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M Chen

Publications and source records attributed to M Chen.

At least 703 records · Page 39Linked to original sources
PubMed1993

Teniposide-resistant CEM cells, which express mutant DNA topoisomerase II alpha, when treated with non-complex-stabilizing inhibitors of the enzyme, display no cross-resistance and reveal aberrant functions of the mutant enzyme.

We have examined the effects of a group of DNA topoisomerase II (topo II) inhibitors, merbarone, aclarubicin, SN22995, RP60475F, and fostriecin, in CCRF-CEM cells and two sublines, CEM/VM-1 and CEM/VM-1-5, that were selected for increasing resistance to teniposide (VM-26). The teniposide-resistant sublines have been termed "at-MDR" for altered topo II-associated multidrug resistance. These topo II inhibitors differ from the "classic" inhibitors such as teniposide in that they do not stabilize DNA-topo II complexes. In this study, we found that our at-MDR cell lines express little or no cross-resistance to these "non-classic" topo II inhibitors. Merbarone and SN22995 inhibited VM-26-mediated DNA-topo II complexes in CEM cells only when they were added before VM-26. Since they did not deplete topo II protein, it suggested that these drugs may inhibit topo II activity before the enzyme binds to DNA, thereby preventing stabilization of VM-26-mediated topo II-DNA complexes. Continuous exposure of CEM cells to merbarone, SN22995, or VM-26 caused G2 arrest, as determined by flow cytometry. Likewise, at-MDR cells continuously treated with VM-26 also arrested in G2. By contrast, treatment of at-MDR cells with either merbarone or SN22995 produced a qualitatively different pattern; the at-MDR cells first accumulated in G2 but then escaped the G2 block and proceeded into mitosis with elongated and intertwined chromosomes but failed to divide. Their DNA was re-replicated, however, and the cells eventually accumulated at the 8N DNA stage. Given that both wild-type and mutant topo II alpha alleles are expressed in the at-MDR cells (B. Y. Bugg, M. K. Danks, W. T. Beck, and D. P. Suttle. Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide. Proc. Natl. Acad. Sci. USA, 88: 7654-7658, 1991), we hypothesize that drugs such as merbarone may inhibit the activity of wild-type topo II alpha, allowing the aberrant activity of the mutant enzyme to be revealed during chromosome condensation and sister chromatid segregation.

Cell Cycle↗
PubMed1993

Cyclic AMP selectively increases renin mRNA stability in cultured juxtaglomerular granular cells.

This study was undertaken to examine the regulation of renin release and gene expression in primary cultures of juxtaglomerular granular (JGG) cells. JGG cells, isolated from mouse kidney, demonstrated high purity and showed regulated renin release in vitro. Changes in steady-state renin mRNA levels were assessed by quantitative polymerase chain reaction techniques, with polymerase chain reaction amplification efficiency monitored by co-amplification of experimental samples with a dilution series of cDNA for a mutant template. When the cells were incubated in the presence or absence of forskolin, isoproterenol, or 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine for 24 h or cholera toxin for 12 h, renin mRNA levels were increased 3.9-, 4.4-, 5.1-, and 3.3-fold, respectively (all, p < 0.05). A significant increase in renin mRNA levels was observed 8 h after treatment with forskolin, but no change was detectable at 4 h. Cycloheximide did not prevent the increase in renin mRNA by isoproterenol. When RNA synthesis was inhibited by incubation with actinomycin D (5 micrograms/ml), renin mRNA levels declined with a half-life of 3.0 +/- 0.8 h. Treatment with forskolin increased renin mRNA half-life to 10.8 +/- 2.7 h (p < 0.025). The half-life of beta-actin, endothelin-1, or the facilitative glucose transporter-1 (GLUT-1) mRNA expressed in the same cells was not altered, although the steady-state levels of GLUT-1 mRNA increased 2.2-fold after treatment with forskolin. These data demonstrate that cAMP increases renin release and mRNA levels in JGG cells in vitro, that the stimulatory effect of cAMP on renin mRNA is delayed but does not require new protein synthesis, and that the increased renin mRNA levels induced by cAMP are due in part to a selective increase in renin mRNA stability.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Interleukin-10 production by human carcinoma cell lines and its relationship to interleukin-6 expression.

