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Biomedical subjects

T Chen

Publications and source records attributed to T Chen.

At least 325 records · Page 18Linked to original sources
PubMed1992

Antibody and peptide probes of interactions between the SH1-SH2 region of myosin subfragment 1 and actin's N-terminus.

The negatively charged residues in the N-terminus of actin and the 697-707 region on myosin subfragment 1 (S-1), containing the reactive cysteines SH1 and SH2, are known to be important for actin-activated myosin ATPase activity. The relationship between these two sites was first examined by monitoring the rates of SH1 and SH2 modification with N-ethylmaleimide in the presence of actin and, secondly, by testing for direct binding of SH1 peptides to the N-terminal segment on actin. While actin alone protected SH1 from N-ethylmaleimide modification, this effect was abolished by an antibody against the seven N-terminal amino acids on actin, F(ab)(1-7), and was greatly reduced when the charge of acidic residues at actin's N-terminus was altered by carbodiimide coupling of ethylenediamine. Neither F(ab)(1-7) nor ethylenediamine treatment reversed the effect of F-actin on SH2 reactivity in SH1-modified S-1. These results show a communication between the SH1 region on S-1 and actin's N-terminus in the acto-S-1 complex. To test whether such a communication involves the binding of the SH1 site on S-1 to the N-terminal segment of actin, the SH1 peptide IRICRKG-NH2(4+) was used. Cosedimentation experiments revealed the binding of three to six peptides per actin monomer. Peptide binding to actin was affected slightly, if at all, by F(ab)(1-7). The antibody also did not change the polymerization of G-actin by the peptides. The peptides caused a small reduction in the binding of S-1 to actin and did not change the binding of F(ab)(1-7).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1992

Lyn, a src-like tyrosine-specific protein kinase, is expressed in HL60 cells induced to monocyte-like or granulocyte-like cells.

During the in vitro differentiation of HL60 cells tyrosine-specific kinases are activated. The expression of lyn, a src-related tyrosine kinase, was studied by analysis of the steady-state levels of its transcript during the cell differentiation process induced by retinoic acid, phorbol 12-myristate 13-acetate and 1,25-dihydroxyvitamin D3. In contrast to an earlier report we observe only a small induction of the lyn-RNA levels compared to uninduced control cells. In unstimulated HL60 cells, the level for the lyn-transcript was comparatively high. A second, minor human lyn-transcript with an estimated size of 3.7 kb which has not been previously described, was identified.

Cell Differentiation↗
PubMed1992

Interleukin-1 beta stimulates cytosolic phospholipase A2 in rheumatoid synovial fibroblasts.

Phospholipase A2 (PLA2) activities in rheumatoid synovial fibroblasts (RSF) stimulated with interleukin-1 beta (IL-1 beta) were investigated. RSF incubated in the presence of IL-1 beta (120 pg/ml) for 18 h secreted 35 fold more PGE2 than did those incubated without IL-1 beta. IL-1 beta treatment did not increase the level of secretory PLA2 (sPLA2) activity or sPLA2 protein in the conditioned medium or subcellular fractions of lysed RSF. In contrast, the cell-associated PLA2 activity increased 3 to 4 fold in IL-1 beta stimulated RSF when compared with the control. The IL-1 beta stimulated, cell-associated PLA2 required submicromolar concentrations of calcium for activity, a characteristic consistent with the calcium sensitivity of cytosolic PLA2 (cPLA2) activity reported in other cell types, such as U937 cells. These findings demonstrate that an elevation in a cytosolic PLA2, rather than a sPLA2, is associated with increased PGE2 production in IL-1 beta stimulated RSF.

Arthritis, Rheumatoid↗
PubMed1992

Myosin subfragment 1 and structural elements of G-actin: effects of S-1(A2) on sequences 39-52 and 61-69 in subdomain 2 of G-actin.

The effect of myosin on the structure of two sequences on G-actin, a loop between residues 39 and 52 and a segment between residues 61 and 69 from the NH2-terminus, was probed by limited proteolytic digestions of G-actin in the presence of the myosin subfragment 1 isozyme S-1(A2). Under the experimental conditions of this work, no polymerization of actin was induced by S-1(A2) [Chen & Reisler (1991) Biochemistry 30, 4546-4552]. S-1(A2) did not change the rates of subtilisin and chymotryptic digestion of G-actin at loop 39-52. In contrast to this, the second protease-sensitive region on G-actin, segment 61-69, was protected strongly by S-1(A2) from tryptic cleavage. The minor if any involvement of loop 39-52 in S-1 binding was confirmed by determining the binding constants of S-1(A2) for pyrene-labeled G-actin (1.2 x 10(6) M-1), subtilisin-cleaved pyrenyl G-actin (0.3 x 10(6) M-1), and DNase I-pyrenyl G-actin complexes (0.3 x 10(6) M-1). Consistent with this, the activity of DNase I, which binds to actin loop 39-52 [Kabsch et al. (1990) Nature 347, 37-44], was inhibited almost equally well by actin in the presence and absence of S-1(A2). These results confirm the observation that DNase I and S-1(A2) bind to distinct sites on actin [Bettache et al. (1990) Biochemistry 29, 9085-9091] and demonstrate myosin-induced changes in segment 61-69 of G-actin.

