this report describes the structures of high-mannose-type N-linked oligosaccharides in glycoproteins synthesized by the microfilariae of Dirofioria immitis. Microfilariae of D. immitis were incubated in vitro in media contaning 2-[(3)H] mannose to allow metabolic radiolabeling of the oligosaccharide moieties of newly synthesized glycoproteins. Glycopeptides were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionation by chromatography on concanavalin A-Sepharose. Thirty eight percent of 2-[(3)H] mannose incorporated into the microfilariae of D. immitis glycopeptides was recovered in high mannose-type asparagine-linked oligosaccharides whech were bound to the immobilized lectin. Upon treatment of 2-[(3)H] mannose labeled glycopeptides with endo-beta-N-acetylglucosaminidase H, the high mannose-type chains were released and their structures were determined by high performance liquid chromatography and exoglycosidase digestion. The major species of high mannose-type chains synthesized by microfilariae of D. immitis have the composition Man(5) GlcNAc(2), Man(6) ClcNAc(2), Man(8) GlcNA(2), and Man(8) GlcNAc(2). Structural analyses indicate that these oligosaccharides are similar to high mannose-type chains synthesized by vertebrates.
parasitology-helminth-nematoda ; Dirofilaria immitis ; mannose ; carbohydrates ; oligosaccharieds ; micrlfilariae ; metabolic labeling ; HPLC ; enzyme digestion ; biochemistry

PURPOSE: Interindividual genetic differences in susceptibility to chemical carcinogens are one of the most important host factors in human cancer. The genetically determined differences in metabolism, related to cytochrome P450 (CYP450) genes have been reported to be associated with various cancer susceptibility. The present study was set up to establish the frequency of the polymorphic genotypes of two CYP450 (CYP2E1/PstI and CYP2E1/DraI) isozymes in Korea, to evaluate a possible increased incidence of the genotype associated with higher cervical cancer risks among Korean cervical cancer patients. MATERIALS AND METHODS: In this study, extracted DNAs from 228 cervical cancer patients and 360 normal healthy controls were analysed with the polymerase chain reaction-restriction fragment length polymosphism (PCR-RFLP) method. RESULTS: In the CYP 2E1 genotypes, detected by PstI or RsaI digestion, there were no statistically remarkable differences between the cervical cancer patients and control groups. And when the cervical cancer patients were divided into subgroups with respect to the age, the frequency of CYP 2E1/PstI polymorphisms in the cervical cancer patients under the 40 years old was not significantly higher compared to the controls or the patients above the 40 years old and, c1/c1 genotype was prominent in this type of polymorphism. The frequency of CYP 2E1/DraI polymorphisms in the cervical cancer patients was not significantly higher compared to the controls, and D/D genotype was prominent in this type of polymorphism. In cervical carcinoma, the polymorphic genotypes of CYP 2E1 were not correlated to other parameters including clinical stage, histological tumor type, and degree of differentiation. CONCLUSION: These results suggest that individuals carrying CYP 2E1/PstI (c1/c1) or CYP 2E1/DraI (D/D) alleles are not genetically susceptible to cervical cancer in Korea.
Adult ; Alleles ; Carcinogens ; Cytochrome P-450 CYP2E1* ; Cytochrome P-450 Enzyme System* ; Cytochromes* ; Digestion ; DNA ; Genetic Predisposition to Disease* ; Genotype ; Humans ; Incidence ; Isoenzymes ; Korea ; Metabolism ; Uterine Cervical Neoplasms*