Recent data indicate a major role for IL-10 in suppressing immune and inflammatory reactions. To date, expression of human IL-10 has been attributed primarily to helper T lymphocytes, activated monocytes, and neoplastic B cells, and was often found to be associated with IL-6 expression. In this study we sought to determine whether non-hematopoietic human tumor cell lines produce IL-10 and, if so, what is the relationship between IL-10 and IL-6. Using ELISA, we determined IL-10 and IL-6 levels in culture supernatants of 48 cell lines established from carcinomas of the kidney, colon, breast and pancreas, malignant melanomas and neuroblastomas. IL-6 protein was secreted by 28 of the tumor cell lines; IL-10 was measurable in 15 cell lines. IL-6 secretion was maximal and most frequent in renal-cancer cell lines, while IL-10 production was found to be highest and most common among cell lines derived from colon carcinomas. IL-10 in conditioned medium of one of the colon carcinoma cell lines (CCL222) was bio-active, as demonstrated in the mouse MC/9 mast-cell-line assay and in human mixed-lymphocyte reactions. In both assays, IL-10 bio-activity was neutralized by an anti-IL-10 monoclonal antibody. Expression of IL-6 and IL-10 was confirmed by RNA analysis using message amplification by PCR and sequencing of amplified cDNA. LPS, IL-1 alpha, and TNF-alpha strongly enhanced the release of IL-6 by RCC cells, but only marginally affected IL-10 production in colon-carcinoma cells. IL-10 secretion by colon-carcinoma cells was moderately stimulated by IFN-gamma and IL-4. Dexamethasone suppressed the release of IL-6, but had no inhibitory effect on IL-10 secretion. Our results demonstrate that tumor cell lines established from certain types of human carcinomas are capable of expressing and releasing IL-6 and/or IL-10, suggesting a role of these cytokines in solid-tumor development and anti-tumor immunity.

Animals↗
PubMed1993

High-level expression of porcine fructose-1,6-bisphosphatase in Escherichia coli: purification and characterization of the enzyme.

A cDNA encoding porcine fructose-1,6-bisphosphatase was isolated from total pig liver RNA. The enzyme's coding sequence was fused to the bacteriophage T7 gene 10 promoter and transcription terminator sequence and expressed in E. coli under control of the T7 RNA polymerase. Induced cells contain elevated levels of fructose-1,6-bisphosphatase enzymatic activity and an abundant 37,000 dalton protein. The enzyme was purified to apparent homogeneity and judged identical to wild-type porcine fructose-1,6-bisphosphatase. The kinetic parameters are similar to those reported for the pig kidney enzyme. The N-terminal amino acid sequence is identical to the predicted sequence and the kinetic parameters are consistent with freedom from proteolysis. As estimated from enzymatic activity and visual inspection of coomassie blue-stained SDS-PAGE gels, porcine fructose-1,6-bisphosphatase constitutes as much as 20% of the soluble protein from the over-expressing E. coli strain.

Amino Acid Sequence↗
PubMed1993

Plasminogen activator inhibitor 1, the primary regulator of fibrinolysis, in normal human cerebrospinal fluid.

The occurrence of type-1 plasminogen activator inhibitor (PAI-1) in human cerebrospinal fluid (CSF) has not previously been reported. As a member of the serpin superfamily of serine protease inhibitors and an acute phase response component, PAI-1 has powerful potential roles in nervous system homeostasis. We have detected this serpin antigen using a polyclonal anti-PAI-1 antibody in normal human CSF. In Western blotting, PAI-1 in several CSF samples appears as a two-band antigen of Mr = 54 and 35 kDa, presumably the intact and proteolytic fragment, respectively. In vitro complex formation studies confirm that the 54 kDa form of PAI-1 interacts with 125I-urokinase after activation with SDS, but the 35 kDa form does not. Quantification of total PAI-1 antigen in 18 normal human CSF samples by ELISA reveals a mean value of 1.0 +/- 0.07 (SEM) micrograms/dL, indicating that a relatively low concentration of the inhibitor occurs in normal human CSF. This information should now allow comparison of PAI-1 levels and activity in various neurologic disorders.

Alzheimer Disease↗
PubMed1993

Serologic response to the Borrelia burgdorferi flagellin demonstrates an epitope common to a neuroblastoma cell line.

Antibodies in sera of 7 patients with neurologic manifestations of Lyme borreliosis and a monoclonal antibody (mAb H9724) to the flagellin of Borrelia burgdorferi have been shown to bind neural tissue. To identify the antibody binding site common to the B. burgdorferi flagellin and the neural tissue, we made recombinant fusion proteins expressing epitopes of flagellin. Antibodies in patients' sera and mAb H9724 bound within an 18-amino acid epitope (residues 208-225) in the central region of flagellin, whereas two other mAbs bound to epitopes mapping elsewhere in the protein. Antibodies in patients sera and mAb H9724 also bound to a human neuroblastoma cell line. Absorption of patients sera with a peptide, EGVQQEGAQQPA, corresponding to amino acids 213-224 of flagellin, inhibited binding to the neuroblastoma cell line. The data suggest that the immune response to a specific B-cell epitope within flagellin, shared by a human neuroblastoma cell line, may be involved in the pathogenesis of neuroborreliosis.

