Biomedical subjects

M J Sun

Publications and source records attributed to M J Sun.

At least 19 recordsLinked to original sources

PubMed2001

Synthetic antigenic decapeptides of human brain acetylcholinesterase cross-immunoreact with peptide-specific antibodies against Torpediniformes narcine timlei acetylcholinesterase.

Antigenic decapeptides of human brain acetylcholinesterase (AChE) were investigated for immunoreactivity with the rabbit anti-Torpediniformes narcine timlei AChE polyclonal antibody (anti-narcine AChE polyclonal antibody). The decapeptides were synthesized using the multipin combinatorial chemical synthesis technique and biotinylated at N-terminals. Rabbit anti-narcine AChE polyclonal antibodies were purified by Protein A-Sepharose CL 4B column chromatography. Enzyme-linked immunosorbent assay (ELISA) was used for the assay of the reaction between the antigen and the antibody. Seven of 11 antigenic synthetic decapeptides of human brain AChE showed obvious immunoreactivity with the rabbit anti-narcine AChE polyclonal antibodies. The similarity of the AChE sequences of humans and Torpedo species were compared thereby with the epitopes indicated. The results indicate that the epitopes of human brain AChE and Torpedo AChEs have been highly conserved during evolution. In view of this, no N-glycosylation site was found in the antigenic decapeptides tested, they all belong to oligopeptide epitopes.

Acetylcholinesterase↗

PubMed2000

High-level expression of human butyrylcholinesterase gene in Bombyx mori and biochemical-pharmacological characteristic study of its product.

The human butyrylcholinesterase (BChE, EC 3.1.1.8) gene was highly expressed in Bombyx mori using baculovirus vector, and the biochemical-pharmacological properties of its product were studied. BChE cDNA was cloned into transfer vector pBn96 and co-transfected with wild-type Bombyx mori nucleopolyhedrovirus (BmNPV) DNA into BmN cells. The recombinant virus with the highest enzyme activity was sorted out and purified. Once the BmN cells or silkworm larvae had been infected with the recombinant virus, recombinant human BChE (rhBChE) could be secreted into the culture medium or the hemolymph of the larvae at levels of 1.5 mg x L(-1) and 35 mg x L(-1), respectively. Western blot and enzymatic staining of the electrophoresis gel of non-denatured protein showed that rhBChE manifested similar antigenicity and enzyme activity to native human BChE (nhBChE). The production of rhBChE in the hemolymph was 23-fold higher than that in BmN cells and about 280-fold that in Chinese hamster overy cells (125 microg x L(-1)). This is the first report of human BChE expression in silkworm with the highest level of yield so far. rhBChE was highly similar to nhBChE in respect to substrate affinity, inhibitor sensitivity, and reactivity of the inhibited enzyme. It is suggested that rhBChE functions as well as nhBChE and has potential practical value.

Animals↗

PubMed2000

Epitopes of human brain acetylcholinesterase.

The main purpose of the present work was to identify B-cell epitopes on human brain acetylcholinesterase (AChE) by the synthetic peptide approach. Five hundred and seventy-four decapeptides comprising amino acids No. n to n+9 (where n denotes the residue number of the 583 amino acids in the primary structure of human brain AChE and is an integer in the range 1-574) were synthesized, using the multipin combinatorial chemical synthesis technique, and biotinylated. Epitopes of human brain AChE were detected by enzyme-linked immunosorbent assay (ELISA) and compared with the predicted epitopes of human AChE by 'Goldkey' software. Among 574 synthetic decapeptides, 47 decapeptides at 11 antigenic regions showed immunoreactivity with mouse anti-human brain AChE polyclonal antibodies. The minimum sequence of epitope was defined at every antigenic region explored. The locations and sequences of the former ten continuous epitopes at the 11 antigenic regions of the human brain AChE had been identified as follows: TPVLVWIY (112-119), RTVLVSMNY (143-151), LLDQRLALQW (173-182), RRATQLAH (246-253), VFRFSFVPV (294 approximately 302), KDEGSYFLVY (332-341), RVYA (424-427), LMRY (476-479), KAPQWPPY (496-503), GLRAQACAFW (523-532). The rate of hits of the predicted epitopes from the software came out at 33%. In our work, the epitopes of human AChE have been mapped by purified polyclonal antibody at eleven distinct sites in the primary structure.

Acetylcholinesterase↗

PubMed1999

Grafting of genetically modified human fetal fibroblasts to produce human butyrylcholinesterase in mice.