Human peritoneal mesothelial cells may have a great potential to secrete chemokines, growth factors, adhesion molecules, and various cytokines stimulated with proinflammatory cytokines during peritoneal infection. In the course of peritonitis, rapid neutrophil cell influx and subsequent monocytic cell influx can be observed. It has been demonstrated that human peritoneal mesothelial cells secrete a C-X-C chemokine, IL-8, which contributes to the recruitment of neutrophil influx during peritoneal infection. However, the production and role of C-C chemokines have not been fully defined in human peritoneal mesothelial cells. This study was performed to evaluate the production of MCP-1 and RANTES and their influence on the chemotaxis of monocytes when human peritoneal mesothelial cells were stimulated with IL-1beta. Mesothelial cells obtained by enzymatic digestion of pieces of human omentum and stimulated with a various doses and times of IL-1beta. The expression of MCP-1 and RANTES mRNA was measured by Northern blot assay and the expression of their proteins was analyzed by ELISA. To evaluate their function, monocytes chemotaxis assay was performed using a 48-well chemotactic chamber. Cultured human peritoneal mesothelial cells appeared to be polygonal at confluence using phase contrast microscope. Indirect immunofluorescent staining demonstrated that the mesothelial cells reacted positively with anti-cytokeratin antibody and anti-vimentin antibody. The expression of MCP-1 and RANTES mRNA increased in response to IL-1beta in time and dose dependent manner. The protein levels of MCP-1 and RANTES with stimulation of 1.0ng/mL of IL-1beta for 24 hours were higher than those without(30.0+/-2.22 vs 3.55+/-0.74ng/105cells and 1.53+/-0.41 vs 0.11+/-0.02ng/105cells respectively, p<0.05, n=6). Chemotaxis assay showed that the supernatants from human peritoneal mesothelial cells with stimulation of IL-1beta for 24 hours had significantly higher chemotaxis of monocytes than those without(71+/-3.4% vs 50+/-2.9%, p<0.05, n=6). Coincubation of supernatants with stimulation and antibodies to MCP-1 or RANTES(20 micro L/mL, 10 micro L/mL, respectively) resulted in a significant inhibition of chemotaxis of monocytes by 33% and 12%(47+/-3.1% and 62+/-3.0% respectively, p<0.05, n=6). Human peritoneal mesothelial cells are capable of the expression of MCP-1 and RANTES mRNA and the production of their proteins in response to IL-1beta. Functionally, mesothelial cells derived Mand RANTES may contribute to the recruitment of monocytes and amplify the inflammatory process. Thus, human peritoneal mesothelial cells play an important role during peritoneal infection.
Antibodies ; Blotting, Northern ; Chemokine CCL5* ; Chemokines ; Chemokines, CC ; Chemotaxis ; Cytokines ; Digestion ; Enzyme-Linked Immunosorbent Assay ; Humans* ; Intercellular Signaling Peptides and Proteins ; Interleukin-8 ; Monocytes ; Neutrophils ; Omentum ; Peritonitis ; RNA, Messenger

Cytokine has been used as an immune stimulator and administered to patients for a treatment of cancer. Interleukin-12 (IL-12) is a potent cytokine which acts through a variety of functions including interferon-gamma production and cytotoxic T-cell activation. Considering the toxicity of high dose systemic IL-12 administration into human, local administration of low dose IL-12 can be a more efficient strategy. In ex vivo therapy, human dermal fibroblast has been considered as a useful vehicle for transferring genes, Here we show that human dermal fibroblast transduced with retrovirus containing IL-12 gene can be manipulated to produce reasonable amount of IL-12 protein. Human dermal fibroblast was isolated from freshly harvested skin specimens by collagenase digestion, grown in primary cultures, and transduced with a retroviral vector containing genes for human IL-12 and a selectable marker Neo(R). Following selection in G418, IL-12 producing fibroblasts were tested for secreted IL-12 level by ELISA. Six specimens of human skin were processed to obtain fibroblasts. ELISA results show that 40-150 units of IL-12 was produced for 24 h from 1x10(6) cells of transduced and selected fibroblast cultures. The primary cultures were maintained for up to nine passages about 108 days. The mean +/- overall time for obtaining enough number of cells was 49 +/- 2 days. The fibroblasts continued to produce IL-12 in culture for 90 days. These preliminary results can be used for the design of ex vivo gene therapy clinical trial using human dermal fibroblast.
Collagenases ; Digestion ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts* ; Genes, Neoplasm* ; Genetic Therapy ; Humans* ; Interferon-gamma ; Interleukin-12* ; Retroviridae ; Skin ; T-Lymphocytes ; Zidovudine

OBJECTIVETo compare the digestibility of main nutrients in genetically modified rice with double antisense starch-branching enzyme gene and parental rice.

METHODSSeven Wuzhishan healthy adult barrows were surgically fitted with a T-cannula at the terminal ileum. After surgery, seven pigs were randomly divided into two groups, and fed genetically modified rice and parental rice by a crossover model. Ileal digesta were collected for analysis of main nutrient digestibility.

RESULTSThe apparent digestibility levels of protein in genetically modified rice and parental rice were 69.50% ± 4.50%, 69.61% ± 8.40%, respectively (t = 0.01, P = 0.994); true digestibility levels of protein were 87.55% ± 4.95%, 87.64% ± 9.40%, respectively (t = 0.01, P = 0.994); fat digestibility levels were 72.86% ± 0.34%, 77.89% ± 13.09%, respectively (t = 0.95, P = 0.378); carbohydrate digestibility levels were 72.92% ± 7.43%, 92.35% ± 5.88%, respectively (t = 4.27, P = 0.005). The apparent and true digestibility of 17 amino acids had no significant difference in the two rice.

