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Biomedical subjects

M D Chang

Publications and source records attributed to M D Chang.

At least 19 recordsLinked to original sources
PubMed1998

Differential activation of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases confers cadmium-induced HSP70 expression in 9L rat brain tumor cells.

We have reported that treatment with CdCl2 at 40-100 microM induces the heat shock proteins (HSPs) in 9L rat brain tumor cells, during which the activation of heat shock factor (HSF) is essentially involved. By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of HSP70 in cells treated with a high concentration of cadmium (i.e. 100 microM) is preceded by the phosphorylation and activation of p38 mitogen-activated protein kinase (p38(MAPK)), while that in cells treated with a low concentration (60 microM) is accompanied by the phosphorylation and activation of extracellular-regulated protein kinases 1 and 2 (ERK1/2). In 100 microM cadmium-treated cells, both HSP70 induction and HSF1 activation are eliminated in the presence of SB203580, a specific inhibitor of p38(MAPK). By contrast, in 60 microM cadmium-treated cells, the processes are not affected by SB203580 but are significantly suppressed by PD98059, which indirectly inhibits ERK1/2 by acting on MAPK-ERK kinase. Taken together, we demonstrate that p38(MAPK) and ERK1/2 can be simultaneously or independently activated under different concentrations of cadmium and that the signaling pathways participate in the induction of HSP70 by acting on the inducible phosphorylation of HSF1. We thus provide the first evidence that both p38(MAPK) and ERK signaling pathways can differentially participate in the activation of HSF1, which leads to the induction of HSP70 by cadmium.

Animals↗
PubMed1998

Involvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells.

Exposure of 9L rat brain tumor cells to 40-100 microM CdCl2 for 2 h leads to an induction of a wide spectrum of heat shock proteins (HSPs). We have demonstrated that induction of the 70-kDa HSP (HSP70) and enhanced expression of its cognate (HSC70) by cadmium are concentration dependent and that the induction kinetics of these HSP70s are different. The increased synthesis of the HSP70s is accompanied by the increase in hsp70 and hsc70 mRNA levels, indicative of transcriptional regulation of the heat shock genes. Electrophoretic mobility shift assay (EMSA) using probes encompassing heat shock element (HSE), TATA, GC, and CCAAT boxes derived from the promoter regions of the heat shock genes shows distinguished binding patterns between hsp70 and hsc70 genes in both control and cadmium-treated cells. The results indicate that, in addition to the HSEs, the basal transcription elements are important in the regulation of the heat shock genes. The binding patterns of the corresponding transcription factors of these elements are examined by EMSA by using extended promoter fragments from respective heat shock genes with sequential addition of excess oligonucleotides encompassing individual transcription elements. Taken together, our results show that the differential induction of hsp70 and hsc70 involves multiple transcription factors that interact with HSE, TATA, GC, and CCAAT boxes.

Animals↗
PubMed1998

Novel glycosylation of HLA-DRalpha disrupts antigen presentation without altering endosomal localization.

The HLA-DR hemizygous B lymphoblastoid cell line, 10.24.6, has a DRA mutation (Pro96-->Ser) that creates a novel glycosylation site at Asn94. The mutant DR molecules are primarily associated with nested fragments of invariant chain (class II-associated invariant chain peptides), and their interaction with HLA-DM is impaired. Here we further analyzed the defect in 10.24.6 cells. Expressing Ser96 mutant DRA cDNA in DRA-null cells recapitulated the 10.24.6 phenotype, indicating that the mutation causes the Ag presentation defect. A mutation to Ala96alpha, which does not introduce an extra glycan, generated a normal phenotype; the critical role of the glycan was further supported by experiments in which N-glycosylation was blocked by tunicamycin. We also evaluated whether the 10.24.6 mutation affected DR3 maturation or trafficking. Metabolic labeling and subcellular fractionation showed that assembly, endosomal transport, and invariant chain proteolysis of mutant DR3 molecules were similar to wild-type. A slight delay in export from the endoplasmic reticulum to the Golgi apparatus in 10.24.6 cells probably did not contribute significantly to the Ag presentation defect, because the abundance of DM and mutant DR in peptide-loading compartments was normal at steady state. Our results indicate that proper localization of these molecules does not depend on their interaction.

