A putative pH-dependent nuclear localization signal in the juxtamembrane region of c-Met

The C-terminal fragment of the c-Met receptor tyrosine kinase is present in the nuclei of some cells irrespective of ligand stimulation, but the responsible nuclear localization signal (NLS) has not been previously reported. Here, we report that two histidine residues separated by a 10-amino-acid spacer (H1068-H1079) located in the juxtamembrane region of c-Met function as a putative novel NLS. Deletion of these sequences significantly abolished the nuclear translocation of c-Met, as did substitution of the histidines with alanines. This substitution also decreased the association of c-Met fragment with importin beta. The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible 'pH-dependency' of this NLS. Indeed, decreasing the cytosolic pH either with nigericin, an Na+/H+ exchanger or pH 6.5 KRB buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin beta, indicating that low pH itself enhanced nuclear translocation. Consistent with this, nigericin treatment also increased the nuclear level of endogenous c-Met in HeLa cells. The putative aberrant bipartite NLS of c-Met seems to be the first example of what we call a 'pH-dependent' NLS.

T Cell Immunoglobulin Mucin Domain (TIM)-3 Promoter Activity in a Human Mast Cell Line

T cell immunoglobulin mucin domain (TIM)-3 is an immunomodulatory molecule and upregulated in T cells by several cytokines. TIM-3 also influences mast cell function but its transcriptional regulation in mast cells has not been clarified. Therefore, we examined the transcript level and the promoter activity of TIM-3 in mast cells. The TIM-3 transcript level was assessed by real-time RT-PCR and promoter activity by luciferase reporter assay. TIM-3 mRNA levels were increased in HMC-1, a human mast cell line by TGF-beta1 stimulation but not by stimulation with interferon (IFN)-alpha, IFN-lambda, TNF-alpha, or IL-10. TIM-3 promoter -349~+144 bp region relative to the transcription start site was crucial for the basal and TGF-beta1-induced TIM-3 promoter activities in HMC-1 cells. TIM-3 promoter activity was increased by overexpression of Smad2 and Smad4, downstream molecules of TGF-beta1 signaling. Our results localize TIM-3 promoter activity to the region spanning -349 to +144 bp in resting and TGF-beta1 stimulated mast cells.

Initial Stability after Bilateral Sagittal Split Ramus Osteotomy Application in Patients with Mandibular Prognathism

Contralateral Neck Lymph Node Metastasis of Early Tongue Cancer: A Case Report

Likely to be the most common oral cancer, squamous cell carcinoma(SCC) of the tongue accounts for about 20% of all oral and pharyngeal cancers. SCC of the tongue frequently arises in the lateral border, and if it metastasize, it occurs on submandibular gland and neck lymph nodes. Location of the primary lesions and neck lymph node metastasis affect the prognosis and decrease survival rate of patients with carcinoma of the tongue. The authors experienced the patient with contralateral neck lymph node metastasis of SCC of the tongue. The patient came to our department with chief complaint of elevated lesion on left lateral border of the tongue. The mass was diagnosed as T2N0M0, Stage II invasive SCC of oral tongue. Computed tomography(CT) & magnetic resonance imaging(MRI) which were taken before the operation showed no significant finding of metastasis. Surgical mass removal and preventive neck dissection on the left side were done. While follow up PET/CT, contralateral neck lymph node metastasis(right side, level II) was detected, and re-operation(Rt. side RND) was done. There are few studies concerning the contralateral neck lymph node metastasis related with SCC of the tongue. The purpose of this report is to introduce the uncommon case of contralateral neck lymph node metastasis occurred in the T2-stage of SCC of the tongue treated by surgical resection.

Establishement of Antibody Selection by Ribosome Display

BACKGROUND: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than 109 members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to 1014 molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. METHODS: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. RESULTS: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. CONCLUSION: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.

Terminal Protein-specific scFv Production by Phage Display

BACKGROUND: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the 65th residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. METHODS: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains 57~80th amino acid residues of TP domain. After isolation of mRNA of heavy-variable region (VH) and light-chain variable region (VL) from the spleen of the immunized mouse, DNA of VH and VL were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. ScFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. RESULTS: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. CONCLUSION: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

Production of Monoclonal Anti-idiotypic Antibody to Monoclonal Anti-DNA Antibody / 대한면역학회지

It has been thought that autoimmune diseases like systemic lupus erythematosus and rhumatoid athritis are closely associated with anti-DNA antibodies (Abs). In studies of the control for anti-DNA Ab generation, an understanding of the regulatory mechanisms by anti-idiotypic Abs that influence the production of anti- DNA Abs would be facillitated by the availability of the hybridomas producing the pairs of DNA-specific and anti-DNA's idiotope-specific monoclonal antibodies (mAbs). We have produced a series of anti-DNA mAbs and then monoclonal anti-idiotypic Ab directed against idiotypic determinant of the 3D8 mAb that has the highest affinity to dsDNA and ssDNA among the anti-DNA mABs that we had obtained. The spleen cells of the MRL-Ipr/Ipr, autoimmune prone, mice were fused with P3X63Ag8.653 myeioma cells to obtain anti-DNA Ab secreting hybridomas. Out of the fourteen clones that showed strong binding to ssDNA, four clones had cross-reactivity with dsDNA whereas none of these clones reacted with left-handed z-DNA. The binding activities of the anti-DNA mAbs to various synthetic polynucleotide sequences were different respectively. Anti-idiotypic mAbs were generated by the fusion of myeloma cells and spleen cells from the Balb/c mice immunized with 3DB-Fab. We have produced two anti-idiotypic mAbs, B7 (IgG2a/k) and 02F3 (IgM/k), which were specific to 3DB-Fab and cloned the variable region of the heavy chain from the 02F3 hybridoma.

The effects of chromium exposure on sister chromatid exchange and concentration of 8-hydroxydeoxyguanosine / 예방의학회지

To elucidate some DNA adducts as a biological marker for workers of chromate pigment, the effects of chromium exposure on the formation of 8-hydroxydeoxyguanosine(8-OH-dG) and sister chromatid exchanges(SCEs) frequency in 38 workers of a pigment plant in Bucheon which utilized lead chromates, were examined. The chromium contents of venous blood and urine were measured as working environmental exposure level. The concentrations of 8-OH-dG in DNA isolated from lymphocytes were determined with high performance liquid chromatography and electrochemical detector and denoted as a molar ratio of 8-OH-dG to deoxyguanosine(dG). The SCEs frequency were analyzed in DNA isolated from lymphocytes. A significant correlation was found between creatinine adjusted urine chromium concentration and the molar ratio of 8-OH-dG to dG(r=0.47, p<0.01). After adjusting the current smoking habit, the correlation coefficient was increased(r=0.62, p<0.05). However, there was no significant correlation between the SCE frequency and chromium exposure. This significant results between molar ratio of 8-OH-dG to dG and chromium exposure are in good agreement with in vitro studies that support the importance of DNA adduct formation for the carcinogenic effect of chromium.