Human diploid fibroblast cultures were established from fetal skin tissue. Enzymic dissociation yielded cultures of higher growth capacity of fibroblasts than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. Transfer of recombinant human butyrylcholinesterase (BChE, EC 3.1.1.8) gene into primary human fibroblasts was achieved successfully using lipofection and retrovirus-mediated transfection. The analysis of drug-resistant colonies suggested the presence of the transcripted BChE mRNA in the cytoplasm of transfected cells. The secreted BChE protein in culture medium was assayed for enzyme activity using butyrylthiocholine as substrate. The genetically modified fibroblasts were mixed with rat tail collagen and transplanted subcutaneously and intraperitoneally to mice. Immunoreactive human BChE appeared in the plasma from the transplanted mice. reaching the top level at day 13. It was not present any longer in most of the mice 20 days later.

Animals↗

PubMed1999

Systematic study of the substituted active C-terminus of hirudin.

Hirudin, a potent clinical thrombin inhibitor from Hirudo medicinalis, consists of 65 amino acids in a single chain. In this paper, we systematically synthesize a series of C-terminal (desulfo hirudin45-65) peptides substituted by 20 natural L-amino acids via the Multipin method. The resulting peptide library is subsequently screened using an alpha-thrombin-mediated fibrinogen clotting assay and alpha-thrombin-induced amidolytic hydrolysis assay.

Amides↗

PubMed1999

Characteristics of recombinant human butyrylcholinesterase.

AIM: To study the biochemical-pharmacological properties of the recombinant human butyrylcholinesterase (rhBChE) and thereby to size up the potential possibility of using it as a detoxifying agent in succinylcholine intoxication. METHODS: CHO-dhfr cells were transfected with plasmids by electroporation. BChE activity was determined colorimetrically by 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) method. Antigenicity was estimated by enzyme-linked immunosorbent assay and Western blot. RESULTS: The maximal expression amounted to 25.83 ng.h-1/10(6) cells. The rhBChE was highly similar to the native human BChE (nhBChE) in terms of its catalytic property, substrate affinity, inhibitor sensitivity, reactivation, stability, and immunoreactivity with anti-nhBChE antibodies. Mice challenged with 1.5 lethal dose of succinylcholine preincubated with rhBChE survived without any symptoms of intoxication. CONCLUSION: The rhBChE and nhBChE exhibit similar biochemical-pharmacological features. It is of potential value in practical use.

Animals↗

PubMed1999

Prophylactic effect of methylene blue against neurotoxicity of sodium nitroprusside.

AIM: To examine the effect of methylene blue (MB) on cytotoxicities of sodium nitroprusside (SNP) and cyclic guanosine monophosphate (cGMP) in cultivated cerebellar neurons. METHODS: The cytotoxicities of xenobiotic SNP and cGMP on cultivated murine cerebellar neurons were examined according to Dessi's method. Toxicity of SNP i.c.v. to mice and the prophylactic effect of i.c.v. MB were investigated with respect to the incidence of seizure and the mortality of mice within 24 h. RESULTS: Ten min treatment of SNP 1 mmol.L-1 decreased the survival rate of murine cerebellar nerve cells from 92% of normal control to 35%. Incubation with cGMP 0.1 mmol.L-1 for 1 h declined the survival rate from 94% of normal to 40%. Injection i.c.v. SNP 20 nmol killed one tenth of the mice in 24 h, and SNP 30 nmol killed 11/13 of the mice. MB (100 nmol) i.c.v. injection protected 11/13 of the mice against seizure and death caused by SNP (30 nmol, i.c.v.), and completely eliminated the toxicity of SNP 20 nmol. CONCLUSION: SNP and cGMP inhibit the vitality of murine neurons in vitro. MB injection i.c.v. markedly antagonizes the dose-dependent neuron-toxic effect of SNP in respect of convulsion and mortality of mice.

Animals↗

PubMed1999

Apoptosis and necrosis induced by sulfur mustard in Hela cells.

AIM: To study the apoptotic effect of sulfur mustard (SM) on Hela cells. METHODS: Exponentially growing Hela cells were treated with SM at various concentrations for 3 h, then apoptosis was examined by electron-microscope, DNA gel electrophoresis, and flow cytometry. RESULTS: SM 1 mumol.L-1 arrested cell growth. After treatment with SM 10-100 mumol.L-1, cells were mainly blocked at G1-phase with apoptosis. Agarose gel electrophoresis of DNA from cells treated with SM revealed "DNA Ladder." About 33% of the Hela cells showed apoptosis 12 h after 3-h treatment with SM 100 mumol.L-1 as determined by flow cytometry and the S-phase cells were more susceptible. However, SM 1000 mumol.L-1 caused marked necrosis in Hela cells. CONCLUSION: SM caused 2 distinct forms of cell death, apoptosis or necrosis, in Hela cells in a concentration-dependent manner.

