• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.206-206
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• 1996

The C-terminus ends of the second putative transmembrane domains of both m1 and m2 muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T), This triplet is repeated as LYT-LYT in m2 receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-TYL) in the sequence of m1 receptors. In this work we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity for determining the distinct differential drug-receptor interaction and cellular function at m1 muscarinic receptor. Mutation of the LYTTYL sequence of m1 receptors to the corresponding m2 receptor LYTLYT sequence, however, did not result in a significant change in the binding affinity of the agonist carbachol or in the affinity of the majority of a series of receptor antagonists which are able to discriminate between wild-type m1 and m2 receptors. Surprisingly, the LYTLYT ml receptor mutant demonstrated markedly enhanced coupling to activation of phospholipase C without a change in its coupling to increased cyclic AMP formation. There was also an enhanced receptor sensitivity in transducing elevation of intracellular Ca$\^$2+/. These changes were not due to alterations in the rate of receptor. desensitization or sequestration, On the other hand, the reverse LYTLYT-LYTTYL mutation in the m2 receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of PI hydrolysis, Our data suggest that the LYTTYL/LYTLYT sequence difference between ml and n12 muscarinic receptors is not involved in determining receptor pharmacology. On the other hand, while these differences might play a role in the modulation of muscarinic receptor coupling to PI hydrolysis, they are not important for specifying coupling of various subtypes of muscarinic receptors to different cellular signaling pathways.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.1
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• pp.19-25
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• 1997

The C-terminus ends of the second putative transmembrane domains of both $M_1$ and $M_2$ muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T). This triplet is repeated as LYT-TYL in $M_1$ receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposedfashion (LYT-LYT) in the sequence of $M_2$ receptors. In our previous work, we investigated the possible significance of this unique sequence diversity for determining the distinct differential receptor function at the two receptor subtypes. However, we found mutation of the LYTTYL sequence of $M_1$ receptors to the corresponding $M_2$ receptor LYTLYT sequence demonstrated markedly enhanced the stimulation of phosphoinositide (PI) hydrolysis by carbachol without a change in its coupling to increased cyclic AMP formation. In this work, thus, the enhanced stimulation of PI hydrolysis in the LYTLYT $M_1$ receptor mutant was further investigated. The stimulation of PI hydrolysis by carbachol was enhanced in the mutant $M_1$ receptor, and this change was not due to alterations in the rate of receptor desensitization or sequestration. The observed larger response to carbachol at mutant $M_1$ receptors was also not due to an artifact resulting from selection of CHO cells which express higher levels of G-proteins or phospholipase C. Our data suggest that although the LYTTYL sequence in $M_1$ muscarinic receptors is not involved in determining receptor pharmacology, mutation of the sequence enhanced the coupling of $M_1$ receptors to the stimulation of phospholipase C.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1070-1083
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• 2015

A gene (GT-SM3B) encoding a thermostable secreted oligoendopeptidase (GT-SM3B) was cloned from the thermophile Geobacillus thermoleovorans DSM 15325. GT-SM3B is 1,857 bp in length and encodes a single-domain protein of 618 amino acids with a 23-residue signal peptide having a calculated mass of 67.7 kDa after signal cleavage. The deduced amino acid sequence of GT-SM3B contains a conservative zinc metallopeptidase motif (His⁴⁰⁰-Glu⁴⁰¹-X-XHis⁴⁰⁴). The described oligopeptidase belongs to the M3B subfamily of metallopeptidases and displays the highest amino acid sequence identity (40.3%) to the oligopeptidase PepF_Ba from mesophilic Bacillus amyloliquefaciens 23-7A among the characterized oligopeptidases. Secretory production of GT-SM3B was used, exploiting successful oligopeptidase signal peptide recognition by Escherichia coli BL21 (DE3). The recombinant enzyme was purified from the culture fluid. Homodimerization of GT-SM3B was determined by SDS-PAGE. Both the homodimer and monomer were catalytically active within a pH range of 5.0–8.0, at pH 7.3 and 40℃, showing the K_m, V_max, and k_cat values for carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala-OH peptidolysis to be 2.17 ± 0.04 × 10^-6 M, 2.65 ± 0.03 × 10^-3 µM/min, and 5.99 ± 0.07 s^-1, respectively. Peptidase remained stable at a broad pH range of 5.0–8.0. GT-SM3B was thermoactive, demonstrating 84% and 64% of maximum activity at 50℃ and 60℃, respectively. The recombinant oligopeptidase is one of the most thermostable M3B peptidase, retaining 71% residual activity after incubation at 60℃ for 1 h. GT-SM3B was shown to hydrolyze a collagenous peptide mixture derived from various types of collagen, but less preferentially than synthetic hexapeptide. This study is the first report on an extracellular thermostable metallo-oligopeptidase.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1862-1867
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• 2007

