• Development and Reproduction
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• v.2 no.1
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• pp.53-62
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• 1998

The lysosomal acid hydrolases including lysosomal acid phosphatase (LAP) are believed to play an important role in intracellular and extracellular degradation. LAP was reported to increase its activity in dedifferentiation stage during urodele limb regeneration. In the paresent study, LAP localization in the Mexican axolotl (Ambystoma mexicanum) limb regenerates was investigated by immunohistochemistry. LAP immunoreactivity with monoclonal antibody against Korean salamander (Hynobius leehii) LAP was observed mainly in the wound epidermis, blastema cells, muscle, and cartilage which were under dedifferentiation process in axolotl limb regenerates. Moreover, LAP immunoreactivity increased gradually during the early phase of lib regeneration and reached the peak level at dedifferentiation stage. However, as redifferentiation begans, LAP immunoreactivity decreased slowly to the basal level. Retinoic acid (RA) which is known to induce skeleton pattern duplication in regenerating urodele limb appears to enhance LAP immunoreactivity. In the RA-treate limg regenerates, LAP immunoreactivity was higher than in the normal regenerates. In addition, the LAP expression period was more extended in the RA treated regenerates than in the normal regenerates. These results suggest that RA is involved in the extension of dedifferentiation state in RA-treated limb regenerate.

• Parasites, Hosts and Diseases
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• v.48 no.4
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• pp.319-324
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• 2010

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4-EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in Echerichia coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.

• Molecules and Cells
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• v.46 no.10
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• pp.592-610
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• 2023

The Hippo kinase cascade functions as a central hub that relays input from the "outside world" of the cell and translates it into specific cellular responses by regulating the activity of Yes-associated protein 1 (YAP1). How Hippo translates input from the extracellular signals into specific intracellular responses remains unclear. Here, we show that transforming growth factor β (TGFβ)-activated TAK1 activates LATS1/2, which then phosphorylates YAP1. Phosphorylated YAP1 (p-YAP1) associates with RUNX3, but not with TEAD4, to form a TGFβ-stimulated restriction (R)-point-associated complex which activates target chromatin loci in the nucleus. Soon after, p-YAP1 is exported to the cytoplasm. Attenuation of TGFβ signaling results in re-localization of unphosphorylated YAP1 to the nucleus, where it forms a YAP1/TEAD4/SMAD3/AP1/p300 complex. The TGFβ-stimulated spatiotemporal dynamics of YAP1 are abrogated in many cancer cells. These results identify a new pathway that integrates TGFβ signals and the Hippo pathway (TGFβ→TAK1→LATS1/2→YAP1 cascade) with a novel dynamic nuclear role for p-YAP1.

• Journal of Life Science
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• v.34 no.4
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• pp.271-278
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• 2024

Redox factor (Ref)-1, a ubiquitously expressed protein, acts as a modulator of redox-sensitive tran- scription factors and as an endonuclease in the repair pathway of damaged DNA. However, the function of Ref-1 in the differentiation of monocytes into macrophages has not been defined. In this study, we investigated the effects of Ref-1 on the monocyte differentiation process using the human monocytic cell line THP-1. The differentiation agent PMA increased cell adhesion over time and showed a sig- nificant increase in phagocytic function but decreased the intracellular amount of Ref-1. Ref-1 inhibitor E3330 and Ref-1 knockdown using the siRNA technique reduced cell adhesion and the expression of differentiation markers, such as CD14, ICAM-1, and CD11b, by PMA stimulation. This means that the role of Ref-1 is absolutely necessary in the initial process of differentiating THP-1 cells stimulated by PMA. Next, the distribution of Ref-1 was examined in the cytoplasm and nucleus of THP-1 cells stimulated with PMA. Surprisingly, PMA stimulation resulted in the rapid translocation of Ref-1 to the nucleus. To prove that movement of Ref-1 to the nucleus is required for monocyte differentiation, a Ref-1 vector with the nuclear localization sequence (NLS) deleted was used. As a result, overexpression of ∆NLS Ref-1, which restricted movement to the nucleus, suppressed the expression of differentiation markers and notably reduced phagocytic function in PMA-stimulated THP-1 cells. In conclusion, these data suggest that the differentiation of monocytic THP-1 cells requires Ref-1 nuclear translocation during the initial process of biochemical events following stimulation from PMA.

• Tuberculosis and Respiratory Diseases
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• v.49 no.3
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• pp.310-322
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• 2000