Actins↗
PubMed1992

Prevention of carryover contamination in the detection of beta S and beta C genes by polymerase chain reaction.

As the polymerase chain reaction (PCR) process becomes a common tool in genetic diagnostic laboratories, prevention of carryover contamination from previous PCR amplifications has become an urgent topic. A PCR carryover prevention technique, utilizing deoxyuridine triphosphate (dUTP) and uracil DNA glycosylase (UDG), has been described recently. We report on its adaptation to a diagnostic system for detecting hemoglobin SS and SC diseases. Excellent amplification was achieved by increasing the dUTP and MgCl2 concentrations. dU-containing DNA could be analyzed by restriction endonucleases to distinguish the beta S from beta A gene using Ddel, but two other restriction enzymes, Bsu361 (replacement for MstII by the manufacturer) and CvnI, can no longer be used. The dU-containing PCR products retained their hybridization specificity with allele-specific oligonucleotide (ASO) probes for the beta A, beta S, and beta C genes. The PCR carryover prevention technique is easy to use and takes only 20 additional minutes. It should be extremely useful to genetic diagnostic laboratories where PCR is repeated daily and carryover contamination may thus lead to misdiagnoses.

Base Sequence↗
PubMed1992

Effect of helium-neon laser irradiation on serum lipid peroxide concentrations in burnt mice.

The effect of helium-neon irradiation on serum lipid peroxide concentrations in mice following 6-7% body surface area burns was investigated in a controlled study. Immediately following injury by an 8-sec 100 degrees C scalding, 25 mice were irradiated by a helium-neon laser at 0.05 J/cm2. A control group of the same size underwent identical treatment but received only sham irradiation. Serum lipid peroxide concentrations increased markedly in the control group at 0.5-4 h (P less than 0.0001, two sample t-test). In the laser treated group, the lipid peroxide concentrations remained relatively constant and were significantly depressed relative to the control group 4 h following burning (P less than 0.0001, two-sample t-test).

Animals↗
PubMed1992

Isolation and characterization of five Drosophila genes that encode an ets-related DNA binding domain.

The recent determination of the site-specific DNA binding properties of several proteins related to the ets oncoprotein has allowed the definition of a novel DNA binding domain, designated the ETS domain. In Drosophila, an ETS domain is present in the early ecdysone-induced E74A protein, which binds DNA in a site-specific manner and interacts with many ecdysone-induced polytene chromosome puffs at the onset of metamorphosis. As a first step toward determining the function of ETS-domain proteins during Drosophila development, we have used PCR amplification with degenerate oligonucleotides to isolate five other ets-related genes. Two of these genes, D-ets-2 and D-elg, have been previously identified. The proteins encoded by these genes are highly related to one another and to the seven identified vertebrate ETS-domain proteins, within the approximately 85-amino-acid DNA binding domain. In situ hybridization to polytene chromosomes revealed that these ets-related genes are not clustered in the genome and that only E74 corresponds to an ecdysone-inducible puff locus. These five ets-related genes are distinguished further from E74 in that they are transcribed through most of development, suggesting that they do not perform a stage-specific function. They are, however, expressed in a variety of patterns in early embryos, suggesting roles in the development of specific cell types. D-ets-2 is expressed in a complex pattern that changes dynamically during early embryogenesis. D-ets-3 and D-ets-6 are expressed in the ventral nervous system. The expression of D-ets-3 is higher in the three thoracic segments and lower in the abdominal segments. The high levels of expression in the thoracic segments are dependent on the presence of the bithorax complex. D-ets-4 and D-elg are expressed at their highest levels in the pole cells, suggesting a role in the development of the germline. This study represents the first effort in any organism to systematically isolate members of the ets gene family. The identification of six independent ets-related genes demonstrates that the ETS-domain proteins constitute a new family of potential transcriptional regulators encoded by the Drosophila genome.

Amino Acid Sequence↗
PubMed1992

Human neutrophil response to recombinant neisserial Opa proteins.

Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.

Amino Acid Sequence↗
PubMed1992

The differentiation pathway of HL60 cells is a model system for studying the specific regulation of some myeloid genes.

During granulopoiesis, certain myeloid genes encoding products of azurophilic granules are specifically down-regulated. The myeloid specific enzyme myeloperoxidase belongs to this group of genes. It is responsible for the production of hypochlorous acid, a potent microbicidal agent which is involved in host defense. During induced differentiation of promyelocytic leukemic HL60 cells to granulocyte- or monocyte-like cells, myeloperoxidase RNA is depressed. We studied this depression process in more detail by limiting the exposure to the inducer phorbol 12-myristate 13-acetate to 24 h. During this time period, no significant decrease in cell number and cell viability could be observed. Analysis of these in vitro differentiated HL60 cells on the protein and RNA levels showed that they can be used under defined conditions as a cell system to study the specific depression of myeloid genes. Under the described conditions, both the transcriptional rate of the myeloperoxidase gene as well as the stability of its transcript was reduced.