Apoptosis↗

PubMed1998

Nitric oxide and soman poisoning.

AIM: To examine whether nitric-oxide (NO) is involved in the toxicity of soman. METHODS: With pretreatments of icv L-arginine (Arg, the substrate of nitric-oxide synthase NOS), NG-nitro-L-arginine methyl ester (NAME, the inhibitor of NOS), the latency of seizure, and the mortality of mice induced by soman poisoning were examined. The activities of brain NOS in soman-intoxicated mice were measured. RESULTS: In case of Arg pretreatments, the latency decreased (P < 0.05) from (5.2 +/- 1.8) min (control) to (4.3 +/- 0.8) min (Arg 160 nmol), and the mortality increased (P < 0.05) from 50% (control) to 81% (Arg 160 nmol). In case of NAME pretreatment, the latency increased (P < 0.01) from (4.0 +/- 1.1) min (control) to (14.5 +/- 5.0) min (NAME 2.20 mumol), and the mortality decreased (P < 0.05) from 87% (control) to 50% (NAME 2.20 mumol). The toxicity of soman in mice was enhanced by Arg and reduced by NAME all in a dose-dependent fashion. NAME antagonized the enhancement of soman poisoning by Arg. Intoxication of mice with soman increased the NOS activity in cerebrum, cerebellum, and hippocampus from 100% to 104% (P < 0.05), 115% (P < 0.01), and 111% (P < 0.01), respectively. CONCLUSION: The onset of seizure and death of mice induced by soman poisoning are related to the NO messenger system.

Animals↗

PubMed1997

Monoclonal antibody 3F3 against conformational epitope of Torpedo acetylcholinesterase.

AIM: To study the type of epitope of native Torpedo acetylcholinesterase (AChE) directed by its monoclonal antibody (McAb) 3F3. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used for the assay of the reaction between antigen and antibody. RESULTS: McAb 3F3 immunoreacted well with the native AChE, but not with the reduced- and alkylated-AChE (RA-AChE) at all. Soman did not interfere the binding of 3F3 with AChE molecule. The synthesized 24-peptide containing the active serine residue of the AChE active center did not react with McAb 3F3. CONCLUSION: 3F3 is a monoclonal antibody against the conformational epitope of Torpedo AChE active center, but dose not occupy the active serine residue of the enzyme.

Acetylcholinesterase↗

PubMed1997

Anionic subsite of active center of Torpedo acetylcholinesterase constructs a part of its conformational epitope.

AIM: To study the structure-activity relationship of Torpedo acetylcholinesterase (AChE) and explore whether the anionic subsite of the active center is a constituent of the conformational epitope of enzyme. METHODS: Using ELISA and enzyme inhibition test to examine the effect of 1-methyl-2-hydroxyiminomethylpyridium chloride (2-PAM), an anionic subsite probe of AChE, on the immunoreactivity between Torpedo AChE and its monoclonal antibody (McAb) 3F3. RESULTS: McAb 3F3 did not react with 2-PAM-AChE complex. 2-PAM decreased the inhibitory rate of McAb 3F3 on AChE in a concentration-dependent fashion, but did not dissociate the McAb 3F3-AChE complex. CONCLUSION: Anionic subsite of the active center of Torpedo AChE constructs a part of its conformational epitope.

Acetylcholinesterase↗

PubMed1996

The Goodpasture autoantigen. Structural delineation of two immunologically privileged epitopes on alpha3(IV) chain of type IV collagen.

