• The Korean Journal of Pharmacology
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• v.32 no.3
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• pp.417-424
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• 1996

The present study was done to examine the involvement of protein kinase C and protein tyrosine kinase in intracellular $Ca^{2+}$ mobilization in C5a-stimulated neutrophils. Although protein kinase C inhibitors, staurosporine and H-7 inhibited intracellular $Ca^{2+}$ release in C5a-stimulated neutrophils, they did not affect $Ca^{2+}$ influx across the plasma membrane and elevation of $[Ca^{2+}]_i$ C5a-induced intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx were inhibited by protein tyrosine kinase inhibitors, genistein and methyl-2,5-dihydroxycinnamate. ADP-evoked elevation of $[Ca^{2+}]_i$ was inhibited by genistein and methyl-2,5-dihydroxycinnamate but was not affectd by staurosporine and H-7. Genistein and methyl-2,5-dihydroxycinnamate reduced the store-regulated $Ca^{2+}$ influx in thapsigargin-treated neutrophils, while the effect of staurosporine and H-7 was not detected. When neutrophils were preincubated wih phorbol 12-myristate 13-acetate, the stimulatory effect of C5a on the elevation of $[Ca^{2+}]_i$ was reduced. These results suggest that protein tyrosine kinase may be involved in control of intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx across the plasma membrane in C5a-activated neutrophils.

• IMMUNE NETWORK
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• v.11 no.3
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• pp.182-189
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• 2011

Background: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-${\gamma}$ ELISPOT assay, cytotoxicity assay and tetramer staining. Results: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of $CD8^+$ and $CD4^+$ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

• Biomedical Science Letters
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• v.13 no.4
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• pp.305-311
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• 2007

cAMP-dependent protein kinase A (PKA) is the best-characterized protein kinases and has served as a model of the structure and regulation of cAMP-binding protein as well as of protein kinases. To determine the function of PKA in development, we employed the yeast two-hybrid system to screen for catalytic subunit of PKA $(C\alpha)$ interacting partners in a cDNA library from mouse embryo. A Testis-brain RNA-binding protein (TB-RBP), specifically bound to $C\alpha$. This interaction was verified by several biochemical analysis. Our findings indicate that $C\alpha$ can modulate nucleic acid binding proteins of TB-RBP and provide insights into the diverse role of PKA.

• YAKHAK HOEJI
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• v.47 no.1
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• pp.31-36
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• 2003

To investigate the effect of protein kinase on melanin production via cAMP-dependent pathway, we measured the melanin amount and tyrosinase activity in B16 melanoma cells stimulated by alpha-melanocyte stimulating hormone (MSH), forskolin and 8-Br-cAMP. MSH, forskolin and 8-Br-cAMP significantly increased both melanin production and tyrosinase activity in B16 cells. Melanin production and tyrosinase activity by MSH are significantly inhibited by cyclic AMP-dependent protein kinase inhibitor (KT5720) and protein kinase C down-regulation treated with PMA. Bisindolmaleimide (1$\mu$M), protein kinase C inhibitor, significantly inhibited melanin production and tyrosinase activity stimulated by MSH, forskolin and 8-Br-cAMP with the following order of potency: MSH＞forskolin＞8-Br-cAMP. Tyrosine kinase inhibitor, genistein and DHC, significantly inhibited both, but the inhibitory effect was more potent in 8-Br-cAMP-stimulated B16 cells than MSH-stimulated cells. NFkB inhibitor (parthenolide) significantly inhibited melanin production and tyrosinase activity. Neither melanin production nor tyrosinase activity induced by MSH, forskolin and 8-Br-cAMP were affected by KN-62 (calmodulin-dependent protein kinase II inhibitor), PD098059 (mitogen-activated protein kinase inhibitor, MAPKK) and worthmannin (phosphatidylinositol 3-kinase inhibitor). These results suggest that both protein kinase C and tyrosine kinase are involved in melanin production by cyclic AMP-dependent pathway and NFkB pathway may play an important role in cyclic AMP-dependent melanin production in B16 melanoma cells.

• KSBB Journal
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• v.24 no.1
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• pp.96-100
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• 2009

One of the major drawbacks associated with the high-level expression of the recombinant proteins in Escherichia coli is the formation of insoluble inclusion bodies in the cytoplasm. Production of recombinant protein at reduced temperature has proven effective in improving the solubility of a number of structurally and functionally unrelated proteins, but a major limitation of using low temperatures for recombinant protein production in E. coli is the reduced rate of synthesis of the heterologous protein caused by the significant reduction of cell growth rate. Here we investigated the effect of co-expression of CspA, a cold-shock protein known to be RNA chaperone at low temperature, on the productivity of recombinant protein at various temperatures by using green fluorescence protein (GFP) as a model recombinant protein. We could observe that the co-expression of CspA enhanced the productivity of GFP at $15^{\circ}C$ by accelerating the growth of E. coli at the temperature. On the other hand, the CspA coexpression didn't affect the cell growth rate as well as the specific GFP production rate at other tested temperatures, $20^{\circ}C$, $25^{\circ}C$, and $37^{\circ}C$.

