Metabolic loading of guanosine induces chondrocyte apoptosis via the Fas pathway

Although the apoptosis of chondrocytes plays an important role in endochondral ossification, its mechanism has not been elucidated. In this study, we show that guanosine induces chondrocyte apoptosis based on the results of acridine orange/ ethidium bromide staining, caspase-3 activation, and sub-G1 fraction analysis. The potent inhibitory effect of dipyridamole, a nucleoside transporter blocker, indicates that extracellular guanosine must enter the chondrocytes to induce apoptosis. We found that guanosine promotes Fas-Fas ligand interaction which, in turn, leads to chondrocyte apoptosis. These findings indicate a novel mechanism for endochondral ossification via metabolic regulation.

Response of Human Renal Cell Carcinoma Cells to Bone Morphogenetic Proteins, and the Expression of Bone Morphogenetic Protein Receptors / 대한비뇨기과학회지

PURPOSE: Bone morphogenetic protein (BMP) is a pleiotropic growth factor, which has been suggested to play a critical role during the development and homeostasis of the kidney. We evaluated the response of the human renal cell carcinoma (RCC) cell lines to BMPs. MATERIALS AND METHODS: We evaluated the growth rate of the human RCC cell lines, 112, 117 and 181, according to the concentrations of BMP-4, -6 and -7, and detected the levels of the BMP receptors (BMPRs) expressed in the cell lines. To demonstrate that the defect in BMP-6 signaling is at the receptor level, BMP-6 resistant cell lines were transfected, with adenovirus containing the constitutively active form of the BMP receptor types II (BMPR-II). After transfection, the cells were transfected with pSBE4, the construct of the BMP-6-responsive luciferase reporter gene, and a luciferase assay performed. RESULTS: The BMP-6 inhibited the proliferation of the 112, but not those of the 117 and 181 cells, in a dose-dependent manner. From Northern blot and immunoblot analyses, it was demonstrated that the 117 and 181 cells had undetectable levels of BMPR-II expression. The levels of luciferase activity, following adenovirus infections, was elevated in both the 117 and 181 cells, suggesting that the down-stream signaling molecules of the BMP-6 was intact in these cell lines. CONCLUSIONS: Taken together, these results demonstrate that the human RCC cell lines 117 and 181 are resistant to the growth inhibitory effects of the BMP-6 due to their decreased levels of BMPR-II expression.

Utility of the Resistive Index Measured by Color Doppler Ultrasonography in Benign Prostatic Hyperplasia / 대한비뇨기과학회지

PURPOSE: To evaluate the specific findings of color Doppler ultrasonography in patient with benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: We examined the resistive index (RI) of 110 patients with BPH, and compared them with 100 controls with no symptom, using color Doppler ultrasonography. The RI was compared with the prostatic volume and maximum flow rate (Qmax). In 20 patients with BPH, the RI was measured following surgical treatment. RESULTS: The RI was significantly higher in the patients than the controls (p<0.001). The BPH patients (n=110) had a mean RI of 0.74, whereas the controls (n=100) had a mean RI of 0.60. There was a significant correlation between RI and Qmax (r=-0.731, p<0.01), and the RI correlated significantly with the prostatic volume (r=0.739, p<0.01). In the 20 BPH patients having undergone TURP, the elevated RI decreased significantly to the normal control levels, from 0.70 to 0.60 (p<0.001), following surgical treatment. CONCLUSIONS: The Doppler RI might be useful as a new parameter in BPH. However, further studies, on its value in representing urodynamic information, will be required.

A Study on CT Attenuation and MR Signal Intensity of Protein Solution

PURPOSE: To correlate CT attenuation and MR signal intensity with concentration of protein solution. MATERIALS AND METHODS: CT and MR examinations of a phantom containing bovine serum albumin solutions of various concentrations ranging from 0 to 55% were performed. CT Hounsfield units(HUs), MR signal intensities, and apparent diffusion coefficients (ADCs) of each albumin solution were measured, and CT HUs and MR signal intensities of the solutions were compared with those of cerebrospinal fluid (CSF), white matter, and cortical gray matter. RESULTS: CT HU increased gradually with increasing albumin concentration. On T1-weighted images(T1WI), signal intensity increased with increasing albumin concentrations of up to 35% but then decreased. On T2-weighted images(T2WI), gradually decreasing signal intensity and increasing albumin concentration were oibserved. Fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted images (DWIs) showed that signal intensity peaked at a concentration of 10% and then gradually decreased. The ADC of the solution gradually decreased as concentration increased. Compared with those of normal brain structures, the CT HUs of solutions at concentrations of over 20% were higher than those of white and gray matter. At T1WI, the signal intensities of 10-45% solutions were similar to or higher than that of the gray matter. At T2WI, the signal intensities of solutions above 25, 35, and 40% were lower than those of CSF, gray matter, and white matter, respectively. FLAIR images showed that the signal intensities of 5-35% solutions were higher than that of gray matter. CONCLUSION: The CT attenuation of albumin solution increased gradually with increasing concentration. MR signal intensities peaked at 35% concentration on T1WI and at 10% on FLAIR and DW images, respectively, and then gradually decreased. T2WI and ADC map images showed gradually decreasing signal intensity and ADC as albumin concentration increased.

