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M J Sun

Publications and source records attributed to M J Sun.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1993

Specificity of Goodpasture autoantibodies for the recombinant noncollagenous domains of human type IV collagen.

Type IV collagen has recently emerged as a family composed of five known chains (alpha 1-alpha 5), each of which contains a carboxyl-terminal noncollagenous domain (NC1) of approximately 230 amino acids. The NC1 domain of the alpha 3(IV) chain is the probable target for autoantibodies in patients with Goodpasture syndrome (GP), as evidenced from studies employing bovine type IV collagen. In the present experiments, the specificity of GP antibodies for the five NC1 domains of human type IV collagen was determined by using recombinant NC1 domains as the antigen. cDNAs encoding each NC1 domain were expressed in E. coli as fusion proteins with a 6-histidine amino-terminal leader. The recombinant NC1 monomers r alpha 1(IV), r alpha 2(IV), r alpha 3(IV), r alpha 4(IV), and r alpha 5(IV) were purified by affinity chromatography to the fusion protein using a nickel resin column, and then characterized by electrophoresis and immunoblot analysis using chain-specific peptide antibodies. The specificity of GP antibodies from four patients to these recombinant proteins was then further evaluated by immunoblot analysis and enzyme-linked immunosorbent assay measurements. The GP antibodies reacted strongly with the r alpha 3(IV) NC1 domain but were not reactive when tested against the other four recombinant monomers. In contrast, neither antisera from patients with two other forms of autoimmune disease (anti-tubular basement membrane disease and Wegener's syndrome) nor normal control sera bound to any of the recombinant NC1 moieties. These results unambiguously establish that GP antibodies are specifically targeted to the NC1 domain of the alpha 3(IV) chain of human type IV collagen. The findings also establish a methodology for large scale preparation of r alpha 3(IV) NC1 domain for use in diagnostic tests and development of therapeutic procedures and offer a strategy for the elucidation of a more complete GP epitope by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1993

Molecular structure of a cross-reactive idiotype on autoantibodies recognizing parenchymal self.

The autoimmune B cell repertoire in anti-tubular basement membrane (alpha TBM) disease is focused on the nephritogenic P1 domain of the 3M-1 target Ag in kidney and normally expresses a disease-modifying cross-reactive Id (IdX). The molecular structure of this Id was determined from a library of rat mAb alpha TBM/alpha 3M-1 by using anchor polymerase chain reactions. Our findings provide the first alignment of V region sequences for rat IgG and reveal that specificity for the P1 domain among alpha 3M-1 antibodies is derived from several recurring germ-line VH genes which have not undergone somatic mutation. The IdX in this repertoire localizes to the H chain on Western blot, and to the CDR3 region as deduced from the cDNA encoding several informative clones. Computer modeling of the Id reveals a conformational structure largely dependent on hydroxyl groups in or near turn position 4 of the H chain CDR3 region. These findings demonstrate that regulatory elements protective of autoimmunity are not encoded in the germ line as IdX, but rather emerge from a recombinatorial diversity engaged by the recognition of parenchymal self.

Amino Acid Sequence↗
PubMed1993

Human cDNA clones transcribed from an unusually high-molecular-weight RNA encode a new collagen chain.

Human collagen (COL) cDNA clones were isolated from a library representing transcripts synthesized by an established rhabdomyosarcoma (RH) cell line. The 0.6-kb insert of the first isolate encodes a discontinuous collagenous sequence not homologous to type I-XVI COL chains. Sequencing of a second clone with a 4-kb insert revealed an open reading frame (ORF) of 2154 nucleotides. The deduced amino acid (aa) sequence begins with an 186-aa noncollagenous region containing seven cysteines (Cys). Several of the Cys and surrounding aa residues can be aligned with those in type XVI, XII and IX COL. Due to the presence of two long interruptions, the 524-aa collagenous region is separated into three subdomains that each have smaller interruptions of 1-6 aa. The protein terminates with an 8-aa noncollagenous peptide including an unusual single Cys which would be expected to form an interchain disulfide bond. Results of Northern blot hybridization suggest that the new COL chain may be uncommonly large since the clone identified a low-abundance RNA at least 12.4 kb in size. The gene coding for RH COL is located on human chromosome 6. It is now important to elucidate the role of this unusual COL in the infrastructure of extracellular matrix.

