death alone, but inhibits cell death induced by overexpression of Amida. Suppression by Arc of cell death caused by Amida.
The proportion of pyknotic nuclei to EGFP positive cells is represented. We have isolated a novel protein designated Amida which interacts with Arc by yeast two-hybrid system, and confirmed that Amida has the ability to associate with Arc in an in vitro reconstruction assay and in transfected cells. Northern blot analysis revealed that Amida has two major transcripts of 1. Many peripheral tissues also contain Amida mRNA less than level of that in the brain, although Arc is expressed at low level.
In the thymus a band of 4 kilobases was also detected in Northern blot analysis, but we have not analyzed it yet. Save to Library. Create Alert. Similar Papers. Figures and Topics from this paper. Citations Publications citing this paper. Arc protein: a flexible hub for synaptic plasticity and cognition. The humility and dynamism that were realized in him by the Dharma were two sides of the same coin. Only a truly humble person can engage in dynamic seeking activities. The core of Buddhism is how Shakyamuni lived, not his ideas and teachings. But after his passing it was often the case that his vital spirit was forgotten and only his ideas and teachings were honored and studied.
The Hinayana developed as follows.
When Shakyamuni died, his disciples lost their beloved and revered teacher. They regarded Shakyamuni as the founder of a new religion. Since they thought that he had left a perfect and definitive teaching, they considered it their mission to faithfully memorize his teachings and preserve them. They did so because they had deep respect for their teacher.
Having deep respect for the teacher is an admirable thing. But at the same time, there is often a danger involved in it. Out of their respect for their teacher, they started to categorize, systematize, and academicize his teachings. They started to dogmatize them. This tradition is called the Hinayana.
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It was in this historical context that the Mahayana, another major Buddhist tradition, appeared in India around the first century B. The Mahayana appeared as a reaction and criticism against the Hinayana, as a revivalist movement. In the eyes of Mahayanists, Hinayanists were attached to lifeless ideas and concepts. Mahayanists believed that the most important thing in Buddhism was not the ideas and concepts, i. In the eyes of Mahayanists, Hinayanists were seeing only the footprints of a rabbit; they were not seeing the dynamic and lively life of the rabbit itself.
Hinayanists were interested in that which Shakyamuni produced, not in the source of inspiration that produced Shakyamuni. Mahayanists were interested in identifying the universal source or basis of the inspiration that awakened and produced Shakyamuni. And they identified it as the Dharma or universal Buddhahood. In order to show this spiritual basis of Shakyamuni in a more concrete human form, Mahayanists created the concept of "Amida"—an ideal human being, a "humble and dynamic" human being who embodies the Dharma.
Mahayanists described this ideal human being in Mahayana texts such as the Sukhavativyuha-sutra henceforth abbreviated as the Larger Sutra. The earliest version of this sutra was composed in India in approximately the first century B. Dharmakara symbolizes the "Innermost Aspiration" Sk.
Purva-pranidhana; Jpn. The "Innermost Aspiration" means the primordial human aspiration—an aspiration that makes humans-humans. It means an aspiration for Buddhahood that is entertained by all human beings. After performing many difficult practices, Dharmakara. Fulfills his "Innermost Aspiration" and becomes Amida Buddha.
The Pure Land tradition that is based on the sutra became one of the major Buddhist traditions in China and Japan. Shinran, the founder of Shin Buddhism, considered the Larger Sutra the most important textual basis of his Buddhism. Now I have discussed the historical background of the appearance of "Amida" in India. With this historical background in mind, let us define "Amida.
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Amida is "a personal symbol. Let me explain this definition by first discussing what Amida is not. Since "Amida" is a fictional character, he is 1 not a god or a divine being and 2 not a historical person. First, Amida is not a god. Just as Hamlet symbolizes certain spiritual qualities of human beings and does not have any superhuman or divine meaning, "Amida" symbolizes certain spiritual qualities of human beings and does not have any superhuman or divine meaning. Second, Amida is not a historical person. Just as Hamlet is a fictional character created by Shakespeare and is not a historical person, Amida is a fictional character created by ancient Indians and is not a historical person.
Hamlet is supposed to be a prince of Denmark but we cannot find his name in the actual chronicle of Denmark. Similarly, there is no actual history of "Amida"; being a symbolic fictional figure, Amida never lived in a specific time and place. Next, let us discuss what "Amida" is, what he symbolizes.
We can say that "Amida" symbolizes two things: 1 Shakyamuni, a historical person, and 2 the Dharma or universal Buddhahood. First, "Amida" symbolizes Shakyamuni, a historical person. We can say that "Amida" symbolizes the "humble and dynamic spirit" of Shakyamuni. As we have seen, Mahayanists created the concept of "Amida" in order to criticize the fossilized doctrines of Hinayanists and restore the vital spirit of Shakyamuni.
Second, "Amida" symbolizes the Dharma or universal Buddhahood. Mahayanists created the concept not only to express the vital spirit of Shakyamuni, but also to show the spiritual basis of Shakyamuni and all human beings. They wanted to show that just as Shakyamuni was awakened and liberated by the Dharma or universal Buddhahood , all human beings are awakened and liberated by it. Thus, as far as our personal attainment of Buddhahood is concerned, this second meaning of "Amida" as a symbol of the Dharma or universal Buddhahood is more important than the first.
The goal in Buddhism is that we personally become Amida Buddhas. The Buddhahood that we are expected to attain in Buddhism is not the historical Buddhahood of Shakyamuni, but the universal Buddhahood that is symbolized in "Amida. However, we can and should identify with the universal aspiration that Dharmakara symbolizes, strive to fulfill it, and become Amida Buddhas. We must realize our deepest reality, our true selves, which is what the realization of Amida Buddhahood means.
Here I want to pose a question concerning the doctrinal relationship between Shakyamuni and the Mahayana. What would Shakyamuni say about the Mahayana? Would he say, "Mahayanists, you have distorted my teachings and deviated from them? Shakyamuni never claimed to have created the Dharma. He identified himself as a person who was awakened and liberated by the Dharma.
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The discovery of the Dharma that had existed before him was called Awakening. He emphasized the importance of relying upon the Dharma. The same thing can be said about Buddhahood. Shakyamuni taught that the Buddhahood that had existed before him awakened and liberated him.