• Molecules and Cells
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• 제26권4호
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• pp.323-328
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• 2008

Eukaryotic cells continuously integrate intrinsic and extrinsic signals to adapt to the environment. When exposed to stressful conditions, cells activate compartment-specific adaptive responses. If these are insufficient, apoptosis ensues as an organismal defense line. The mechanisms that sense stress and set the transition from adaptive to maladaptive responses, activating apoptotic programs, are the subject of intense studies, also for their potential impact in cancer and degenerative disorders. In the former case, one would aim at lowering the threshold, in the latter instead to increase it. Protein synthesis, consuming energy for anabolic processes as well as for byproducts disposal, can be a significant source of stress, particularly when difficult-to-fold proteins are produced. Recent work from our and other laboratories on the differentiation of antibody secreting cells, revealed a regulatory circuit that integrates protein synthesis, secretion and degradation (proteostasis), into cell lifespan determination. The apoptotic elimination - after an industrious, yet short lifetime - of terminal immune effectors is crucial to maintain immune homeostasis. Linking proteostasis to cell death, this paradigm might prove useful for biotechnological purposes, and the design of novel anti-cancer therapies.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.65-76
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• 2024

Bortezomib (BTZ) is a proteasome inhibitor used to treat multiple myeloma (MM). However, the induction of peripheral neuropathy is one of the major concerns in using BTZ to treat MM. In the current study, we have explored the effects of BTZ (0.01-5 nM) on rat neural stem cells (NSCs). BTZ (5 nM) induced cell death; however, the percentage of neurons was increased in the presence of mitogens. BTZ reduced the B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein ratio in proliferating NSCs and differentiated cells. Inhibition of βIII-tubulin (TUBB3) degradation was observed, but not inhibition of glial fibrillary acidic protein degradation, and a potential PEST sequence was solely found in TUBB3. In the presence of growth factors, BTZ increased cAMP response element-binding protein (CREB) phosphorylation, brain-derived neurotrophic factor (Bdnf) transcription, BDNF expression, and Tubb3 transcription in NSCs. However, in the neuroblastoma cell line, SH-SY5Y, BTZ (1-20 nM) only increased cell death without increasing CREB phosphorylation, Bdnf transcription, or TUBB3 induction. These results suggest that although BTZ induces cell death, it activates neurogenic signals and induces neurogenesis in NSCs.

• IMMUNE NETWORK
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• 제1권3호
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• pp.187-195
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• 2001

The expression of recombinant proteins fused to 26 kDa glutathione S-transferase (GST) extracted from Schistosoma japonicum represents an attractive system for purifiying proteins of interest in a single step using GST-affinity chromatography. In addition, the GST-tag is used conveniently for detecting fused proteins since its high solubility as well as its relatively small size rarely interferes with the biological activity of the fused protein. In this regard, the GST system is frequently applied for tracing fusion proteins in both prokaryotic and eukaryotic cells to elucidate the physiological interactions and functional compartments of proteins. To provide a further tool in analyzing GST-fusion proteins, a new monoclonal antibody, with a high specificity to the S. japonicum GST was produced. Methods: BALB/c mice were immunized both with recombinant S. japonicum GST proteins, and by the fusion of splenocytes from these mice with myeloma cells. From this, a new anti -GST monoclonal antibody, termed SARAH, was generated. The specificity and reactivity of this antibody was confirmed by ELISA and by Western blot analysis. Results: SARAH showed a high reactivity to recombinant GST and GST fusion protein but not with native mammalian GST proteins as derived from other species including humans, cows, rabbits and rats. The applicability of SARAH was further demonstrated by confocal laser scanning microscopy, where GST proteins that were expressed transiently in mouse fibroblast cells, were specifically detected without interference of endogenous GST. Conclusion: SARAH is new monoclonal antibody with a high specificity to recombinant GST proteins but not to endogenous GST in mammalian cells.

• BMB Reports
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• 제33권1호
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• pp.92-95
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• 2000

The HIV-1 p24(202-221) sequence ETINNEEEWDRVHPV HAGP contains a B-cell epitope with the earliest immune response and the highest antibody titer against anti-mouse sera obtained by immunization with p24 antigens. A novel mouse monoclonal antibody (mAb) was generated against the immunodominant B-cell epitope of the HIV-1 p24 capsid protein, p24(202-221). BALB/c mice were immunized with the four branched multiple antigenic peptide (MAP) containing the HIV-1p24(202-221) sequence, and antibody-secreting hybridoma were produced by fusion of mouse splenocytes with P3X63Ag8.653, mouse myeloma cells. One clone which produced the antigen-specific mAb named KI-24 (Isotype IgG1, light chain: ${\kappa}$) was identified. mAb KI-24 was highly specific for both the p24(202-221) and p24 proteins when analyzed by ELISA and Western blotting. Since p24(202-221) also contains a cytotoxic T-lymphocyte epitope, this specfic peptide epitope and the monoclonal antibody with specific reactivity against the p24 protein and p24(202-221) can be used in peptide vaccine development and p24 antigen detection from HIV patients.