Amino Acid Sequence↗
PubMed1993

The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis.

p37 and p40 are two cloned gene products of the five-subunit human cellular DNA replication factor activator 1 (A1) protein complex (also called replication factor C). Here, we describe the solubilization, purification, and characterization of these two proteins that were overproduced in Escherichia coli. Using a nitrocellulose filter binding assay, we demonstrated that the purified A1 p37 protein associated with DNA preferentially at the primer terminus, a property resembling that of the A1 complex. We also show that in the presence of relatively high levels of salt, the recombinant p37 protein alone activated DNA polymerase epsilon but not polymerase delta in catalyzing the elongation of DNA chains. The p40 protein specifically associated with cellular p37 and proliferating-cell nuclear antigen (PCNA) present in HeLa cell cytosolic extract. The addition of purified p40 protein abolished the in vitro polymerase delta-catalyzed DNA elongation reaction dependent on both PCNA and A1. However, this inhibition was reversed by excess polymerase delta, suggesting a specific interaction between the polymerase and the p40 protein. Thus, while p37 binds DNA at the primer end and has a specific affinity for pol epsilon, p40, which binds ATP, interacts with PCNA and pol delta. These activities are essential for the DNA elongation reactions that lead to the synthesis of leading-strand DNA and the maturation of Okazaki fragments.

Autoantigens↗
PubMed1993

Site-specific mutagenesis of the metal binding sites of porcine fructose-1,6-bisphosphatase.

Mutations in the metal binding sites of porcine fructose-1,6-bisphosphatase were carried out by site-specific mutagenesis based on the crystal structure of the enzyme. The mutant and wild-type enzymes have been characterized by circular dichroism spectrometry and initial-rate kinetics. One of the mutant forms of fructose-1,6-bisphosphatase (Glu280Gln) is associated with a single metal site, whereas two other mutants (Glu97Gln and Asp118Asn) ligate two metal ions. The mutant enzymes exhibit very large decreases in kcat relative to the wild-type enzyme; however, other kinetic parameters, such as Km values, are not greatly altered. Metal binding cooperativity and binding affinity is decreased in the mutants compared to wild-type fructose-1,6-bisphosphatase. Mutations in the metal binding sites greatly enhance the enzyme's affinity for AMP, a potent regulator of fructose-1,6-bisphosphatase activity. The results of these investigations are fully consistent with predictions made on the role of specific amino acid residues at the metal binding sites in porcine fructose-1,6-bisphosphatase from X-ray diffraction studies [Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W.N. (1993) Biochemistry, 32, 1844-1857].

Animals↗
PubMed1993

Reduced activation of transcriptional factor AP-1 among peripheral blood T cells from elderly humans after PHA stimulation: restorative effect of phorbol diesters.

In the present study, we evaluated if aging influences the activation and characteristics of transcriptional factor AP-1 in human T cells. Using gel mobility shift assays, the activation of AP-1 was quantified in peripheral blood T cells from 11 elderly (mean 74 year) and young (mean 33 year) subjects following stimulation with PHA, PMA, or PHA plus PMA. The results showed that the activation of AP-1 was significantly reduced in PHA-stimulated T cells from the group of elderly subjects when compared to T cells from young subjects (P < 0.05). Even though PHA-stimulated T cells from 8 of the elderly subjects had pronounced impairments in the activation of AP-1, additional signals provided by costimulation with PMA frequently restored AP-1 activation to more normal levels. Other experiments demonstrated that the AP-1 complexes expressed by stimulated T cells of elderly and young subjects exhibited similar properties in gel shift assays with competing unlabeled AP-1 oligonucleotides and with blocking antibodies specific for Fos and Jun. Thus, these data suggest that the activation of AP-1 can be reduced in human T cells during aging and that these reductions may often be related to impairments in signal transduction rather than represent an absolute loss in the ability to express AP-1.

Adult↗
PubMed1993

Separation of the rabbit ciliary body epithelial layers in viable form: identification of differences in bicarbonate transport.

