Comparative study of new bone formation capability of zirconia bone graft material in rabbit calvarial

PURPOSE: The purpose of this study was to compare the new bone formation capability of zirconia with those of other synthetic bone grafts. MATERIALS AND METHODS: Twelve rabbits were used and four 6-mm diameter transcortical defects were formed on each calvaria. Each defect was filled with Osteon II (Os), Tigran PTG (Ti), and zirconia (Zi) bone grafts. For the control group, the defects were left unfilled. The rabbits were sacrificed at 2, 4, and 8 weeks. Specimens were analyzed through micro computed tomography (CT) and histomorphometric analysis. RESULTS: The Ti and Zi groups showed significant differences in the amount of newly formed bone between 2 and 4 weeks and between 2 and 8 weeks (P < .05). The measurements of total bone using micro CT showed significant differences between the Os and Ti groups and between the Os and Zi groups at 2 and 8 weeks (P < .05). Comparing by week in each group, the Ti group showed a significant difference between 4 and 8 weeks. Histomorphometric analysis also showed significant differences in new bone formation between the control group and the experimental groups at 2, 4, and 8 weeks (P < .05). In the comparison of newly formed bone, significant differences were observed between 2 and 4 weeks and between 2 and 8 weeks (P < .05) in all groups. CONCLUSION: Zirconia bone graft material showed satisfactory results in new bone formation and zirconia could be used as a new synthetic bone graft material.

Development of Human Antibody Inhibiting RNase H Activity of Polymerase of Hepatitis B Virus Using Phage Display Technique

BACKGROUND: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. METHODS: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. RESULTS: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of 4.46 X 10(9) cfu was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of 4.5 X 10(-7) M and 1.9 X 10(-7) M, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. CONCLUSION: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

Listeria seeligeri Gene Encoding Heme-containing Catalase Produces No Activity in Streptococcus pneumoniae

Streptococcus pneumoniae is a facultative anaerobe lacking catalase enzyme and requires exogenous catalase supplemented to culture media for aerobic growth. We introduced a catalase gene (kat) of Listeria seeligeri into S. pneumoniae and tried to see if this listerial kat gene was expressed within the pneumococcal host. To clone the listerial kat gene in the pneumococcal chromosome, a non-replicating plasmid pAHA-LSt3, along with its original promoter region was used for integration the chromosome via homologous recombination. One of three resulting transformants was confirmed to contain the kat gene and designated as EHS2. In addition, the kat gene was subcloned in Escherichia coli in frame to the lac promoter of a shuttle vector to generate pDL-Kat, which was subsequently used for pneumococcal transformation. Four identical recombinants were identified to contain the plasmid with the kat gene. By performing RT-PCR, it was observed that the listerial kat gene was indeed transcribed within pneumococcal recombinants from its original promoter in the chromosome of EHS2 and from the lac promoter in the plasmid pDL-Kat. In contrast to the E. coli kat+ recombinants, however, the pneumococcal kat+ recombinants failed to reveal any catalase activities detectable by ferricyanide staining on non-denaturing PAGE. When the pDL-Kat plasmid DNA purified from pneumococci was allowed to transform E. coli again, many kat+ recombinants were obtained, ruling out the possibility of the defective kat E. coli transformants gene within pneumococci. The observation that the listerial kat gene in pneumococci was unable to produce the functional catalase enzyme, which requires a heme group at its active site and a cofactor NADPH, suggests pneumococcal defect in heme production.

An Epidemiologic Study on Sudden Deaths of Cattle Occurred in Kyongju / 한국역학회지

PURPOSE: This study was conducted to provide the baseline data for the epidemiologic and microbiologic investigation for the etiology of sudden deaths of cattle in Sara-Ri, Seo Myun, Kyongju. METHODS: This survey was performed between April 11 and 22, 1994. Epidemiologic investigation consisted of interview of the residents, as well as pathologic and microbiologic test on tissues and blood samples from cardiac puncture. RESULTS: The dead numbers of cattle were 149 in 35 households during about 20 years. The cows(63.9%) were more than bulls(36.1%) and most of them were raised in playpen(95.7%). The first death occurred in 1974, and then number of deaths increased until 1994. Besides the age of cattle at death was over two years old (88.3%), most of them(69.4%) died within one hour after onset of noticeable symptom by the farmers. The most common symptom of cattle at death was 'sudden death after screaming(71.1%)' and 'seizure (33.3%)'. Colonies from blood of case 3 showed double hemolysis in blood agar plate. The microbiologic test results in the culture of Clostridium perfringens. The pathological features were characterized as most of renal tubules revealed coagulative necrosis. Some gram-positive bacilli are scattered in interstitium. CONCLUSIONS: Above results suggest C. perfringens as a possible pathogen of this ourbreak in livestock. The possibility of human infection, although nonfatal, and lack of vaccination against C. perfringens raises a need for stronger preventive action toward this communicable disease of cattle on this village.

Introduction of a Catalase Gene into Streptococcus Pneumoniae

No Abstract Available.

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 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.

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.