Amino Acid Sequence↗
PubMed1993

Purification and properties of acetylcholinesterase from human brain.

Acetylcholinesterase from human caudate nucleus and partial thalamus was purified by using Con A-Sepharose, short-arm and long-arm ligand Sepharose affinity chromatographies. SDS-PAGE of the purified AChE under the reduced condition showed one main band, corresponding to a molecular weight of 66 kD. The purified AChE with a specific activity of 3384 U/mg protein represented 20% activity of the homogenate supernatant. Analysis of purified AChE by gradient slab PAGE and DISC-PAGE with activity staining revealed the existence of monomer, dimer, tetramer, hexamer and octomer of the enzyme. The isoelectric point of AChE ranged between pH 5.6 and 6.0. Con A-Sepharose affinity chromatography retained most of the applied AChE activity implying that the enzyme is a kind of glycoprotein. The isolated human brain AChE had no cross-immunoreactivity with 3F3 and weak cross-immunoreactivity with 2G8 and 1H11 anti-Torpedo AChE antibodies. Balb/c mice were immunized with human cerebellum AChE purified with Con A and short-arm ligand affinity chromatographies. The antiserum produced showed strong cross-immunoreactivity with Torpedo AChE but weak cross-immunoreactivity with human RBC membrane AChE. The purified human brain striatum AChE was reduced and alkylated, and then hydrolyzed by immobilized TPCK-treated trypsin. Trypsin peptides in the hydrolysate was separated by RP-HPLC. Several large peptide peaks and numbers of small peaks were observed. The large peaks showed obvious immunoreactivity with the mouse anti human cerebellum AChE antiserum.

Acetylcholinesterase↗
PubMed1992

Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines.

Fibroblasts in parenchymal organs potentially contribute extracellular matrix to local fibrogenic processes. This contribution, in some circumstances, may be initiated by cytokines disseminated from inflammatory lesions. Different populations of fibroblasts, however, might respond distinctively to this cytokine bath depending on the microenvironment in which they reside. We have begun to explore this issue using syngeneic, low-passage fibroblasts cultured in serum-free media that were derived originally from the dermis (DFBs) and from tubulointerstitium (TFBs) of the kidney. Our findings indicate that, while fibroblasts from each compartment appear similar at the ultrastructural level, there are a variety of functional differences which distinguish their proliferative response, and their collagen secretory response (types I, III, IV, and V) following challenge with various doses of immune-relevant cytokines (TGF beta, EGF, IL-1, IL-2 and gamma IFN) in culture. DFBs, for example, express more surface EGF receptors than do TFBs, and, as a consequence, exhibit a more robust proliferative response to EGF in serum-free media. Unstimulated DFBs also secrete more collagen types I and III than TFBs, while unstimulated TFBs secrete more types IV and V. The expression of these collagens in TFBs was confirmed by Northern blot hybridization. When these sets of fibroblasts were further stimulated by cytokines, some of the cytokines not only differentially effect the secretion of various species of collagens within the same group of cells, but also between cells from populations which are anatomically distinct. DFBs, furthermore, at mid-level doses of cytokine, demonstrated a general trend towards less secretion of all types of collagen (particularly for TGF beta, EGF, and IL-2), while TFBs seemed less repressive. In TFBs the cytokine-induced responses for collagen types I and III tended to be discordant, and for types I and IV EGF inhibited, while TGF beta stimulated the secretory process. These findings speak collectively for the presence of a functional heterogeneity among organ-based populations of syngeneic fibroblasts in normal tissues.

Animals↗
PubMed1992

Isolation and characterization of cDNA from renal tubular epithelium encoding murine Rantes.