Resurrection of antibody as a therapeutic drug

Currently 18 monoclonal antibodies were approved by FDA for inj ection into humans for therapeutic or diagnostic purpose. And 146 clinical trials are under way to evaluate the efficacy of monoclonal antibodies as anti-cancer agents, which comprise 9% of clinical trials in cancer therapy field. When considering a lot of disappointment and worries existed in this field during the past 15 years, this boom could be called as resurrection. Antibodies have several merits over small molecule drug. First of all it is easier and faster in development, as proper immunization of the target proteins usually raises good antibody response. The side effect s of antibodies are more likely to be checked out in immunohistomchemical staining of whole human tissues. Antibody has better pharmacokinetics, which means a longer half-life. And it is non-toxic as it is purely a natural drug. Vast array of methods was developed to get the recombinant antibodies to be used as drug. The mice with human immunoglobulin genes were generated. Fully human antibodies can be developed in fast and easy way from these mice through immunization. These mice could make even human monoclonal antibodies against any human antigen like albumin. The concept of combinatorial library was also actively adopted for this purpose. Specific antibodies can be screened out from phage, mRNA, ribosomal library displaying recombinant antibodies like single chain Fvs or Fabs. Then the coding genes of these specific antibodies are obtained from the selected protein-gene units, and used for industrial scale production. Both naive and immunized libraries are proved to be effective for this purpose. In post-map arena, antibodies are receiving another spotlight as molecular probes against numerous targets screened out from functional genomics or proteomics. Actually many of these antibodies used for this purpose are already human ones. Through alliance of these two actively growing research areas, antibody would play a central role in target discovery and drug development.

Mass Production and Characterization of Anti-HBsAg Human Antibody B7 Fd

We expressed anti-HBsAg human antibody fragment (B7 Fd) using pRSET-A vector and BL21(DE3)pLysS strain of E. coli. B7 Fd is composed only of variable domain (VH) and CH1 constant domain of IgG heavy chain molecule. This Fd molecule was solubilized using guanidine salt and then expressed in the form of inclusion body and successfully refolded into functional antibody molecule by rapid dilution in refolding buffer. B7 Fd reacted with d epitope of hepatitis B virus surface antigen (HBsAg). Its affinity was determined by competition enzyme-linked immunosorbent assay (competition ELISA). The K value of B7 Fd was 3.3 * 10.

Expression, Characterization and Chain Shuffling of an Anti-HBsAg Phage Antibody

In our previous report an anti-HBsAg human monoclonal antibody was generated using antibody phage display library technique. Using pComb3 filamentous phagemid vector, Fab molecule was expressed in fusion form to a phage coat protein in the periplasm of E. coli. A clone of HBsAg binder was selected after panning and designated as B7. In order to select the clones with higher affinity and to examine which chain contributes most to the affinity of B7, the light and heavy chain of B7 was sequentially deleted and replaced with new library. HBsAg-binders were selected and tested by EIA (enzyme immunoassay). It was revealed that the affinity of B7 depends only on the heavy chain of Fd. B7 Fd was constructed without light chain and specificity and affinity was further confirmed by western blot analysis. This human monoclonal Fd antibody was found to react with d antigenic determinant of HBsAg as the original clone did. The nucleotide sequence analysis revealed that VH of B7 could be classified into Kabat's subgroup II and human IgG heavy chain family IV. The CH1 of B7 was IgG1.

Production of Mouse Single Chain Fv Antibody to Surface Protein of Hepatitis B virus using Antibody Phage Display Library

In this study, we are to produce the single chain variable fragment (scFv) antibodies against surface protein of hepatitis B virus (HBV) using antibody phage display technique. Balb/c mice were immunized with preS1 and cDNAs of heavy and light chains of splenic B cells from immunized mice were prepared using RT-PCR. Two cDNAs were linked with (64S) linker DNA under recombination PCR to produce single chain Fv DNA. After digestion of scFv DNA with Sp 1 and Not 1, the digested DNA was ligated into pCANTAB 5E and electroporated into E. coli XL1-Blue to prepare scFv-library. The size of library was 1 * 10' pfu/ml. Phage antibodies (phabs) against preS1 were rescued with M13K07 helper phages, and preS1-binders were selected through 3 times of panning using 96 well microtitre plates. Phage antibody clones were assayed directly for the ability to bind preS1 by ELISA. And then 7 phage antibody clones had high ELISA signals against preS1. Phabs from preS1-specific pMsc-17 had the strongest ELISA signal to preS1. Phabs from pMsc-17 were used for Western blot to preS1 and the results revealed that it was specific to preS1. To prepare the soluble scFv antibody, phabs from pMsc-17 were transfected into non-suppressor E. coli HB2151, and grown under 1 mM IPTG. Soluble scFv antibody was mainly accumulated in the periplasmic space, but small amount of antibody was secreted into culture media.

Production of Human Fab Monoclonal Antibody to Surface Protein, preS1, of Hepatitis B Virus using Antibody Phage Display Library / 대한면역학회지

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. By cloning human Ig gene segments from the B cells of volunteer into pComb3 phagemid vector, antibody library was created of filamentous phage particles displaying Fab fragments on their surface after being rescued with M13KO7 helper phages. The size of library was 7x10' pfu. Phage antibodies (phabs) were panned against biotinylated preS1 using streptavidine coated Dynabead. The soluble Fab antibodies were prepared from phagemid colonies and assayed directly for the ability to bind preS1 by ELISA. And then 3DW and SGW specific to preS1 which have both heavy and light chain to form Fab fragment, were selected. The soluble Fab antibody from 3DW was expressed highly at the concentration of 0.1 - 1.0 mM of IPTG, and 5 hours postinduction. The soluble antibodies from 3DW and SGW showed their relative affinities of 2x10' M ', and Sx10 M ', respectively, and the specificities to preS1 on ELISA. Our results suggest that antibody phage display library is very useful method to generate the human monoclonal antibody and that the human Fab monoclonal antibodies specific to preS1 selected in this study open the way to treat hepatitis B as a component of passive irnmunotherapeutics.

Establishment of I-131, Tc-99m labeling methods to in-house anti-CEA antibodies and evaluation of the immunological characteristics / 대한핵의학회잡지

No abstract available.