• IMMUNE NETWORK
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• v.6 no.2
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• pp.76-85
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• 2006

Background: Chemotaxis is one of the cardinal functions of leukocytes, which enables them to be recruited efficiently to the right place at the right time. Analyzing chemotactic activities is important not only for the study on leukocyte migration but also for many other applications including development of new drugs interfering with the chemotactic process. However, there are many technical limitations in the conventional in vitro chemotaxis assays. Here we applied a new optical assay to investigate chemotactic activities induced in differentiated HL-60 cells. Methods: HL-60 cells were stimulated with 0.8% dimethylformamide (DMF) for 4 days. The cells were analyzed for morphology, flow cytometry as well as chemotactic activities by a time-lapse videomicroscopic assay using a chemotactic microchamber bearing a fibronectin-coated cover slip and an etched silicon chip. Results: Videomicroscopic observation of the real cellular motions in a stable concentration gradient of chemokines demonstrated that HL-60 cells showed chemotaxis to inflammatory chemokines (CCL3, CCL5 and CXCL8) and also a homeostatic chemokine (CXCL12) after DFM-induced differentiation to granulocytic cells. The cells moved randomly at a speed of $6.99{\pm}1.24{\mu}m/min$ (n=100) in the absence of chemokine. Chemokine stimulation induced directional migration of differentiated HL-60 cells, while they still wandered very much and significantly increased the moving speeds. Conclusion: The locomotive patterns of DMF-stimulated HL-60 cells can be analyzed in detail throughout the course of chemotaxis by the use of a time-lapse videomicroscopic assay. DMF-stimulated HL-60 cells may provide a convenient in vitro model for chemotactic studies of neutrophils.

• Molecules and Cells
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• v.46 no.4
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• pp.191-199
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• 2023

The Golgi apparatus modifies and transports secretory and membrane proteins. In some instances, the production of secretory and membrane proteins exceeds the capacity of the Golgi apparatus, including vesicle trafficking and the post-translational modification of macromolecules. These proteins are not modified or delivered appropriately due to the insufficiency in the Golgi function. These conditions disturb Golgi homeostasis and induce a cellular condition known as Golgi stress, causing cells to activate the 'Golgi stress response,' which is a homeostatic process to increase the capacity of the Golgi based on cellular requirements. Since the Golgi functions are diverse, several response pathways involving TFE3, HSP47, CREB3, proteoglycan, mucin, MAPK/ETS, and PERK regulate the capacity of each Golgi function separately. Understanding the Golgi stress response is crucial for revealing the mechanisms underlying Golgi dynamics and its effect on human health because many signaling molecules are related to diseases, ranging from viral infections to fatal neurodegenerative diseases. Therefore, it is valuable to summarize and investigate the mechanisms underlying Golgi stress response in disease pathogenesis, as they may contribute to developing novel therapeutic strategies. In this review, we investigate the perturbations and stress signaling of the Golgi, as well as the therapeutic potentials of new strategies for treating Golgi stress-associated diseases.

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.87-93
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• 2011

S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50), a key enzyme for polyamines biosynthesis, was tightly regulated for homeostatic levels. Carnation SAMDC gene (CSDC9) has an small upstream open reading frame (uORF) of 54 amino acids in 5'-leader sequence. To explore the functional mechanism of uORFs in controlling translation, we used a GUS reporter gene driven with the 35S promoter and uORF region of SAMDC gene for making transgenic tobacco plants. In our experiment, there were a translational inhibition of its downstream GUS ORF by SAMDC uORF sequence or SAMDC uORF protein. Expecially, translational inhibition was most effective in point-mutated construct, in which the start codon was changed. Therefore, this results suggested the ribosomal stalling might be involved in this translational inhibitory process. The frame shift in amino acid sequence of SAMDC uORF with start codon and stop codon resulted in a moderate increasing in GUS activity, suggesting the native amino acid sequence was important for a function as a translational inhibitor. Also, we showed that the production of GUS protein was significantly inhibited in the presence of the small uORF using histochemical analysis of GUS expression in seedlings and tobacco flowers. Importantly, the small uORF sequence induced a real peptide of 5.7 kDa, which was provided the presence of SAMDC uORF peptide band using an in vitro transcription/translation system. The peptide product of uORF might interact with other components of translational machinery as well as polyamines, which was resulted from that polyamine treatment was inhibited GUS protein band in SDS-PAGE experiment.

• Journal of Plant Biotechnology
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• v.33 no.1
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• pp.65-73
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• 2006

Regulatory mechanism for endogenous levels of castasterone (CS) and its biosynthetic precursors in shoots of maize was investigated by the use of enzyme solution prepared from the plant tissue. When [$^2H_0$]- and [$^2H_6$]-CS was used as substrates, [$^2H_0$]-26-norCS and [$^2H_3$]-28-norCS were identified as products, indicating that [$^2H_0$]- and [$^2H_6$]-CS are differently metabolized into [$^2H_0$]-26-norCS and [$^2H_3$]-28-norCS by C-26 and C-28 demethylation, respectively. This suggests that both C-26 and C-28 demethylation can be involved in CS catabolism. In fact that C-28 demethylation only occurred when isotope labeled substrate was used, however, C-26 demethylation is thought be a natural reaction occurred in the maize shoots. When 6-deoxoteasterone (6-deoxoTE) was used, 6-deoxo-26-norTE and 3-dehydro-6-deoxo-26-norTE as well as 6-deoxo-3-dehydroTE and 6-deoxotyphasterol (6-deoxoTY) were identified as enzyme products. When 6-deoxoTY was added, 6-deoxo-26-norTY as well as 6-deoxo-3-dehydroTE and 6-deoxoTE was identified as products. These indicate that C-26 demethylation of 6-deoxoTE, 6-deoxo-3-dehydroTE and 6-deoxoTY as well as a reversible C-3 epimerization from 6-deoxoTE to 6-deoxoTY intermediated by 6-deoxo-3-dehydroTE are operative in the maize shoots, demonstrating that endogenous levels of biosynthetic precursors of CS are also controlled by C-26 demethylation. Therefore, it is thought that C-26 demethylation is an important and a common deactivation process which functions to maintain steady state levels of endogenous brassinosteroids in the maize shoots.