Antigen Presentation↗
PubMed1998

Involvement of p38 mitogen-activated protein kinase signaling pathway in the rapid induction of the 78-kDa glucose-regulated protein in 9L rat brain tumor cells.

We have previously shown that treatment with okadaic acid (OA) followed by heat shock (HS) (termed OA --> HS treatment) leads to rapid transactivation of the 78-kDa glucose-regulated protein gene (grp78) in 9L rat brain tumor cells. A cAMP-responsive element-like (CRE-like, TGACGTGA) promoter sequence and a protein kinase A signaling pathway are involved in this induction, and activation of both CRE binding protein (CREB) and activating transcription factor-2 (ATF-2) is required in the above process. Herein, we report that transactivation of grp78, as well as phosphorylation/activation of ATF-2, can be completely annihilated by SB203580, a highly specific inhibitor of p38 mitogen-activated protein kinase (p38(MAPK)). Activation of p38(MAPK) by OA --> HS is also substantiated by its own phosphorylation as well as the phosphorylation and activation of MAPK activating protein kinase-2 in cells subjected to this treatment. The involvement of p38(MAPK) in the activation of ATF-2, which leads to the transactivation of rat grp78, is confirmed by electrophoretic mobility shift assay using a probe containing the CRE-like sequence as well as by transient transfection assays with a plasmid containing a 710-base pair stretch of the grp78 promoter. Together with our previous studies, these results led us to conclude that phosphorylation/activation of CREB upon OA --> HS treatment is mediated by cAMP-dependent protein kinase, whereas that of ATF-2 is mediated by p38(MAPK). The transcription factors may bind to each other to form heterodimers that in turn transactivate grp78 by binding to the CRE-like element. This suggests that distinct signaling pathways converge on CREB-ATF-2, where each subunit is individually activated by a specific class of protein kinases. This may allow modulation of grp78 transactivation by diverse external stimuli.

Animals↗
PubMed1997

Rapid induction of the Grp78 gene by cooperative actions of okadaic acid and heat-shock in 9L rat brain tumor cells--involvement of a cAMP responsive element-like promoter sequence and a protein kinase A signaling pathway.

We have demonstrated that treatment with 200 nM okadaic acid (OA) for 1 h followed by a 15-min heat shock (HS) at 45 degrees C (termed OA-->HS treatment) leads to a rapid transactivation of grp78, the gene for the 78-kDa glucose-regulated protein, in 9L rat brain tumor cells. The level of Grp78 mRNA rose 15-fold in 60 min after the combined treatment. Nuclear extracts from cells subjected to OA-->HS treatment, compared to those of treatment with OA or HS alone, exhibited an increased binding activity toward an oligonucleotide probe containing the cAMP-responsive element-like (CRE-like, TGACGTGA) regulatory element in electrophoretic mobility shift assays (EMSA). The binding resulted in the formation of two protein-EMSA probe complexes exhibiting different association and dissociation rates in kinetic studies. The protein factors in the upper band (complex I) and lower band (complex II) were identified as the activating transcription factor-2 (ATF-2) and the CRE binding factor 1 (CREB-1), respectively, by antibody interference assays. In addition, the identity of CREB-1 was confirmed by supershift analysis. The binding activity, as well as the transactivation of the grp78 gene, can be abolished by a 1-h treatment with the cAMP-dependent protein kinase (PKA) inhibitor but not with protein kinase C or Ca2+/calmodulin-dependent protein kinase II inhibitors. Accumulation of steady-state level of ATF-2 was observed and was also modulated by treatment with H-89, a PKA inhibitor. From these results, we conclude that the CRE-like element plays an important role in the rapid transactivation of the grp78 gene and that the PKA signaling pathway is involved. In addition, PKA-mediated transcriptional regulation of grp78 in OA-->HS treatment is through regulation of protein phosphorylation as well as de novo synthesis of ATF-2.