An individual's immune response is critical for host protection from many different pathogens, and the responsiveness can be assessed by the amount of cytokine production upon stimulating bacterial components such as lipopolysaccharide (LPS). The difference between individuals in their peripheral blood mononuclear cells (PBMC) responsiveness to LPS, a Gram-negative endotoxin, was investigated from 27 healthy individuals. We observed a large variation in $IFN{\gamma}$ production among different individuals. The PBMC of the consistently three highest and three lowest $IFN{\gamma}$ producers were investigated. Since previous studies described that a single point mutation in the coding region of TLR2 and TLR4 is linked to the individual responsiveness to pathogenic bacterial infections, we first examined the known point mutations in the coding region of $TLR2^{Pro681His}$, $TLR4^{Pro714His}$ located in the cytoplasmic regions of the Toll-like domain as well as $TLR4^{Asp299Gly}$ located in the extracellular region. None of these mutations were associated with an individual's responsiveness to LPS, despite the presence of $TLR4^{Asp299Gly}$ mutation. Further investigation revealed that the variation of PBMC responsiveness to LPS among healthy individuals was due to constitutive expression levels of TLR4 and TLR2. This result is consistent with an aging-related low expression of Toll-like receptors in the mouse model of LPS responsiveness. The present study therefore suggests that the constitutive expression levels of TLR2 and TLR4 may contribute to the individual response to LPS.

• IMMUNE NETWORK
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• v.5 no.3
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• pp.172-178
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• 2005

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. However, it is known that the induction of immune response to CEA is very difficult because CEA is a self-antigen expressed in fetal cells and weakly expressed in normal colorectal epithelial cells. To enhance anti-tumor immunity specific for CEA, recombinant CEA protein was modified using listeriolysin O (LLO) for endosomal lysis and trans activator of transcription (Tat) domain for transducing extracellular proteins into cytoplasm. Methods: After immunization using dendritic cells pulsed with Tat-CEA, both Tat-CEA and LLO, and both Tat-CEA and Tat-LLO, antibody titer to CEA and LLO, cytotoxic T lymphocyte activity and the frequency of IFN-${\gamma}$ producing T lymphocytes were measured. Results: Immunization using DC pulsed with both Tat-CEA and Tat-LLO protein showed the increasement of production of CEA-specific antibody in serum, cytotoxic T lymphocyte activity, the frequency of IFN-${\gamma}$ secreting T cells, compared with DC pulsed with both Tat-CEA and LLO. Furthermore the ratio of CD8+T cell to $CD4^+$ cell among CEA-specific T cells was increased in group pulsed with both Tat-CEA and Tat-LLO. Conclusion: These results suggested that DC vaccine using Tat-LLO could be used for the development of effective immunotherapy for the treatment of tumor.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.7
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• pp.4279-4282
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• 2013

Background: The activation and inactivation of receptor tyrosine kinases are tightly regulated to ensure faithful replication of cells. After having transduced extracellular growth activating signals, activated EGFR is subjected to downregulation either by clathrin mediated endocytosis or c-Cbl mediated proteasome degradation depending on the ligand concentration. c-Cbl is an ubiquitin ligase which requires a phosphorylated tyrosine residue at position 1045 in the cytoplasmic domain of EGFR to interact and add ubiquitin molecules. While activating mutations in exons 19 and 21 have been associated with the development of several cancers, the status of mutations at tyrosine 1045 coding exon 27 of EGFR remain to be investigated. Consistently, defective phosphorylation at 1045 has been associated with sustained phosphorylation of EGFR in non-small lung carcinomas. Hence in the present study we investigated the genetic status of the tyrosine 1045 coding site within exon 27 of EGFR gene to explore for possible occurrence of mutations in this region, especially since no studies have addressed this issue so far. Materials and Methods: Tumor chromosomal DNA isolated from thirty five surgically excised oral squamous cell carcinoma tissues was subjected to PCR amplification with intronic primers flanking the tyrosine 1045 coding exon 27 of EGFR gene. The PCR amplicons were subsequently subjected to direct sequencing to elucidate the mutation status. Results: Sequence analysis identified no mutations in the tyrosine 1045 codon of EGFR in any of the thirty five samples that were analyzed. Conclusions: The lack of identification of mutation in the tyrosine 1045 codon of EGFR suggests that mutations in this region may be relatively rare in oral squamous cell carcinomas. To the best of our knowledge, this study is the first to have explored the genetic status of exon 27 of EGFR in oral squamous cell carcinoma tissue samples.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.sup3
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• pp.293-297
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• 2016