CONCLUSIONCarbohydrate digestibility in genetically modified rice was significantly lower than that in non-genetically modified rice, other main nutrients digestibility in the two rice have substantial equivalence.

1,4-alpha-Glucan Branching Enzyme ; metabolism ; Animals ; Carbohydrate Metabolism ; Digestion ; Food ; Ileum ; metabolism ; Intestinal Absorption ; Oryza ; chemistry ; Plants, Genetically Modified ; chemistry ; Starch ; metabolism ; Swine ; metabolism

Electrical stimulation among several factors that influence bone remodeling has been studied by many investigators with great enthusiasm in orthodontic field. The action mechanisms of Pulsed Electromagnetic Field (PEMF) are different from those of the conventional electrode application method in that PEMF induces endogenous current in the living tissues. PEMF is known to have the healing effect in nonunion of bone and osteoporosis. It is widely used in orthopaedic scopes and the possibility of using the method in clinical orthodontics is also conceivable. But the exact mechanisms by which the PEMF exerts its effects are not clearly understood. Therefore, the author wanted to see the effect of PEMF on five groups of rat calvarial cells obtained by sequential enzyme digestion method, and observed the changes in enzyme activation, collagen synthesis and 3H-thymidine incorporation. The results were as follows: 1. Under the effect of PEMF, there were no changes in the alkaline phosphatase activity in five groups of cell populations. 2. Both the PEMF group and the PTH with PEMF group showed no changes in acid phosphatase activities and there were no differences between two experimental groups. 3. Under the effect of PEMF, there was significant increase of collagen synthesis in the group V cell population. 4. Under the effect of PEMF, there were significant increases of 3H-thymidine incorporation in the group IV and V cell populations.
Acid Phosphatase ; Alkaline Phosphatase ; Animals ; Bone Remodeling ; Collagen ; Digestion ; Electric Stimulation ; Electrodes ; Electromagnetic Fields* ; Enzyme Activation ; Humans ; Magnets* ; Orthodontics ; Osteoporosis ; Rats* ; Research Personnel

BACKGROUND: Epidemiological studies have identified that positive family history and frequent exposures to environmental toxins such as 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) are of prime causative factors for PD. These toxins are mainly metabolized by MAO-B and CYP2D6. Thus, an individual with inherited defect in xenobiotic metabolism could have a higher susceptibility to PD. We performed this study to investigate a possible allelic association of MAO-B and CYP2D6 known to be involved in metabolism of dopamine and other drugs such as debrisoquine in PD. METHODS: We studied polymorphism of MAO-B and CYP2D6 genes in 69 sporadic idiopathic PD patients (31 males and 38 females) and 41 age-matched healthy control (20 males and 21 females) using genomic DNA extracted from peripheral blood white cell with polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism (RFLP). RESULTS: There were eight different alleles of various numbers of GT repeats within the second intron of MAO-B. The frequency of (GT)20 allele was the highest (44.7%) in PD, while the frequencies of (GT)14 allele and (GT)19 allele were the highest in control groups. Furthermore, the odds ratios of (GT)16 allele and (GT)20 allele were 4.93 (95% confidence interval 0.6-107.63) and 6.15 (95% confidence interval; 2.52-15.51), respectively, suggesting a higher susceptibility to PD in (GT)20 allelic group (p<0.001). Polymorphism of CYP2D6 was also examined by PCR amplification followed by digestion with restriction enzymes. However, we were unable to identify G to A substitution at the junction of intron 3 and exon 4 nor base pair deletion in exon 5 from PD and control groups, which have been reported previously. CONCLUSIONS: These results suggest that the MAO-B gene polymorphism could serve as a determinant of genetic susceptibility to PD at least in Korean population. But the susceptibility may not be directly associated with polymorphism of CYP2D gene examined in this study.
Alleles ; Base Pairing ; Cytochrome P-450 CYP2D6* ; Cytochrome P-450 Enzyme System* ; Cytochromes* ; Debrisoquin ; Digestion ; DNA ; Dopamine ; Epidemiologic Studies ; Exons ; Genetic Predisposition to Disease ; Humans ; Introns ; Male ; Metabolism ; Monoamine Oxidase* ; Odds Ratio ; Parkinson Disease* ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