Activating Transcription Factor 1↗
PubMed1997

Estradiol down-regulates LPS-induced cytokine production and NFkB activation in murine macrophages.

PROBLEM: In vivo and in vitro studies have indicated that estradiol can affect cytokine production in different cell types. This study examines whether estradiol affects inflammatory cytokine production by murine splenic macrophages. METHODS: Mouse splenic macrophages were first treated with 17 beta-estradiol, followed by lipopolysaccharide (LPS) stimulation. The production of cytokines by macrophages with or without estradiol treatment was determined at both the protein and mRNA levels. The nuclear factor-kB (NFkB) activity of activated mouse splenic macrophages was also evaluated by electrophoretic mobility shift assay. RESULT: Our results show that 17 beta-estradiol decreases LPS-induced IL-1 alpha, IL-6, and TNF-alpha production but not IL-10, IL-12, and macrophage inflammatory protein (MIP) production by splenic macrophages. Furthermore, inhibition of cytokine production by 17 beta-estradiol was associated with a decreased LPS-induced NFkB-binding activity. CONCLUSION: Because cytokines are important mediators of immune function, the alteration of cytokine production by 17 beta-estradiol may thus have a profound effect on the outcome of immune response during inflammation.

Animals↗
PubMed1997

Oligoclonality of CD8+ T cells in breast cancer patients.

Substantial evidence has suggested that T cells play an important role in antitumor immunity. T cells with cytotoxic activity against tumors have been isolated from in vitro culture of tumor-infiltrated lymphocytes of cancer patients. In addition, clonal expansions of T cells have been identified in lesions of tumors by using a PCR-based CDR3 analysis of T cell receptors (TCR). Since the CDR3 region of the T cell receptor directly interacts with the antigen-MHC complex and is thus highly polymorphic, a dominant CDR3 length in a particular TCR V beta population will indicate the clonal expansion of a specific T cell clone. Utilizing this technique, we have analyzed the T cell repertoire in lymph nodes (LNs) and peripheral blood of 20 breast cancer patients. Our results show that in most cases, peripheral blood mononuclear cells (PB-MCs) and LN express dominant CD8+ T cell clones in different V beta gene families, and the number of dominant clones is higher in PBMC than in the LN. Furthermore, in 7 out of 16 patients' lymph nodes, there is a dominant V beta 18 T cell clonal expansion in the CD8+ T cell subset. The frequency of an oligoclonal expansion of V beta 18 CD8+ T cells in non-breast cancer lymph nodes is 1 out of 9, but no obvious motif in the CDR3 region of V beta 18 TCR can be identified. The prevalence of the clonal dominance found in breast cancer is discussed in the context of a possible tumor-related antigen stimulation.

Adult↗
PubMed1995

Mutational analysis of two DR alpha residues involved in dimers of HLA-DR molecules.

Crystallographic analysis of HLA-DR1 molecules reveals a "dimer of dimers" with two reciprocal salt bridges between Glu 88 and Lys 111 of the two DR alpha chains. To determine whether these amino acids are critical for Ag presentation, we generated a panel of human B cell transfectants expressing DR alpha chains with mutations at residues 88, 111, or both. The mutant DR alpha chains, paired with endogenous DR3 beta chain, form cell surface dimers that retain epitopes recognized by a panel of anti-DR3 Abs. Replacement of Glu 88 with Ala (88A) selectively eliminates the ability to activate an alloreactive (anti-DR3) T cell clone. Mutant DR molecules with Lys substituted for Glu 88 (88K) fail to activate an alloreactive, an Ag-specific, and a peptide-specific T cell line. The DR alpha 88 mutants bind an exogenously supplied DR3-specific peptide and the mutant DR molecules migrate as dimers on SDS-PAGE, implying that their defective Ag presentation is not due to an inability to bind antigenic peptides. In contrast, substitution of Lys 111 with either Ala (111A) or Glu (111E) does not abrogate Ag presentation. Further, the defect introduced by Glu 88 to Lys mutation (88K) is not overcome by compensatory Lys to Glu mutation at position 111 (111E). Taken together, these results indicate an important functional or structural role for position 88 of the DR alpha chain, but argue against a requirement for interaction between DR alpha 88 and 111 during Ag-specific T cell stimulation.