Background : Phospholipase C(PLC) plays an important role in cellular signal transduction and is thought to be critical in cellular growth, differentiation and transformation of certain malignancies. Two second messengers produced from the enzymatic action of PLC are diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3). These two second messengers are important in down stream signal activation of protein kinase C and intracellular calcium elevation. In addition, functional domains of the PLC isozymes, such as Src homology 2 (SH2) domain, Src homology 3 (SH3) domain, and pleckstrin homology (PH) domain play crucial roles in protein translocation, lipid membrane modificailon and intracellular memrane trafficking which occur during various mitogenic processes. We have previously reported the presence of PLC-${\gamma}1$, ${\gamma}2$, ${\beta}1$, ${\beta}3$, and ${\delta}1$ isozymes in normal human lung tissue and tyrosine-kinase-independent activation of phospholipase C-${\gamma}$ isozymes by tau protein and AHNAK. We had also found that the expression of AHNAK protein was markedly increased in various mstologic types of lung can∞r tissues as compared to the normallungs. However, the report concerning expression of various PLC isozymes in lung canærs and other lung diseases is lacking. Therefore, in this study we examined the expression of PLC isozymes in the paired surgical specimens taken from lung cancer patients. Methods : Surgically resected lung cancer tissue samples taken from thirty seven patients and their paired normal control lungs from the same patients, The expression of various PLC isozymes were studied. Western blot analysis of the tissue extracts for the PLC isozymes and immunohistochemistry was performed on typical samples for localization of the isozyme. Results : In 16 of 18 squamous cell carcinomas, the expression of PLC-${\gamma}1$ was increased. PLC-${\gamma}1$ was also found to be increased in all of 15 adenocarcinoma patients. In most of the non-small cell lung cancer tissues we had examined, expression of PLC-${\delta}1$ was decreased. However, the expression of PLC-${\delta}1$ was markedly increased in 3 adenocarcinomas and 3 squamous carcinomas. Although the numbers were small, in all 4 cases of small cell lung cancer tissues, the expression of PLC-${\delta}1$ was nearly absent. Conclusion : We found increased expression of PLC-${\gamma}1$ isozyme in lung cancer tissues. Results of this study, taken together with our earlier findings of AHNAK protein-a putative PLD-${\gamma}$, activator-over-expression, and the changes observed in PLC-${\delta}1$ in primary human lung cancers may provide a possible insight into the derranged calcium-inositol signaling pathways leading to the lung malignancies.

• Applied Microscopy
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• v.29 no.4
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• pp.479-491
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• 1999

This study was performed to investigate the distribution and differentiation of choline acetyltransferase (ChAT)-immunoreactive cells in the basal nucleus of Meynert of the postnatal and adult rat forebrains, utilizing techniques of immunocytochemistry. According to the cell shape and the ratio of long axis vs short axis of cell soma, the ChAT-immunoreactive nerve cells in the basal nucleus of Meynert of the adult rat were classified into six types. In the adult rat, the frequency distributions (FD) of round, oval, elongated, fusiform, triangular and polygonal cells were 9.4%, 35.5%, 32.1%, 5.9%, 9.1% and 8.0%, respectively. The FD of oval and round nerve cells on the postnatal day (PND) 14 were observed to be 18.7% and 51.5%, respectively. Those were shown to be progressively decreased during developmental process to the adult. Also, those of elongated and triangular nerve cells on the PND 21 were observed to be 30.4% and 10.1%, respectively. Those were shown to be same phenomenon a,1 those in the round and oval cells. Meanwhile, those of the triangular and polygonal nerve cells were progressively increased from the early postnatal stage to the adult. The total mean volumes of ChAT-immunoreactive cell somata in the PND 7 rat were the lowest $(1,083{\mu}m^3)$ and those in the PND 21 rat were shown to be the highest $(5,045{\mu}m^3)$. But in the adult, those were decreased to $(2,731{\mu}m^3)$. Those in the PND 21 rat were shown to be about 84.7% larger than those in the adult. On the electron micrography, the cell organelles such as ribosomes, polysomes, rough endoplasmic reticula (RER) and mitochondria were well developed in the PND 21 rat forebrains, but Golgi complexes were shown to be proliferating phase. Especially, ribosomes, polysomes and RER were immunoreactive in the tissues treated with 0.05% triton X-100. According to the observations in the present study, it is considered that the ChAT-immunoreactive nerve cells in the basal nucleus of Meynert of the rat forebrains are differentiated throughout the following processes of changes during the postnatal development: 1) increase of cell soma volumes with the differentiation of tell organelles and neurites, 2) increase in the FD of differentiated tell types and 3) cell schrinkage without cell loss. The ribosomes, polysomes and RER are considered to be closely related to the intracellular localization and biosynthesis of the ChAT but not Colgi complex.

• Journal of Life Science
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• v.27 no.10
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• pp.1191-1198
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• 2017

Vesicles and organelles are transported along microtubule and delivered to appropriate compartments in cells. The intracellular transport process is mediated by molecular motor proteins, kinesin, and dynein. Kinesin is a plus-end-directed molecular motor protein that moves the various cargoes along microtubule tracks. Kinesin 1 is first isolated from squid axoplasm is a dimer of two heavy chains (KHCs, also called KIF5s), each of which is associated with the light chain (KLC). KIF5s interact with many different binding proteins through their carboxyl (C)-terminal tail region, but their binding proteins have yet to be specified. To identify the interacting proteins for KIF5A, we performed the yeast two-hybrid screening and found a specific interaction with Ras-GTPase-activating protein (GAP) Src homology3 (SH3)-domain-binding protein 2 (G3BP2), which is involved in stress granule formation and mRNA-protein (mRNP) localization. G3BP2 bound to the C-terminal 73 amino acids of KIF5A but did not interact with the KIF5B, nor the KIF5C in the yeast two-hybrid assay. The arginine-glycine-glycine (RGG)/Gly-rich region domain of G3BP2 is a minimal binding domain for interaction with KIF5A. However, G3BP1 did not interact with KIF5A. When co-expressed in HEK-293T cells, G3BP2 co-localized with KIF5A and was co-immunoprecipitated with KIF5A. These results indicate that G3BP2, which was originally identified as a Ras-GAP SH3 domain-binding protein, is a protein that interacts with KIF5A.