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Biomedical subjects

B Boyle

Publications and source records attributed to B Boyle.

At least 19 recordsLinked to original sources
PubMed2001

Repression of the defense gene PR-10a by the single-stranded DNA binding protein SEBF.

The potato pathogenesis-related gene PR-10a is transcriptionally activated in response to pathogen infection or elicitor treatment. Characterization of the cis-acting elements of the PR-10a promoter revealed the presence of a silencing element between residues -52 and -27 that contributes to transcriptional regulation. In this study, we have isolated a silencing element binding factor (SEBF) from potato tuber nuclei that binds to the coding strand of the silencing element in a sequence-specific manner. The consensus binding site of SEBF, PyTGTCNC, is present in a number of PR genes and shows striking similarity to the auxin response element. Mutational analysis of the PR-10a promoter revealed an inverse correlation between the in vitro binding of SEBF and the expression of PR-10a. SEBF was purified to homogeneity from potato tubers, and sequencing of the N terminus of the protein led to the isolation of a cDNA clone. Sequence analysis revealed that SEBF is homologous with chloroplast RNA binding proteins that possess consensus sequence-type RNA binding domains characteristic of heterogeneous nuclear ribonucleoproteins (hnRNPs). Overexpression of SEBF in protoplasts repressed the activity of a PR-10a reporter construct in a silencing element-dependent manner, confirming the role of SEBF as a transcriptional repressor.

Amino Acid Sequence↗
PubMed1999

Thalidomide stimulates T cell responses and interleukin 12 production in HIV-infected patients.

We performed a placebo-controlled study to evaluate the effects of immunomodulatory treatment with thalidomide on HIV levels, TNF-alpha levels, and immune status of 31 HIV-infected individuals, after temporary suppression of viral replication with antiretroviral drugs. Treatment with a combination of zidovudine and lamivudine (ZDV/LMV) for 14 days resulted in a median decline in plasma viremia of 1.94 log10 RNA equivalents/ml. After discontinuation of ZDV/LMV, thalidomide therapy (200 mg/day for 4 weeks) did not retard the prompt return of HIV titers to the pretreatment levels, and had no effect on plasma levels of TNF-alpha. In contrast, thalidomide treatment resulted in significant immune stimulation. We observed increased levels of plasma soluble IL-2 receptor, soluble CD8 antigen, and IL-12 (p < 0.01 for all parameters), as well as increased cutaneous delayed-type hypersensitivity reactions to recall antigens (p < 0.01) in thalidomide-treated patients. These changes were associated with a median increase in HIV titer of 0.2 log10 RNA equivalents/ml in the thalidomide-treated group (p < 0.05), which resolved after stopping the drug. Further studies were performed in vitro to elucidate the mechanism of thalidomide-induced immune stimulation. When purified T cells from HIV-infected individuals were stimulated by immobilized anti-CD3 in the presence of thalidomide, a costimulatory effect of the drug was observed, resulting in increased production of IL-2 and IFN-gamma, and increased T cell-proliferative responses. Further experiments showed that thalidomide increased IL-12 production by antigen-presenting cells in a T cell-dependent manner. Our findings suggest a potential application for thalidomide as a novel immune adjuvant in HIV disease.

Adult↗
PubMed1999

Two mutations identified in the androgen receptor of the new human prostate cancer cell line MDA PCa 2a.

PURPOSE: We have characterized the androgen receptor (AR) in a new human prostate cancer cell line, MDA PCa 2a, that has recently been established from a bone metastasis of a patient whose cancer exhibited androgen-independent growth. MATERIALS AND METHODS: Androgen responsiveness of these cells was assessed by measuring the effect of DHT and R1881 on cell growth and PSA secretion. Scatchard analysis was used to characterize the affinity and abundance of AR protein. Using a PCR based strategy, genomic DNA of the entire coding region of AR gene was sequenced to identify possible mutations. RESULTS: These cells express abundant AR (Nmax = 685 +/- 149 fmol./mg. protein), but the AR binding affinity (Kd) for DHT is only 25 nM, approximately 50-fold lower affinity than the mutated AR in LNCaP prostate cancer cells (Kd = 0.5 nM) or the wildtype AR in MCF-7 breast cancer cells (Kd = 0.4 nM). Two mutations, L701H and T877A, were identified in the ligand binding domain of the AR gene. Compared with LNCaP cells, the new cell line is significantly less responsive to DHT and R1881 as well as to other androgens such as testosterone, androstenedione, and DHEA. Similar to LNCaP cells, the ligand specificity of the AR in MDA PCa 2a cells appears to be relaxed and non-androgens such as progesterone and estradiol act as agonists although with less potency than in LNCaP cells. Interestingly, in the absence of androgens, the new cell line expresses 15-fold higher baseline levels of PSA than LNCaP. CONCLUSIONS: Two mutations were identified in the AR gene of the MDA PCa 2a cell line that are likely responsible for the decreased androgen sensitivity and altered ligand specificity observed in these cells. Thus, this new cell line with partial androgen responsiveness and PSA expression can serve as a functionally relevant model system of bone metastatic prostate cancer, and can be used to investigate the role of AR mutations in prostate cancer and its progression to androgen independence.