The family of type IV collagen comprises six chains numbered alpha1 through alpha6. The alpha3(IV) NC1 domain is the primary target antigen for autoantibodies from patients with anti-basement membrane disease and Goodpasture syndrome. Earlier peptide studies suggested that the last 36 amino acids of the alpha3 NC1 domain probably contains one recognition site for Goodpasture autoantibodies, and an algorithm analysis of secondary structure from a later study predicted a second possible upstream epitope near the triple helix junction. We have used several analytic approaches to evaluate the likelihood of two immunologic epitopes for the Goodpasture antigen. In our first set of studies, peptide antibodies directed against these two putative regions co-inhibited Goodpasture autoantibodies binding to denatured human alpha3(IV) NC1 monomer by nearly 80%, with the helix-junction region of the alpha3 NC1 domain contributing 26% of the binding sites and the C-terminal region contributing the remaining 50%. Second, both of these candidate regions are normally sequestered within the associated alpha3(IV) NC1 hexamer but become more visible for binding by anti-peptide antibodies upon their dissociation, a property that is shared by the Goodpasture autoantibodies. Third, segment deletions of recombinant alpha3 NC1 domain further confirmed the presence of two serologic binding sites. Finally, we looked more closely at the C-terminal binding region of the alpha3(IV) NC1 domain. Since the lysines in that region have been previously advanced as possible contact sites, we created several substitutions within the C-terminal epitope of the alpha3 NC1 domain. Substitution of lysines to alanines revealed lysines 219 and 229 as essential for antibody binding to this distal site; no lysines were present in the NC1 part of the helix-NC1 junction region. Substitutions involving arginine and cysteines to alanines in the same C-terminal region did not produce significant reductions in antibody binding. In summary, our findings characterize two Goodpasture epitopes confined to each end of the alpha3 NC1 domain; one is lysine-dependent, and the other is not. We propose, as a hypothetical model, that these two immunologically privileged regions fold to form an optimal pathogenic structure within the NC1 domain of the alpha3 chain. These sites are subsequently concealed by NC1 hexamer assembly of type IV collagen.

Anti-Glomerular Basement Membrane Disease↗

PubMed1996

Lipoperoxidative injury to macrophages by oxidatively modified low density lipoprotein may play an important role in foam cell formation.

Both oxidatively and malondialdehyde modified low density lipoprotein (Ox-LDL and MDA-LDL) could be recognized by the scavenger receptor and induce intracellular cholesteryl ester accumulation of macrophage. The cholesteryl ester accumulation caused by MDA-LDL could be largely cleared by high density lipoprotein (HDL3), but that caused by Ox-LDL could not be. Further studies showed that Ox-LDL and MDA-LDL all could decrease the binding capacity of HDL3 and increase intracellular thiobarbituric acid reactive substances (TBARS). When macrophages were first cultured with MDA-LDL and then in medium without LDL, the decreased binding capacity of HDL3 was somewhat recovered and the intracellular TBARS did not increase any more. However, if macrophages were first cultured with Ox-LDL, the binding capacity of H DL3 continued to decrease and intracellular TBARS continued to increase. There was a negative correlation (r = -0.81, P < 0.01) between the decreased binding capacity of HDL3 and the increased intracellular TBARS caused by Ox-LDL. These results imply that lipid peroxidative injury to macrophages caused by Ox-LDL play an important role in foam cell formation.

Animals↗

PubMed1995

Comparative distribution of the alpha 1(IV), alpha 5(IV), and alpha 6(IV) collagen chains in normal human adult and fetal tissues and in kidneys from X-linked Alport syndrome patients.

We have shown previously that the 5' ends of the genes for the alpha 5(IV) and alpha 6(IV) collagen chains lie head-to-head on Xq22 and are deleted in patients with Alport syndrome (AS)-associated diffuse leiomyomatosis. In this study, we raised a rabbit anti-human alpha 6(IV)chain antibody, demonstrated its specificity by the analysis of recombinant NC1 domains af all six type IV chains, and studied the distribution of the alpha 6(IV) chain in relation to the alpha 1(IV) and alpha 5(IV) chains in human adult and fetal tissues involved in AS and diffuse leiomyomatosis. The alpha 6(IV) chain colocalizes with the alpha 5(IV) chain in basement membranes (BMs) of many tissues, but not in glomerular BM. These data exclude the alpha 6(IV) chain as a site for AS mutations. The head-to-head genomic pairing of the alpha 5(IV) and alpha 6 (IV) genes implies coordinate transcription of the two genes. Differential localization of the alpha 5(IV) and alpha 6(IV) chains shows that the two chains are not always coordinately regulated. The alpha 6(IV) chain, together with the alpha 3(IV)-alpha 5(IV) chains, was absent from all renal BMs in eight patients with X-linked AS while the alpha 1(IV) and alpha 2(IV) chains were increased. The data support the existence of two independent collagen networks, one for the alpha 3(IV)-alpha 6(IV) chains and one for the alpha 1(IV) and alpha 2(IV) chains.

Adult↗

PubMed1994

Characterization of a cis-acting regulatory element which silences expression of the class II-A beta gene in epithelium.