Antigen Presentation↗
PubMed1995

Osteopetrotic (op/op) mice deficient in macrophages have the ability to mount a normal T-cell-dependent immune response.

The osteopetrotic (op/op) mouse possesses an inactivating mutation in the CSF-1 gene that is associated with a lack of certain mononuclear phagocyte populations. To examine the effects of these deficiencies on T-cell-dependent immune functions, the responses of op/op and normal mice to a T-cell-dependent antigen, ovalbumin, and to a foreign histocompatibility antigen were compared. The ability of op/op mice (1) to induce antigen-specific proliferation of naive T-cells, (2) to generate cytotoxic T-lymphocytes, (3) to supply spleen cells to serve as stimulators in a mixed lymphocyte reaction, and (4) to produce IgM and IgG antibodies was indistinguishable from normal mice. These data are consistent with the involvement of specific macrophages, or other cells whose development is independent of CSF-1, in these immune responses.

Animals↗
PubMed1995

Expression and purification of two isozymes of clavaminate synthase and initial characterization of the iron binding site. General error analysis in polymerase chain reaction amplification.

Clavaminate synthase is an Fe(2+)-, O2-, and alpha-ketoglutarate-dependent oxygenase that catalyzes three transformations in the biosynthesis of the important beta-lactamase inhibitor clavulanic acid. The genes from Streptomyces clavuligerus encoding two isoenzymes of clavaminate synthase have been over-expressed in Escherichia coli to give soluble proteins whose reactions, kinetic properties, and molecular masses are in excellent agreement with the wild-type isozymes. Preliminary investigation of the active site of clavaminate synthase was undertaken using diethyl pyrocarbonate and N-ethylmaleimide. Each was inhibitory to catalytic activity. Protection from inactivation in the presence of these reagents by Fe2+, O2, and alpha-ketoglutaric acid was thwarted by the rapid self-inactivation of the enzyme in the absence of substrate. However, protection was achieved when Co2+, a potent competitive inhibitor of clavaminate synthase 2 with respect to Fe2+, was substituted. This is consistent with the presence of histidine and cysteine, respectively, at or near the active site and possibly involved in iron binding. In the course of constructing the expression vector, a simply applied general error analysis of the polymerase chain reaction was formulated to calculate the proportion of correctly replicated DNA and guide the design of experiments using this method.

Base Sequence↗
PubMed1995

Analysis of HLA-DMB mutants and -DMB genomic structure.

The HLA-DM locus encodes class II-like A and B chains and apparently regulates the antigen presentation function of conventional major histocompatibility complex (MHC) class II molecules. Here we describe the HLA-DMB mutations in three presentation defective B lymphoblastoid cells lines (B-LCL), 7.19.6, 10.6.6, and 10.78.6, which express DMB transcripts of abnormal length. Mutant 7.19.6 has a C-->T point mutation that introduces a 5' splice site into exon 3 of DMB. The independently derived mutants, 10.6.6 and 10.78.6, each harbor a G-->A mutation in exon 3 and also lack an identical downstream segment of RNA. Mapping of DMB intron/exon borders, using a genomic clone, revealed that the segment missing in mutants 10.6.6 and 10.78.6 represents the fourth exon of DMB; no mutations were found within exon 4 in either 10.6.6 or 10.78.6, however. In addition, the DMB gene was found to have a six exon genomic structure, typical of MHC class II B genes.

Amino Acid Sequence↗
PubMed1995

Replacement of the DR alpha chain with the E alpha chain enhances presentation of Mycoplasma arthritidis superantigen by the human class II DR molecule.