Actins↗
PubMed1992

[Studies of cockroachcidal toxin].

A cockroachcidal bacterial isolate CW-W-90-3 was selected by egg yolk agar plate. The isolate produced phospholipase C (PLC) which was pathogenic to the nymph of cockroach. The conditions for production of high level PLC indicated that using LB medium supplement Tween-80 or minimal medium could effectively increase the activity of PLC. The optical phase for production of PLC was in the period of 12-18 hours and below pH8.0. The activity of PLC was reduced along with the culture time until 48 hours. The PLC was resistant to heat. The partially purified PLC from the culture supernatant was assayed by using cockroach nymphes and produced 71.74% mortality.

Animals↗
PubMed1991

IL-3-induced generation of alkylacylglycerol and diacylglycerol in an IL-3-dependent cell line.

The hematopoietic growth factor IL-3 promotes the proliferation and development of several hematopoietic lineages. Inasmuch as protein kinase C has been suggested to mediate the response of IL-3, we examined the accumulation of diradylglycerols (DG) in response to IL-3 in CFTL-12 cells, a murine mast cell line that requires IL-3 for growth. Exposure of CFTL-12 cells to IL-3 resulted in the conversion of [3H]myristate-labeled lipids to DG. Mass analysis of the DG of CFTL-12 cells cultured in the presence of IL-3 showed that 58% was the ether-linked form, alkylacylglycerol, and 42% was diacylglycerol. The levels of both alkylacylglycerol and diacylglycerol declined when CFTL-12 cells were withdrawn from IL-3 and became quiescent. Stimulation of quiescent cells with IL-3 produced an acute increase in the mass of both alkylacylglycerol and diacylglycerol, consistent with phosphatidylcholine as a significant source. The effects of PMA on the generation of DG were examined to explore the role of protein kinase C activation in the response to IL-3. PMA stimulated an increase in DG accumulation that was not augmented by the simultaneous addition of IL-3. Down-modulation of protein kinase C by long term PMA treatment reduced, but did not eliminate, the IL-3-stimulated increase in DG, suggesting that protein kinase C activation results in an amplification of the initial accumulation of DG. These results indicate a role for DG, generated through the hydrolysis of phosphatidylcholine, in the induction of protein kinase C activity and the events leading to cell proliferation in response to IL-3.

Acylation↗
PubMed1991

Myeloperoxidase is a primary response gene in HL60 cells, directly regulated during hematopoietic differentiation.

The expression of myeloperoxidase was studied in three human myeloid leukemic cell lines. The myeloperoxidase transcript was strongly expressed in promyelocytic HL 60 cells, whereas much lower levels were detected in immature monocytic U 937 cells. Phorbol-12-myristate-13-acetate induction resulted in inhibition of myeloperoxidase expression within 24 hrs. This regulatory event could not be blocked by cycloheximide. Furthermore, cycloheximide did not superinduce myeloperoxidase mRNA levels in KG 1, HL 60 and U 937 cells, arguing against the existence of a negative gene regulator for myeloperoxidase. Therefore, the myeloperoxidase gene can be classified as a primary response gene.

Blotting, Northern↗
PubMed1991

Interactions of myosin subfragment 1 isozymes with G-actin.

The polymerization of G-actin by myosin subfragment 1 (S-1) isozymes, S-1(A1) and S-1(A2), and their proteolytically cleaved forms was studied by light-scattering, fluorescence, and analytical ultracentrifugation techniques. As reported previously, S-1(A1) polymerized G-actin rapidly while S-1(A2) could hardly promote the assembly reaction (Chaussepied & Kasprzak, 1989a; Chen and Reisler, 1990). This difference between the isozymes of S-1 was traced to the very poor, if any, ability of G-actin-S-1(A2) complexes to nucleate the assembly of actin filaments. The formation of G-actin-S-1(A2) complexes was verified in sedimentation velocity experiments and by fluorescence measurements using pyrene-labeled actin. The G-actin-S-1(A2) complexes supported the growth of actin filaments and accelerated the polymerization of actin in solutions seeded with MgCl2-, KCl-, and S-1(A1)-generated nuclei. The growth rates of actin-S-1(A2) filaments were markedly slower than those for actin-S-1(A1) filaments. Proteolytic cleavage of S-1 isozymes at the 50/20-kDa junction of the heavy chain greatly decreased their binding to G-actin and thus inhibited the polymerization of actin by S-1(A1). These results are discussed in the context of G-actin-S-1 interactions.

Actins↗