Search PubMed⌕ Search

Biomedical subjects

B Choy

Publications and source records attributed to B Choy.

6 recordsLinked to original sources
PubMed2000

Immortalized human adult articular chondrocytes maintain cartilage-specific phenotype and responses to interleukin-1beta.

OBJECTIVE: To develop a reproducible immortalized human chondrocyte culture model for studying the regulation of chondrocyte functions relevant to arthritic diseases in adult humans. METHODS: Primary adult articular chondrocytes were immortalized with a retrovirus expressing a temperature-sensitive mutant of SV40-large T antigen (tsTAg). The established tsT/AC62 chondrocyte cell line was examined in monolayer and alginate culture systems. The levels of messenger RNA (mRNA) encoding cartilage matrix proteins and interleukin-1beta (IL-1beta)-inducible mRNA were analyzed by reverse transcriptase-polymerase chain reaction. Matrix protein synthesis was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-sulfate-labeled proteoglycans and Western blotting of type II collagen and aggrecan. Type II collagen (COL2A1)-luciferase reporter gene expression was analyzed by transient transfection. Phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38 mitogen-activated protein kinase (p38 MAPK), and activating transcription factor 2 (ATF-2) were detected by Western blotting. RESULTS: The tsT/AC62 cells expressed TAg at the permissive temperature (32degrees C), and the loss of TAg at 37 degrees C and 39 degrees C correlated with decreased cell proliferation. Cells in alginate culture deposited abundant alcian blue-stainable matrix and continued to proliferate at 32 degrees C. Preferential retention of aggrecan was observed in the cell-associated matrix, while biglycan and decorin were secreted into the medium of monolayer and alginate cultures. The levels of COL2A1 and aggrecan mRNA were increased after transfer from monolayer to alginate culture at 32 degrees C. Treatment with IL-1beta decreased COL2A1 and aggrecan mRNA levels and increased the levels of matrix metalloproteinases 1, 3, and 13 mRNA, as well as those of cyclooxygenase 2, type I collagen, and secretory phospholipase A2 type IIA mRNA, but not those of inducible nitric oxide synthase mRNA. IL-1beta also stimulated phosphorylation of p38 MAPK, SAPK/JNK, and ATF-2. The p38 MAPK-selective inhibitor, SB203580, partially reversed IL-1beta-induced inhibition of COL2A1 mRNA levels and COL2A1-luciferase reporter gene expression. CONCLUSION: The tsT/AC62 cells provide a reproducible model that mimics the adult articular chondrocyte phenotype, particularly in alginate culture, and demonstrates characteristic responses to IL-1beta. These studies also show, for the first time, that p38 MAPK is one of the signals required for IL-1beta-induced inhibition of COL2A1 gene expression. Availability of this model will permit identification of signals that regulate cytokine responses, and will also provide rational strategies for targeting these pathways.

Adult↗
PubMed1999

Transcriptional repression by the Caenorhabditis elegans germ-line protein PIE-1.

In the early Caenorhabditis elegans embryo, maternally expressed PIE-1 protein is required in germ-line blastomeres to inhibit somatic differentiation, maintain an absence of mRNA transcription, and block phosphorylation of the RNA polymerase II large subunit (Pol II) carboxy-terminal domain (CTD). We have determined that PIE-1 can function as a transcriptional repressor in cell culture assays. By fusing PIE-1 sequences to the yeast GAL4 DNA-binding domain, we have identified a PIE-1 repression domain that appears to inhibit the transcriptional machinery directly. A sequence element that is required for this repressor activity is similar to the Pol II CTD heptapeptide repeat, suggesting that the PIE-1 repression domain might target a protein complex that can bind the CTD. An alteration of this sequence element that blocks repression also impairs the ability of a transgene to rescue a pie-1 mutation, suggesting that this repressor activity may be important for PIE-1 function in vivo.

Amino Acid Sequence↗
PubMed1995

A role for activator-mediated TFIIB recruitment in diverse aspects of transcriptional regulation.

BACKGROUND: Transcription by RNA polymerase II in eukaryotic cells requires the ordered assembly of general transcription factors on the promoter to form a preinitiation complex. Transcriptional activator proteins (activators) stimulate transcription by increasing the rate and/or extent of preinitiation complex assembly. We have shown previously that acidic activators increase the stable association of TFIIB on the promoter, a process we refer to as 'recruitment'. In this study, we provide evidence that diverse activators facilitate TFIIB assembly by a related mechanism. We then investigate the activator-mediated assembly of TFIIB with regard to two aspects of transcription: the distance-dependence of activator function, and reinitiation. RESULTS: We have previously described amino-acid-substitution mutants of TFIIB that are able to support an activator-independent basal level of transcription but do not respond to acidic activators. We now show that these mutants also do not respond to other classes of activators. We demonstrate that this defect is due to a failure of the activators to recruit the mutant TFIIB to the promoter. Activators often lose activity as their distance from the initiation site is increased. We show that this impaired transcriptional activity correlates with a decrease in TFIIB recruitment. Finally, we find that following the initiation of transcription, TFIIB dissociates from the promoter, requiring the activator-mediated reassembly of TFIIB in the preinitiation complex for each new round of transcription. CONCLUSION: We have provided evidence that diverse activators recruit TFIIB to the promoter by a related mechanism. This central step in transcriptional activation is sensitive to promoter architecture, and is required for each new round of transcription.

Gene Expression Regulation↗
PubMed1993

Eukaryotic activators function during multiple steps of preinitiation complex assembly.

Eukaryotic activator proteins (activators) stimulate transcription by increasing assembly of the preinitiation complex. We have developed methods to quantify the stable assembly of general transcription factors into transcriptional complexes in response to activators. We show that activators function during at least two stages of preinitiation complex assembly: first, to recruit the general transcription factor TFIIB, and then at a second step, after TFIIB entry. It is at this second step that the TATA-box binding protein associated factors act. This step also seems to be critical for activators to stimulate transcription synergistically.

Cell Nucleus↗
PubMed1985

Comparison of anti-inflammatory compounds in the carrageenan induced paw edema model and the reversed passive Arthus model utilizing the same animal.

In order to better define antiinflammatory activity in new agents, a test was devised utilizing both carrageenan induced paw edema and the reversed passive Arthus reaction in the same animal. The model of carrageenan induced rat paw edema is a standard laboratory assay used to predict classical "aspirin-like" antiinflammatory molecules. The reversed passive cutaneous Arthus reaction involves precipitating antigen-antibody complexes, complement and infiltrating polymorphonuclear leukocytes (PMN's) and can be used to identify agents that affect one or more of these factors specifically. Antiinflammatory compounds were given orally one hour prior to the administration of carrageenan and goat anti-rat serum. Comparisons were made between several non-steroidal compounds and the steroid hydrocortisone. All of the compounds tested gave good carrageenan activity, but only hydrocortisone produced significant Arthus lesion inhibition in this assay.

Animals↗