We have been interested in identifying proinflammatory molecules which might play a role in attracting monocytes and T cells to the kidney. Some of the new intercrines are potential candidates. In this report we have isolated cDNA encoding murine Rantes (MuRantes) from renal tubular epithelium (MCT cells). MuRantes is a 91 amino acid member of the -C-C- or intercrine beta subgroup of the Scy superfamily. The amino acid sequence for mature MuRantes was deduced from its coding cDNA and was found to be 90% homologous to its mature human counterpart (HuRantes). MCT epithelium expresses a single mRNA transcript for MuRantes of approximately 1100 bp. The MuRantes protein could be detected in cell lysates of MCT epithelium by western blotting and in the cytoplasm of MCT cells by immunofluorescence using a polyclonal antibody generated against HuRantes fusion protein. A search protocol using MuRantes-specific primers and cDNA amplification revealed that mRNAs for MuRantes are expressed additionally in syngeneic mesangial cells (MMC cells), whole kidney, liver, and spleen, as well as in nephritogenic antigen-specific CD4+ helper and CD8+ effector T cells. cDNA amplification studies also demonstrated a significant elevation in mRNA transcripts encoding MuRantes in response to the stimulation of MCT epithelium with TNF alpha and IL-1 alpha in culture, but not with TGF beta, gamma IFN, or IL-6. Our findings indicate that proximal tubular epithelium is an authentic source of MuRantes, and that transcripts encoding MuRantes are responsive to the modulating influence of paracrine factors having a known role in the development of parenchymal injury.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of a major nephritogenic domain in the autoantigen of anti-tubular basement membrane disease.

Anti-tubular basement membrane (alpha TBM) disease is a form of primary interstitial nephritis mediated by autoimmune T cells and alpha TBM antibodies. In mice and humans the nephritogenic immune response is directed to a glycoprotein (3M-1) found along the proximal tubule of the kidney. We have isolated cDNAs from an expression library that encodes for the common framework domain of the 3M-1 antigen. This common domain was once related evolutionarily to a family of intermediate filament-associated proteins. Northern hybridization revealed that all isoforms of 3M-1 range between 1700 and 1900 base pairs and in situ hybridization studies indicate that transcripts are found in tubular epithelium. Candidate peptide fragments were deduced and synthesized from the sequence encoding this common framework domain, and one of the peptide residues was able to bind a monoclonal 3M-1-reactive alpha TBM antibody, stimulate the growth of 3M-1-reactive helper T cells, and induce nephritogenic effector T cells capable of producing interstitial nephritis. Our results indicate that a unique, immunodominant region of the 3M-1 antigen is an informative participant in the emergence of autoimmune injury to certain basement membranes.

Amino Acid Sequence↗
PubMed1991

Expression of homeobox genes in a proximal tubular cell line derived from adult mice.

We have been studying the expression of several homeobox genes in cultures of proximal tubular epithelium (MCT cells) harvested from adult mus musculus. Hox genes 2.1, 2.3, and 3.3, in particular, are all expressed at low levels in resting MCT cells. The expression of Hox 2.1 and 3.3 were not influenced by mitogenic (epidermal growth factor: EGF, and platelet-derived growth factor: PDGF) nor by hypertrophogenic cytokines (angiotensin II: Ang II) in serum-free media. Transcripts for Hox 2.3, however, were elevated in MCT cells by Ang II. EGF, and serum treatment, as early as 30 minutes after their addition, whereas no change, or slight reductions were observed with transforming growth factor beta (TGF beta), PDGF, and gamma-interferon (gamma IFN). Hox 2.3 was also super-induced by serum, in the presence of cycloheximide, in cells rested previously in serum-free media, suggesting that new protein synthesis was not required for expressive augmentation. The induction of Hox 2.3, moreover, was not specific for tubular epithelium, since the gene could be activated in tubulointerstitial fibroblasts after treatment with EGF. These experiments collectively represent a first report regarding the characterization of transcripts encoding homeoboxes in adult cells derived from renal tissue. The putative DNA-binding properties of homeobox proteins in general, the prompt and rapid induction of Hox 2.3 by morphogenic cytokines in tubulointerstitial cells, and the observed effect of cycloheximide on this gene, all indicate that Hox 2.3 might have a role in the general activation of mature somatic cells, as an immediate early event. probably in the capacity of a nuclear trans-acting factor.