Mycoplasma arthritidis mitogen (MAM) is produced by an organism which can cause chronic proliferative arthritis in rodents. MAM possesses a typical superantigenic activity; it has the ability to activate a large panel of T cells which express specific V beta segments of the T-cell receptor. The presentation of MAM to T cells by antigen-presenting cells is mediated primarily through its binding to the major histocompatibility complex (MHC) class II E alpha chain in mice and the DR alpha chain in humans. However, MAM is much less active for human peripheral blood lymphocytes than for mouse splenocytes. It was suggested that a difference in MAM binding affinity between human and mouse class II molecules may account for their different MAM activities. To examine this possibility, we generated a panel of B-cell transfectants whose DR molecule is composed of either the DR alpha or the E alpha chain paired with a DR3 beta chain. The ability of these transfectants to present MAM to human peripheral T cells was analyzed. Our data show that transfectants expressing E alpha DR beta chimeric molecules have higher MAM-presenting activity than transfectants expressing wild-type DR alpha DR beta molecules, while the latter have higher activity in stimulating DR3-alloreactive T cells. Since both types of transfectants present MAM to T cells expressing the same T-cell receptor V beta gene families, the higher MAM-presenting activity of the E alpha transfectant is not due to its ability to interact with a different set of T cells. Furthermore, both the E alpha 1 and E alpha 2 domains contribute to this increased affinity for MAM binding. Taken together, our data suggest that there may be multiple MAM binding sites on the E alpha and DR alpha chains and residues unique to the E alpha chain may provide additional affinity for MAM.

Antigen Presentation↗
PubMed1995

Osmolar regulation of endothelin signaling in rat renal medullary interstitial cells.

We tested the hypothesis that endothelin (ET) responsiveness in the renal medulla is modulated by ambient osmolarity. Cultured renal medullary interstitial cells (RMICs) were incubated from 3 to 24 h in isosmolar culture medium (300 mOsm/kg H2O) or media rendered hyperosmolar (600 mOsm/kg H2O) by the addition of urea. Under hyperosmolar conditions, the peak of ET-evoked Ca2+ transient was blunted by 45-58% (P < 0.02) and PGE2 accumulation decreased from 16- to 2-fold above basal values (P < 0.001). To explore whether hyperosmolar conditions blunt intracellular signaling via modulation of receptor number or expression, kinetics of ET binding and Northern blot analysis of ETA receptor mRNA was performed. Under hyperosmolar conditions, ETA receptor density was reduced by 84% versus isosmolar conditions (238 +/- 12 vs. 1450 +/- 184 fmol/mg) (P < 0.01). In contrast to the ligand binding studies, ETA receptor mRNA was increased by 58% (P < 0.05) in cells grown under hyperosmolar versus isosmolar media. These observations indicate that in the hyperosmolar setting, ET-evoked intracellular signaling is blunted in RMICs due to ET receptor downregulation. Since ETA receptor mRNA is increased under hyperosmolar conditions, we conclude that ET receptor downregulation is the consequence of either decreased translation of message, increased degradation of receptor peptide, or increased internalization of specific receptor sites.

Animals↗
PubMed1993

Mouse placental macrophages have a decreased ability to present antigen.

Large numbers of macrophages can be found in an animal's uteroplacental unit. This high concentration of macrophages suggests they must play an important role during placental development. To gain a better understanding of the functional capacity of placental macrophages, we have obtained a highly enriched placental macrophage culture and have derived several cell lines from this population. Both placental macrophages and cell lines show colony-stimulating factor 1-dependent growth, express Fc receptors, and can perform Fc-receptor-mediated phagocytosis. In addition, they express macrophage markers Mac-1, F4/80, and CD14. Although placental macrophages express major histocompatibility complex class II molecules constitutively, they display a decreased ability to present protein antigens to T cells. Since primary fetal liver macrophages of the same gestational stage also show a decreased ability to present antigens, this phenomenon may reflect a developmental stage of macrophages.