Class II major histocompatibility complex (MHC) genes encode for alpha/beta chain pairs that are constitutively expressed principally on mature B cells and dendritic cells in mice. These gene products are easily induced on macrophages with cytokines, and may also aberrantly appear on the surface of epithelium during immune injury. The appearance of class II determinants in parenchymal tissue potentially renders these somatic cells capable of antigen presentation to circulating CD4+ T lymphocytes, and their absence may be protective for normal tissues expressing self-antigens. The low surface class II expression observed on parenchymal cells generally correlates with low levels of mRNA, suggesting that transcription rate is a major element in class II regulation. To understand the transcriptional mechanism maintaining low basal surface expression of class II in somatic cells, we transiently transfected mini-gene reporter constructs to study the regulation of the murine A beta promoter in a cultured renal epithelial cell line. We describe here a negative cis-acting regulatory region located between -552 and -489 bp upstream of the A beta cap site that silences the transcriptional activity of the A beta promoter in epithelial cells in an orientation-dependent manner, and is also able to silence a heterologous promoter. This region is not active in class II-expressing B cells (BAL-17) in culture, but is functional in two other murine class II-negative cell lines, fibroblasts and thymoma T cells. Using competition electrophoretic mobility shift assays, we have localized the core protein binding site within this region to an 8-10-bp response element, designated A beta NRE, at -543 to -534 bp. A nuclear extract from BAL-17 cells does not bind to this element. Mutation of this site abrogates the transcriptional silencing activity of the region. We conclude that the transcription of class II-A beta in parenchymal cells, and some lymphocytes, can be actively repressed by an upstream silencing element.

Animals↗

PubMed1994

A monoclonal antibody marker for Alport syndrome identifies the Alport antigen as the alpha 5 chain of type IV collagen.

The nephropathy of Alport syndrome is associated with unique abnormalities of glomerular basement membranes and is caused in many families by mutations in the X-chromosomal gene COL4A5, which encodes the alpha 5 chain of type IV collagen. We have previously reported that Alport epidermal and glomerular basement membranes fail to bind a monoclonal antibody, Mab A7, that reacts with normal epidermal and glomerular basement membranes, and that this abnormality is unique to Alport syndrome. The molecule in normal tissues that reacts with Mab A7 was termed the "Alport antigen". In the present study we used recombinant carboxyterminal noncollagenous (NC1) domains of the alpha 1, alpha 2, alpha 3, alpha 4 and alpha 5 chains of type IV collagen to determine the molecular identity of the Alport antigen. Mab A7 was found to bind specifically to the NC1 domain of the alpha 5 chain of type IV collagen, by ELISA and immunoblotting studies. This finding provides a molecular explanation for the utility of Mab A7 as a marker for the Alport basement membrane defect. Mab A7 can identify the Alport basement membrane defect in those patients in whom COL4A5 mutations prevent incorporation of alpha 5(IV) into basement membranes.

Antibodies, Monoclonal↗

PubMed1994

COL4A5 gene deletion and production of post-transplant anti-alpha 3(IV) collagen alloantibodies in Alport syndrome.

Mutations in the COL4A5 gene encoding the alpha 5(IV) chain of type IV collagen have been implicated as the primary defect in X-linked Alport syndrome. Several kinds of mutations have been reported so far, spanning point mutations to complete gene deletions. About 5% of Alport patients, who undergo renal transplantation, develop anti-glomerular basement membrane (GBM) nephritis, causing loss of allograft function. In one such patient, COL4A5 gene deletion was recently identified. In the present study, the GBM constituent, targeted by the anti-GBM alloantibodies from the patient who had complete COL4A5 gene deletion was identified. Its identity was determined on the basis of circulating antibody binding to various GBM constituents, domains of bovine type IV collagen and recombinant NC1 domain of human type IV collagen. These results establish, for the first time, the absence of the alpha 5(IV) chain in Alport GBM and, in the same patient, the production of an alloantibody that is targeted to a different chain of type IV collagen, the alpha 3(IV) chain. These findings provide further support for the hypothesis that: (1) anti-alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) chain in Alport GBM, as reflected by the production of anti-alpha 3(IV) alloantibodies.

Basement Membrane↗

PubMed1994

Epitopes recognized by anti-reduced and alkylated acetylcholinesterase antibodies.

Peptides of the reduced and alkylated acetylcholinesterase (RA-AChE) from the electric organ of Torpediniformes Torpedo torpedo subjected to bromo-cynogen (CNBr) cleavage or/and peptic digestion conserved well the antigen-antibody reactivity with anti-RA-AChE monoclonal antibodies E9, F6, and F12, whereas peptides produced by CNBr and tryptic treatments lost all the reactivity. Periodate oxidation of the RA-AChE or glycopeptidase digestion of the CNBr cleaved RA-AChE did not change the antigen-antibody reactivity. It implied that the epitopes recognized by the 3 anti-RA-AChE monoclonal antibodies are all peptide determinants rather than carbohydrate determinants.

Acetylcholinesterase↗