Animals↗
PubMed1991

Analysis of the cDNA sequence encoding MHC-A beta in tubular epithelium from mouse kidney.

Class II gene products of the major histocompatibility complex (MHC) are not expressed usually in abundance on normal epithelium. The cell surface visibility of such proteins for the immune system is thought to be limited protectively in order to minimize inflammation consequent to the recognition of self-antigens in parenchymal structures by T lymphocytes. In the current experiments we investigated whether the previously recognized sparseness of A beta on the surface of tubular epithelial cells might be accounted for by a protein coding difference deduced from the primary structure of its transcript compared with sequence from lymphoid cells that normally express A beta in generous amounts. We demonstrate, however, using clones obtained from a cDNA library prepared from tubular epithelium harvested from H-2s (A beta/alpha+; E beta/alpha-) mice susceptible to autoimmune interstitial nephritis, that the nucleotide sequence encoding the class II A beta chain in cells from both compartments is essentially identical. Our findings suggest that there is no primary structural aberrancy in the coding region of parenchymal A beta that would contribute to its low expression. The protective tolerance afforded by reduced numbers of class II molecules in normal tissues is, therefore, more likely the result of repressive regulatory processes.

Animals↗
PubMed1991

Lysis of herpes simplex virus-infected cells by lymphokine-activated killer cells.

Activation of human peripheral blood mononuclear cells (PBMC) or murine splenocytes with recombinant interleukin-2 (rIL-2) has been found to be able to generate cells which are lytic in vitro for a variety of cells infected by herpes simplex virus (HSV). Lymphokine-activated killer (LAK) cells were generated by the incubation of human PBMC or murine splenocytes with 500 u/ml of rIL-2 for 3 to 4 days in a CO2 incubator at 37 degrees C. These cells were lytic to cultured tumor cell lines, yet sparing normal primary cell cultures as has been reported. Human PBMC infected with HSV, however, were susceptible to the lytic effect of these LAK cells. From 30 to 50 percent lysis of HSV-infected human PBMC was observed when the effector-to-target ratios were 40:1 and 100:1 respectively by four hr 51Cr-release assays. Similar cytolytic effect was observed when mouse embryonic fibroblasts (MEF) were infected with HSV and used as targets when rIL-2-activated mouse splenocytes were used as effectors. The cytolytic effects of LAK cells on the HSV-infected cells can be demonstrated even after lowering of NK activities by the treatment of effector cells with anti-NK antibody Leu-11b plus complement. Cytolytic effects were not restricted to the autologous system, and could be demonstrated in the heterologous system as well. Human LAK cells killed not only HSV-infected autologous PBMC but also infected mouse embryonic fibroblasts. However, mouse LAK cells exhibit species-specific cytotoxicity. In conclusion, it was demonstrated that LAK cells are cytolytic to HSV-infected cells in both human and murine systems.

Animals↗
PubMed1990

[Aerobic metabolism of VX and mixed function oxidases].

In our preliminary study, it has been found that VX oxidase exists in the microsome fraction of rat liver and the catalytic reaction needs the participation of molecular oxygen and coenzyme I or II. In this paper, the data showed that deoxycholate inactivated both the mixed function oxidase and VX oxidase. The specific inhibitor proadifen of the mixed function oxidase also profoundly inhibited VX oxidase activity. The complex of VX and cytochrome P-450 exhibited typical difference spectrum of type I. Aniline competitively inhibited the inactivation of VX catalyzed by microsomes. These results indicate that VX is one of the substrates of mixed function oxidase. VX oxidase in the rat liver cells is exactly the mixed function oxidase.

Aerobiosis↗
PubMed1989

Eruption effects of microdontia--a case report.

A patient presented at the Department of Orthodontics, Medunsa Dental Hospital, complaining of "crooked teeth". On clinical examination it was evident that several teeth were unerupted. The absence of a normal eruption pattern of certain teeth as well as delayed eruption of others, resulted in a malocclusion.

Adolescent↗
PubMed1988

Clonotypic heterogeneity in experimental interstitial nephritis. Restricted specificity of the anti-tubular basement membrane B cell repertoire is associated with a disease-modifying crossreactive idiotype.