Animals↗
PubMed1992

Two isozymes of clavaminate synthase central to clavulanic acid formation: cloning and sequencing of both genes from Streptomyces clavuligerus.

Clavaminate synthase (CS) is an alpha-ketoglutarate-dependent oxygenase central to the biosynthesis of clavulanic acid, a potent inhibitor of beta-lactamases. CS catalyzes the oxidative cyclization/desaturation of proclavaminic acid to clavaminic acid in a two-step process involving the intermediacy of dihydroclavaminic acid [Salowe, S. P., Krol, W. J., Iwata-Reuyl, D., & Townsend, C. A. (1991) Biochemistry 30, 2281-2292]. During the purification of CS to homogeneity from Streptomyces clavuligerus [Salowe, S. P., Marsh, E. N., & Townsend, C. A. (1990) Biochemistry 29, 6499-6508], two forms of the enzyme capable of carrying out the complete reaction having very similar molecular weights and kinetic properties were isolated by Mono-Q chromatography. The gene for each has been cloned, sequenced, and found to be significantly homologous (87% identity). The two genes so isolated, cs1 and cs2, have open reading frames of 975 and 978 nucleotides, respectively, encoding proteins of M(r) 35,347 and 35,774. These genes are located in different loci of the genome separated by > 20 kbp. This separation is large for a natural product biosynthetic pathway in bacteria where gene duplication and limited divergence are typically observed to occur within narrower confines of a gene cluster. Sequence comparisons made between cs1/cs2 and other genes encoding iron-dependent proteins involved in penicillin and cephalosporin biosynthesis in the same organism show minimal homology. Further sequence alignments made to other non-heme iron oxygenases reveal unexpected dissimilarity within the alpha-ketoglutarate-dependent class itself. The limited data available suggests evolutionary convergence among these proteins.

Amino Acid Sequence↗
PubMed1990

A murine macrophage line of the H-2d/f haplotype can activate H-2k suppressor T cells.

Little is known about the molecular basis for activation of suppressor T cells. In this report we describe two macrophage cell lines, BAC1.2.SC8 and its variant progeny B26, that differ in their ability to activate suppressor T cells. The SC8 line is derived from a (BALB/c x A.CA)F1 (H-2d/f) mouse and is haploid with respect to I-Ed. It is capable of activating I-Ed-restricted helper T cells as well as poly-(Glu50Tyr50)-specific I-Ed-restricted suppressor cells. The B26 variant can activate H-2d-restricted helper T cells but activates H-2k-restricted suppressor cells. The I-Ed molecules of SC8 and of B26 have identical amino acid sequences. This suggests that suppressor T cells either recognize posttranslational modifications of the I-E molecule or that there is another accessory molecule that helps determine the major histocompatibility complex restriction in the activation of suppressor T cells.

Animals↗
PubMed1987

Idiotypic analysis of myeloma proteins: anti-DNA activity of monoclonal immunoglobulins bearing an SLE idiotype is more common in IgG than IgM antibodies.

The anti-idiotype 3I which recognizes a determinant on kappa-chains of anti-DNA antibodies in SLE patients also recognizes a determinant on kappa-chains of 82/706 myeloma proteins tested. Twenty-nine of these 82 proteins bind to double-stranded DNA, including two monoclonal IgM, one monoclonal IgA, and 26 monoclonal IgG proteins. DNA binding is much more frequent in the IgG than in the IgM myeloma proteins (p less than 0.001), and is also associated with cationic antibody charge. Two-dimensional gel electrophoresis reveals markedly increased charge heterogeneity of both heavy and light chains of the monoclonal IgG as compared with the monoclonal IgM proteins. We postulate that the increased charge heterogeneity of the IgG-associated 3I-reactive kappa-light chains may reflect somatic mutation, and that DNA specificity within the 3I idiotype system arises by somatic mutation of germ-line genes found in normal individuals. DNA binding may be associated with those mutations that give rise to a cationic immunoglobulin charge.

Antibodies, Monoclonal↗