OBJECTIVES: Toll-like receptors (TLRs) detect microbial infections and they can directly induce innate host defense responses. TLR 2 has been shown to be primarily involved in the recognition of peptidoglycans and lipoteichoic acid of gram positive bacteria. TLR 4 recognizes lipopolysaccharides and lipoteichoic acids from both gram-negative and gram-positive bacteria. Both mutations lead a reduced capacity to elicit inflammation and they increase the risk for gram-positive and negative infections. This study was performed to investigate the expressions of TLR 2 and 4 and their mutations in patients suffering with otitis media and middle ear effusion. METHODS: Middle ear fluid samples were collected from 40 otitis media effusion (OME) patients who had ventilating tubesinserted. Bacteria in the effusion fluid were detected by standard bacterial culture. The secreted IgG, IgA and IgM were measured by Enzyme-linked immunosorbent assay. TLR 2 and 4 were assessed by performing RT-PCR. The genomic DNA from each patient was isolated from the middle ear fluid samples that were collected from 60 OME patients, and the presence of mutations was determined by performing restriction digestion and DNA sequencing analysis. RESULTS: Among the 40 middle ear fluid samples, bacteria were detected in 13 middle ear fluid samples. The amounts of IgM, IgA, and IgG were 151.20+/-60.94 ng/mL, 21.59+/-7.96 ng/mL and 11.55+/-16.98 ng/mL, respectively. TLR 2 and 4 were expressed in the middle ear fluid and the expression of TLR 2 was higher than that of TLR 4. However, there was no correlation between the expressions of TLR 2 and 4, and the concentration of immunoglobulin or the presence of bacteria (P>0.05). There ware no mutations of TLR 2 (Arg753Gln, Arg677Trp) and TLR 4 (Asp299Gly, Thr399Ile). CONCLUSION: TLR 2 and 4 were expressed in all the middle ear fluid samples of OME, but the mutations of TLR 2 and 4 were not detected. TLR 2 and 4 may play a vital role in the immunological responses of patients with OME.
Bacteria ; Digestion ; DNA ; Ear, Middle ; Enzyme-Linked Immunosorbent Assay ; Gram-Positive Bacteria ; Humans ; Immunoglobulin A ; Immunoglobulin G ; Immunoglobulin M ; Immunoglobulins ; Inflammation ; Lipopolysaccharides ; Otitis ; Otitis Media ; Otitis Media with Effusion ; Peptidoglycan ; Sequence Analysis, DNA ; Stress, Psychological ; Teichoic Acids ; Toll-Like Receptors

OBJECTIVE: To investigate the association between endometriosis and polymorphisms of N-acetyl transferase 2 (NAT2), glutathione S-transferase M1 (GSTM1), and cytochrome P450 (CYP) 1A1 genes in Korean infertile patients. MATERIALS AND METHODS: A total of 303 infertile patients who had undertaken diagnostic laparoscopy during January, 2001 through December, 2003 at Samsung Cheil Hospital enrolled in this study. The patients were grouped according to laparoscopic findings: minimal to mild endometriosis (group I: n=147), moderate to severe endometriosis (group II: n=57), normal pelvic cavity (n=99). Peripheral blood was obtained and genomic DNA was extracted. The genotypes of each genes were analyzed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). For NAT2, RFLP was used to detect the wild type (wt) and mutant (mt) alleles, enabling classification into slow (mt/mt) or fast (wt/wt or wt/mt) acetylation genotypes. For GSTM1, PCR was used to distinguish active (+/- or +/+) from null (-/-) genotypes. For CYP1A1, MspI digestion was used to detect the wild type (A1A1), heterozygote (A1A2) or mutant (A2A2) genotypes. RESULTS: The genotype frequencies of NAT2 slow acetylator was 12.8%, 10.9%, 12.8% in group I, group II and control, respectively. The genotype frequencies of GSTM1 null mutation was 55.3%, 41.8%, 53.2% in group I, group II and control, respectively. The genotype frequencies of CYP1A1 MspI polymorphism was 16.3%, 9.1%, 18.1% in group I, group II and control, respectively. No significant difference was observed between endometriosis and normal controls in the genotype frequencies of the NAT2, GSTM1, CYP1A1 MspI polymorphism. CONCLUSION: The NAT2, GSTM1, CYP1A1 gene polymorphism may not be associated with the susceptibility of endometriosis in Korean women.
Acetylation ; Alleles ; Classification ; Cytochrome P-450 CYP1A1 ; Cytochrome P-450 Enzyme System* ; Cytochromes* ; Digestion ; DNA ; Endometriosis* ; Female ; Genotype ; Glutathione Transferase* ; Glutathione* ; Heterozygote ; Humans ; Laparoscopy ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Transferases*

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.
Animals ; Antibodies ; B-Lymphocytes ; Bacteriophages* ; Blotting, Western ; Cell Surface Display Techniques ; Clone Cells ; Culture Media ; Digestion ; DNA ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis* ; Isopropyl Thiogalactoside ; Mice* ; Periplasm ; Polymerase Chain Reaction ; Recombination, Genetic ; Single-Chain Antibodies*