Experimental anti-tubular basement membrane (anti-TBM) disease is an autoimmune interstitial nephritis elicited in susceptible rodents after immunization with renal tubular antigen. The nephritogenic antigen in the immunizing preparation is 3M-1, a 48,000 Mr noncollagenous glycoprotein. The hallmarks of the renal lesion are the presence of anti-TBM antibodies (anti-TBM-Ab) and a dense mononuclear cell infiltrate. The anti-TBM B cell repertoire in this disease was analyzed using a library of 22 anti-TBM mAbs generated in a prototypically susceptible Brown Norway rat. These anti-TBM mAbs were all demonstrated to be 3M-1 specific and their characterization formed the basis for the following observations: (a) The size of the anti-TBM B cell population is estimated at 58 distinct clones; (b) by competitive inhibition criteria, all anti-TBM mAbs recognize the same (or spatially close) epitope(s) on 3M-1. This focused recognition was maintained in spite of considerable variability in affinity. Epitopic dominance could also be demonstrated in human polyclonal anti-TBM antisera from a patient with anti-TBM disease; and (c) a crossreactive idiotype was documented, and antisera directed toward this set of variable region determinants was shown to be effective as a prophylactic regimen to abrogate disease, and as a therapeutic modality to arrest the progression of disease; (d) analysis of VH gene families suggested biased usage of Q52- and 7183-like families, although at least three gene families are used in the anti-TBM-Ab response. Thus, the anti-TBM B cell compartment in BN rats is moderately large, but is primarily focused to a single epitope on the nephritogenic antigen and is associated with a disease-modifying crossreactive idiotype.

Animals↗
PubMed1987

Murine interstitial nephritis. VII. Suppression of renal injury after treatment with soluble suppressor factor TsF1.

We prepared soluble suppressor T cell factor (TsF1) from donor spleens harvested from mice primed with tubular antigen-derivatized lymphocytes to analyze both its functional interactions with a larger suppressor T cell network and its influence on the nephritogenic effector T cell response producing interstitial nephritis to a parenchymal antigen. Our findings indicate that TsF1 is antigen-specific, genetically restricted by I-J in its direct mediation of suppression, and capable of inhibiting the development of interstitial lesions. TsF1 also provides an inducing signal for the activation of effector Ts-2 suppressors following presentation by accessory cells. The induction of a Ts-2 effect, however, requires that the factor-presenting cell and the recipient of such cells share homology at I-J, and that the TsF1, the precursor Ts-2 cells, and the recipient of the Ts-2 effect share the same Igh-V allotype. Finally, the results of this current report clearly demonstrate a possible therapeutic role for soluble suppressor factors in the management of interstitial renal disease.

Animals↗
PubMed1987

Effector T cell differentiation in experimental interstitial nephritis. I. The development and modulation of effector lymphocyte maturation by I-J+ regulatory T cells.

Because mice susceptible to interstitial nephritis use different effector T cells than nonsusceptible mice, we analyzed the differentiation process of the effector T cell repertoire by using an in vitro culture technique. In the presence of helper T lymphocytes, accessory cells, IL 2, tubular antigen, and precursor effector cells, both Lyt-2+ nephritogenic effector cells and L3T4+ nonnephritogenic effector cells can be initially induced in both susceptible and nonsusceptible strains within 3 days of culture. In nonsusceptible mice, however, the Lyt-2+ nephritogenic cell is inhibited from further development and disappears, whereas in susceptible mice, its presence is preserved with a resulting effect of tissue destruction. This selection of effector T cell preference is regulated by I-J+ T lymphocytes which are co-functionally expressed with effector cell expansion. Unlike precursor effector lymphocytes, however, the maturation of the regulatory process requires a subset of I-J+ accessory cells and structurally intact tubular antigen. Our findings indicate, therefore, that both susceptible and nonsusceptible mice have the potential for the expression of interstitial nephritis, but nonsusceptible mice are formally protected from autoimmunity by the regulation of lymphocyte preference.

Animals↗