TRAIL, tumor necrosis factor (TNF)-related apoptosis-inducing ligand belongs to one of important cytokine superfamilIES, tumor necrosis factor ($TNF{\alpha}$). TRAIL-2 receptor agonists activate several cell signaling pathways in cells in different manners and could lead to apoptosis or necrosis. Agonistic egg yolk antibodies like IgY which have been developed in a selective manner could activate TRAIL death receptors such as TRAIL-2 (DR5) and thus apoptosis signaling. We here investigated induction of apoptosis in human breast cancer cells (MCF7 cell line) by an IgY produced against an 21 aminoacid epitope of the human TRAIL-2 receptor. As the first step a small peptide of 21 aminoacids choosen from the extracellular domain of DR5 protein was produced with a peptide synthesizer. After control assays and confirmation of the correct amino acid sequence, it was injected to hens immunized to achieve high affinity IgYs. At the next step, the produced IgYs were extracted and examined for specificity against DR5 protein by ELISA assay. Subsequently, the anticancer effect of such IgYs was determined by MTT assay in the MCF7 human breast cancer cell line. The produced peptides successfully immunized hens and the produced antibodies which accumulated in egg yolk specifically recognized the DR5 protein. IgYs exerted significant toxicity and killed MCF7 cells as shown by MTT assay.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.24
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• pp.10875-10878
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• 2015

The epidermal growth factor receptor (EGFR) is an important surface receptor with N-glycans in its extracellular domain, whose glycosylation is essential for its function, especially in tumor cells. Here, we demonstrated that polylactosamine is markedly increased in H7721 hepatocellular carcinoma cells after treatment with EGF, while it apparently declined after exposure to all-trans retinoic acid (ATRA). In the study of the enzymatic mechanism of this phenomenon, we explored changes in the expression of poly-N-acetyllactosamine (PLN) branching glycosyltransferases using RT-PCR. Among the four glycosyltransferases with altered expression, GnT-V was most elevated by EGF, while GnT-V and B3GNT2 were most declined by ATRA. Next, we conducted co-immunoprecipitation experiments to test whether B3GNT2 and EGFR associate with each other. We observed that EGFR is a B3GNT2-targeting protein in H7721 cells. Taken together, these findings indicated that the altered expression of B3GNT2 will remodel the PLN stucture of EGFR in H7721 cells, which may modify downstream signal transduction.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.225-230
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• 2004

A plasminogen kringle domain 1 to 3, rKl-3, was expressed in Escherichia coli under the control of T7 promoter. For the cost-effective production of rKl-3, the induction process was analyzed and optimized. Induction characteristics with lactose were analyzed in terms of induction time and inducer concentration in various culture conditions including batch and high-cell-density fed-batch cultures. In the fed-batch culture, the induction around 6 h after initiation of the DO-stat fed-batch culture resulted in the highest expression level of rKI-3 among the induction points examined. The highest demand of oxygen at this point was crucial for the maximum expression level of rKI-3. As the lactose concentration increased, the expression level also increased, though the expression level showed a plateau above a concentration of 14 mM of lactose. Lactose acted less specifically than IPTG since most of it was hydrolyzed to glucose and galactose. However, using lactose, the cell growth and the maximum expression level of rKl-3 increased by 20% and 24%, respectively, compared with those using IPTG in the fed-batch culture. The lactose seemed to be hydrolyzed by intracellular and extracellular $\beta$-galactosidase liberated by cell lysis at the same time. Residual concentration of glucose was maintained to a a limit of detection by high performance liquid chromatography, and galactose was not consumed by the host strain Escherichia coli BL2l(DE3).

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.82-82
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• 1995

4-lBB molecule is expressed on the surface of activated CD4$\^$+/ and CD8$\^$+/ T cells. We generated a panel of anti-4-1 B5 murine mAbs using a fusion protein consisting of the extracellular domain of human 4-1 BB fused to Glutathione S-transferase. The binding activity against cell surface 4-1 BB molecule was assessed by flow cytometry analysis. These studies showed that several anti-4-1 BB mAbs bound to 10-30% of CD4$\^$+/ and CD8$\^$+/T cells in PHA or Con A stimulated PBLs, although these mAbs interacted with only, l-2% of CD4$\^$+/ and CD8$\^$+/ T cells in normal PBLs, indicating the specificity of mAbs to the 4-l BB molecule on activated CD4$\^$+/ and CD8$\^$+/ T cells. Next, we examined the effect of an anti-4-l BB mAb (4B4-1-1) on allogeneic mixed lymphocyte reactions (MLRs). The data indicated that the antibody significantly inhibited the proliferative response at higher concentrations. When tested with several T cell mitogens, the antibody had no stimulatory or inhibitory effects on the mitogen-mediated T cell proliferation. These data suggest that 4-1 BB molecule may play a role in the regulation of antigen-mediated immune response.