Sections of whole ciliary (CB) dissected from Dutch belted rabbits were incubated for 2 hr at 36 degrees C in a 30 microM Ca2+ Ringer's. This incubation resulted in the spontaneous dissociation of the two cell layers comprising this epithelium, each remaining firmly cohesive with its own basement membrane. The inner limiting membrane with its adherent non-pigmented epithelium (NPE) was then mechanically removed from the surface exposing the apical surface of the pigmented epithelium (PE). Ultrastructural examination revealed no noxious effects in most cells although gross morphological changes in the NPE cells were noted. The newly separated layers were loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. Most cells of both layers acquired stable BCECF fluorescence indicating viability. To achieve a preliminary evaluation of differences in PE and NPE bicarbonate transport, dye-loaded tissues were perfused in a flow-through chamber which was mounted on a microscope equipped for quantitative epifluorescence. The intracellular pH (pHi) of groups of cells (5-10) was derived from the ratio of emission intensities generated by excitations at 490 and 440 nm. In Hepes-buffered Ringer's the pHis for the PE and NPE were 7.20 +/- 0.10 and 7.33 +/- 0.14 (+/- S.D., n = 6), respectively. Replacement of 28 mM Hepes by 28 mM HCO3-/5% CO2 led to a 0.13 pHi increase in the PE and a decrease of 0.27 U in the NPE. The pHi responses of the two cell layers to removal and/or reintroduction of Na+ and/or Cl- were also highly dissimilar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

ZO-1 in corneal epithelium; stratal distribution and synthesis induction by outer cell removal.

The tight junctions (TJs) present exclusively in between the superficial cells (SC) are an important component for the barrier and ion secretory functions of the corneal epithelium. Electrophysiological studies have demonstrated that digitonin-induced devitalization of overlying cells induces the de novo generation of a paracellular barrier between the intrastratal cells becoming exposed to the outer surface. It was also shown that the closer the exposed cells to the SC position the higher their competency for this activity. We have now examined the spatial and quantitative distribution of ZO-1, a protein closely associated with the cytosolic face of TJs in the rabbit. Immunohistology showed: (a) no detectable ZO-1 in basal cells; (b) incipient punctuate accumulations in the wing cells; (c) numerous foci and a diffuse cytosolic staining in the squamous (SQ) cells; and (d) strong staining at the apical TJ locations in the superficial SQ layer. Immunoblot analysis of separated layers showed a SQ:basal cell ZO-1 concentration ratio in excess of 100. Devitalization of the SQ layers induced strong ZO-1 synthesis in the newly exposed wing cells where electron microscopy showed time-dependent development of TJs. A similar ZO-1 increase was observed in the basal cells after removal of all suprabasal cells by a low [Ca2+] incubation method. These results provide a biochemical correlate to the previous electrophysiological findings and show that expression of ZO-1 in the corneal epithelium is intimately related to the development of the superficial cell phenotype.

Animals↗
PubMed1993

The roles of signal peptide and mature protein in RNase (barnase) export from Bacillus subtilis.

Barnase, an extracellular RNAse from Bacillus amyloliquefaciens is secreted post-translationally from B. subtilis. The rate of secretion of barnase from B. subtilis was improved by replacement of the barnase signal peptide with a heterologous signal peptide. However, the barnase signal peptide exported Escherichia coli alkaline phosphatase faster than mature barnase. Heat shock of B. subtilis cells did not significantly alter the export of barnase using the barnase signal peptide. The slow rate of export of barnase from B. subtilis is due to both the signal peptide and the mature protein sequence rather than either alone.

Amino Acid Sequence↗
PubMed1993

Altered DNA topoisomerase II in multidrug resistance.

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II alpha gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Rapid and simple subtyping of the HLA-DRB3 gene in Graves' disease by using temperature-gradient gel electrophoresis.

A HLA-DRB3-subtyping system that uses TGGE for analyzing DRB3 alleles was developed. The polymorphic second exon of the HLA-DRB3 gene was amplified from four homozygous typing cell lines, 223 healthy individuals, and 102 patients with Graves' disease by using the PCR. The PCR products were electrophoresed in a temperature gradient from 35 degrees C to 70 degrees C, and the resulting fragments were visualized by silver staining. Four DRB3 alleles (HLA-DRB3*0101, *0201, *0202, and *0301) were distinguished from one another by the migration of the corresponding homoduplex with the exception that DRB3*0201 was indistinguishable from DRB3*0202. The latter two alleles, however, were resolved by the artificial heteroduplexing approach. Arginine in position 74 of the DRB3 gene product (i.e., HLA-DRB3*0101) was significantly more frequent in Graves' patients than in controls. The relative risk conferred by the presence of the DRB3*0101 was 15.8 (p < or = 0.001). The presence of arginine in position 74 contributed to an etiologic fraction of 75% in our study population. The PCR-TGGE technique is a simple, nonisotopic method, which may be useful in rapid screening of large populations for HLA disease markers.

Alleles↗