• 한국미생물·생명공학회지
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• 제14권3호
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• pp.219-223
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• 1986

사람 선유아세포 인터페론의 정제에 사용되는 단 clone성 항체생산 세포주를 조작하기 위하여 BA-LB/C mouse의 복강과 꼬리정맥을 통하여 HuIFN-$\beta$를 면역화시키고 그 비장세포(spleen cells) 와 NS-O 세포주를 세포융합 시켰다. 융합된 1300 hybrids를 ELISA방법으로 선별하고 soft agarose 방법과 limiting dilution방법으로 subcloning하여 높은 항체를 생성하는 것으로 판명된 11 hybrids를 재선별 하였다. 재선별된 11 hybrids 각각의 항체형 (Ig type)을 조사하고 최종 Protein A-sepharose와 친화성이 높은 IgG 2a/형의 clone # 4-1-19와 clone # 551-4-1을 선별하여 배양된 세포를 각각 nude(nu/nu) mouse 및 BALB/c mouse 복강에 접종배양 하였다. 이들 mouse복강액으로 부터 얻은 ascites fluid를 protein A-sepharose를 이용한 affinity column분획으로 항체를 정제하였으며 ascites fluid $m{\ell}$당 약 4mg의 정제된 항체를 얻을 수 있었고 SDS-polyacrylamide gel상에서 전기영동 시킨 결과 분자량 14-16만 dalton으로 추정되는 항체를 확인할 수 있었다.

• Biomolecules & Therapeutics
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• 제5권4호
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• pp.344-350
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• 1997

The purpose of the present study was to produce and characterize a monoclonal antibody against human ${\beta}_2$-adrenergic receptor. Male BALB/c mice were immunized with glutathione S-transferase (GST) fusion protein of the C-terminal portion of the human ${\beta}_2$-adrenergic receptor which was expressed in E.Coli. The immunized splenocytes were fused with myeloma SP2/0-Agl4 cells. The resulting hybridomas were screened for the production of a monoclonal antibody which can recognize human ${\beta}_2$-adrenergic receptor, and then subcloned by limiting dilution. The resulting monoclonal antibody was named as mAb$\beta$CO2. The mono-clonal antibody $\beta$CO2 was determined as IgM subtype and then purified by anti-mouse IgM-agarose affinity chromatography. The results of ELISA, Western blot, and immunocytochemistry showed that mAb$\beta$CO2 recognized human ${\beta}_2$-adrenergic receptor in the ${\beta}_2$-adrenergic receptor-GST fusion protein and human spider-moid carcinoma cell line A431 with highly specific immunoreactivity, The monoclonal antibody $\beta$CO2 may provide useful tools for the study of the $\beta$-adrenergic receptor of human and other species including rats.

• 통합자연과학논문집
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• 제10권2호
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• pp.95-104
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• 2017

Proviral Integration site of Moloney (Pim) murine Leukemia virus kinases is a serine/threonine specific protein kinase. It is largely involved in cell survival and proliferation. Pim-1 phosphorylates multiple cellular substrates to inhibit apoptosis and promote cell cycle progression. Over expression of Pim-1 kinase is observed in a range of malignancies and various solid cancers. High level of Pim-1 expression is seen in myeloma, acute myeloid leukemia, prostate cancer and liver carcinomas. Hence, Pim-1 is considered as an interesting cancer target. In the present study, we have performed region-focused CoMFA study on a series of imidazopyridazine derivatives as Pim-1 kinase inhibitors. A statistically acceptable region-focused CoMFA model ($q^2=0.571$; ONC=3; $r^2=0.909$) was developed. The model was then validated using Bootsrapping and progressive sampling. The contour map highlighted the regions favorable to increase the activity. Bulky substitutions in $R^2$ position of the phenyl ring could increase the activity. Similarly, small negative substitution in the $R^1$ position of the Pyridine ring could increase the activity considerably. Our results will be useful to design novel Pim-1 kinase inhibitors of this series.