• International Journal of Industrial Entomology and Biomaterials
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• v.9 no.1
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• pp.149-153
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• 2004

Here we report the molecular cloning of a LIM protein cDNA of the CRP (cysteine-rich protein) family from the mulberry longicorn beetle, Apriona, geramri. The A. germari LIM protein cDNA contains an open reading frame of 276 bp encoding 92 amino acid residues with a calculated molecular weight of approximately 10 kDa. The A. germari LIM protein contains the cysteine-rich consensus sequence of LIM domain and the glycine-rich consensus sequence observed in cysteine-rich protein family 1 (CRP1). The potential nuclear targeting signal is retained. The deduced amino acid sequence of the A. germari LIM protein cDNA showed 81 % identity to both Bombyx mori muscle LIM protein (Mlp) and Drosophila melanogaster Mlp60A and 77% to Epiblema scudderiana Mlp. Northern blot analysis showed that A. germari LIM protein is highly expressed in epidermis and muscle, and less strongly in midgut, but not in the fat body.

• Preventive Nutrition and Food Science
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• v.4 no.1
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• pp.23-27
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• 1999

To evaluate the quality of fish extracts with spicy vegetables (garlic, onion and ginger) in suppressing fishy oder, fish extracts of crucian carp, loach, bastard halibut and jacopever were processed at 100 $^{\circ}C$ for 6 hours, and their in vitro and in vivo protein qualities were determined . Protein and total lipid contents were closely related to the degree of discarding floated lipid on fish extracts and the kinds of added apicy vegetables . Boiling (10$0^{\circ}C$) , appeared to improve in vitro protein qualities slightly more than hydrocooking (11$0^{\circ}C$), but those with mild processing tended to result in better protein qualities than high temperature cooking (136-14$0^{\circ}C$). Spicy vegetables did not have remarkable effects on improving in vitro protein quality parameters. Fish extracts with 10% ginger were generally higher in in vitro protein quality than with the other vegetables . In spite of higher in vivo protein digestibility of fish extracts containing spicy vegetables processed under mild conditions(10$0^{\circ}C$), PERs of those extracts were not higher htan those of extranct processed at high temperature.

• Animal cells and systems
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• v.1 no.1
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• pp.135-142
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• 1997

Random sequencing of expressed sequence tags in roots of Chinese cabbage led to isolation of a partial cDNA clone, BR77, which encoded a putative protein kinase. Using the BR77 cDNA as a probe, we isolated a full-length cDNA encoding the Brassica campestris protein kinase 1 (Bcpk1). The Bcpt1 cDNA contained one open reading frame encoding a polypeptide of 439 amino acids. The putative polypeptide consisted of a short N-terminal region and a protein kinase catalytic domain. The catalytic domain of Bcpkl showed a high homology to cAMP- and calcium- phospholipid-dependent subfamilies of serine/threonine protein kineses. Eleven major catalytic domains in protein kineses were well conserved in Bcpk1. However, Bcpk1 contained a unique nonhomologous intervening sequence between subdomains VII and VIII, which was not found in protein kineses of animals and lower eukaryotes. Genomic DNA gel blot analysis showed that Bcpt1 genes might be present as three copies in the Chinese cabbage genome. These imply that Bcpk1 belongs to a plant-specific serine/threonine protein kinase subfamily.

• The Korean Journal of Pharmacology
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• v.32 no.1
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• pp.103-112
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• 1996

Roles of protein kinase C and protein tyrosine kinase in the activation of neutrophil respiratory burst, degranulation and elevation of cytosolic $Ca^{2+}$ in platelet-activating factor (PAF)-stimulated neutrophils were investigated. Superoxide and $H_2O_2$ production and myeloperoxidase and acid phosphatase release in PAF-stimulated neutrophils were inhibited by protein kinase C inhibitors, staurosporine and H-7 and protein tyrosine kinase inhibitors, genistein and tyrphostin. The PAF-induced elevation of $[Ca^{2+}]_i$ in neutrophils was inhibited by staurosporine, genistein and methyl-2,5-dihydroxycinnamate. Staurosporine inhibited both intracellular $Ca^{2+}$ release and $Mn^{2+}$ influx in PAF-stimulated neutrophils. Genistein and methyl-2,5-dihydroxycinnamate inhibited $Mn^{2+}$ influx induced by PAF, whereas their effects on intracellular $Ca^{2+}$ release were not detected. In neutrophils preactivated by PMA, the stimulatory effect of PAF on the elevation of $[Ca^{2+}]_i$ was reduced. Protein kinase C and protein tyrosine kinase may be involved in respiratory burst, lysosomal enzyme release and $Ca^{2+}$ mobilization in PAF-stimulated neutrophils. The elevation of $[Ca^{2+}]_i$ appears to be accomplished by intracullular $Ca^{2+}$ release and $Ca^{2+}$ influx which are differently regulated by protein kinases. Preactivation of protein kinase C appears to attenuate the stimulatory action of PAF on intracellular $Ca^{2+}$ mobilization.

• Journal of the Korean Society of Food Science and Nutrition
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• v.22 no.6
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• pp.823-832
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• 1993

This study was made to determine alleviating effects of vitamin C(Vt C) on the gramoxone toxicity in rats with respect to the growth gain, feed effeciency ratio, total lipid contents, lipid peroxication and protein patterns in rat liver. Growth gain, feed efficiency ratio and liver weight in the gramoxone group were decreased significantly as compared with the control group, while those values obtained in the gramoxone-Vt C group were increased as compared with the gramoxone group. Lipid contents and TBA value of rat liver of the gramoxone group were increased significantly as compared with the control group. However, those values of the gramoxone+Vt C group were decreased as compared with the gramoxone group. No significant differences were observed in the liver protein patterns between the gramoxone and the control group. The contents of liver protein were decreased significantly in the gramoxone group fed for 2 weeks as compared with the control group. The change of the liver protein patterns, such as the decrease of high molecular weight protein and the increase of low molecular weight protein were observed in the gramoxone+Vt C group.