• 한국임상수의학회지
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• 제29권1호
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• pp.98-102
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• 2012

A 1.5-year-old male Golden Retriever was presented with worsening lameness of two month duration. Abnomral findings of blood works and serum chemistry included anemia, thrombocytopenia, hypercalcemia and hyperglobulinemia. Radiography revealed osteolysis of polyostotic regions including right femur and tibia, bilateral ilium, and spinous processes from the 13th thoracic vertebra to 5th lumbar vertebra. Enlarged multiple lymph nodes and mixed echo pattern of muscular region ventral to vertebra were observed with ultrasonography. Because concentrations of both parathyroid hormone and parathyroid hormone related peptide were all within reference ranges, humoral hypercalcemia by tumor was ruled out and extensive osteolysis was considered as the cause of hypercalcemia. Based on radiographic and ultrasonographic study, lymphoma, multiple myeloma and osteomyelitis were included in differential diagnosis. Fungal serologic test was negative. Monoclonal gammopathy was not found on serum protein electrophoresis. Cytological and histopathological examinations of the lytic lesions revealed neoplastic lymphoid proliferation, and B cell type clonal expansion was detected by polymerase chain reaction for the antigen receptor gene rearrangement. The case was diagnosed as B cell lymphoma involving polyostotic regions.

• 한국어업기술학회:학술대회논문집
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• 한국어업기술학회 2000년도 춘계수산관련학회 공동학술대회발표요지집
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• pp.446-446
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• 2000

Specific polyclonal and/or monoclonal antibodies (MAbs) to immunoglobulins (Igs) and their subunits have proved to be valuable tools in immunological research and in immunological assays. In this study, we developed and characterized MAbs against flounder serum Igs. To obtain the pure flounder serum Igs, mouse IgG (mIgG) was immunized to flounder. Flounder Igs were purified by using mIgG-agarose affinity column chromatography. The structure of purified flounder Ig was observed, on denatured SDS-PAGE, to be composed of two heavy chains (77 and 72 kd) and two light chains (28 and 26 kd). MAbs were produced by fusion of myeloma cells (SP2/0) with Balb/c mouse spleen cells previously primed with the flounder Igs. Finally, three hybridoma clones, FIM 511, FIM 519 and FIM 562 were established to recognize both 2 heavy chains, 26 kd of light chain and 28 kd of light chain, respectively. On the other hand, the flounder immune sera collected on the weekly basis were tested on ELISA and immunoblot analysis whether boosting effect is present in flounder humoral immune system. As a result, the secondary immune response in flounder was ascertained on ELISA, but not on immunoblot analysis. Further, we observed an alteration of serum protein levels following immunization. Our MAbs and basic information on flounder humoral immune system obtained in this study will be helpful to control and monitor the efficiency of fish vaccines and therapeutic process of flounder diseases.

• Animal cells and systems
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• 제1권1호
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• pp.157-164
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• 1997

Ly-6E.1 antigen was proposed as a regulatory molecule of T lymphocyte activation, a hematopoietic stem cell marker, a memory cell marker, and an adhesion molecule. Though there were several reports suggesting the presence of Ly-6 ligand, the characterization of the ligand was not yet performed, As an attempt to screen the expression of Ly-6E.1 ligand, we prepared a probe for detecting Ly-6E.1 ligand by producing a fusion protein between Ly-6E.1 and $hlgC_{r1}$, A mammalian cell expression vector with Ly-6E.$1/hlgC_{r1}$ chimeric cDNA was transfected in SP2/0-Ag14 myeloma cells, and stable transfectants were selected. The fusion protein was produced as a dimer and maintained the epitopes for monoclonal antibodies specific for Ly-6E.1 and for anti-human lgG antibody. The purified fusion protein through Gammabind G column was used for FACS analyses for the expression of Ly-6E.1 ligand. The fusion protein interacted with several cell lines originating from B cells, T cells, or monocytes. The fusion Protein also strongly stained bone marrow, lymph node, and spleen cells, but thymic cells weakly, if any. The staining was more obvious in C57BL/6 $(Ly-6^b)$ than Balb/c $(Ly-6^a)$ mice. These results suggest that the interaction of Ly-6E.1 with Ly-6E.1 ligand may function both in the stem cell environment and in the activation of mature lymphocytes. The fusion protein may be a valuable tool in characterization of biochemical properties of the Ly-6E.1 ligand and, further, in isolating its cDNA.