Cell Division↗
PubMed1998

Sequential and structural homology between intracellular pathogenesis-related proteins and a group of latex proteins.

The intracellular pathogenesis-related proteins have been identified in a broad range of flowering plants. Some display quite different patterns of expression, in many cases unrelated to the pathogenic response. Nevertheless, these proteins are all very similar and in most cases share more than 35% sequence identity. In this report we investigate the significance of a rather weak similarity between the intracellular pathogenesis-related (IPR or PR-10) proteins and a group of proteins identified in the latex of opium poppy and in Arabidopsis, among others. A sequence analysis held together with the recently published three-dimensional structure of Bet v 1, an IPR protein from birch pollen, strongly suggests sequential and structural homology between the two protein families.

Amino Acid Sequence↗
PubMed1994

Effect of tamoxifen on the multidrug-resistant phenotype in human breast cancer cells: isobologram, drug accumulation, and M(r) 170,000 glycoprotein (gp170) binding studies.

We have performed isobologram analyses of the ability of tamoxifen (TAM) to alter the response to Adriamycin (ADR) and vinblastine (VBL) in human breast cancer cells. MCF-7 cells express functional receptors for estrogen and progesterone but do not express detectable levels of M(r) 170,000 glycoprotein (gp170). CL 10.3 and MCF-7ADR cells are MCF-7 variants which express gp170. CL 10.3 but not MCF-7ADR cells express functional steroid hormone receptors. Tamoxifen (1-2.5 microM) interacts synergistically with ADR and VBL in CL 10.3 and MCF-7ADR cells. TAM increases the cytotoxicity of VBL and ADR and the intracellular levels of [3H]VBL by approximately 2-3-fold. TAM also prevents the binding of [3H]azidopine to gp170. The ability of TAM to concurrently increase the cytotoxic effects of ADR and VBL, increase VBL accumulation, and inhibit the binding of azidopine to gp170 strongly implies that the synergistic effects of TAM are mediated through its effects on gp170. TAM produces an antagonistic to additive interaction with ADR and VBL in MCF-7 cells, and at high concentrations (5 microM) the synergy apparent in CL 10.3 and MCF-7ADR cells is lost. While TAM clearly has a significant potential for use as a chemosensitizing agent, the design of clinical trials may require careful consideration.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

13-cis-retinoic acid-mediated growth inhibition of DU-145 human prostate cancer cells.

The purpose of this study was to examine the effects of 13-cis-retinoic acid (13-cis-RA) on the growth regulation of DU-145 human prostatic cancer cells. The results of these experiments demonstrate that cell growth and metastatic potential of DU-145 cells were significantly inhibited by 13-cis-RA (10 microM). In order to elucidate the possible molecular mechanisms of 13-cis-RA action on prostate cancer cells, we examined the expression of nuclear receptor genes (hRXR alpha) and found that 13-cis-RA treated cells showed higher mRNA expression for hRXR alpha nuclear receptors compared to untreated cells. To elucidate further the possible biochemical mechanisms associated with these alterations, we analyzed the phosphorous metabolites by MR spectroscopy and found that the major metabolites were PME, (PC, PE) and DPDE (UDP-GalNAc, UDP-GLcNAc). The DU-145 cells and xenografts, which were both treated with 13-cis-RA, showed a two-fold decrease in DPDE's, compared to their controls. The higher resolution spectra of perfused cells revealed that phosphocholine levels were twice as high in 13-cis-RA-treated DU-145 cells as compared to untreated cells. These investigations demonstrate for the first time that 13-cis-RA inhibits the growth of human prostatic cancer cells, and this inhibition is associated with an increase in hRXR alpha nuclear receptor gene expression and alterations in phosphorous metabolites detected by 31P MR spectroscopy.

Animals↗
PubMed1992

Metastasis-associated alterations in phospholipids and fatty acids of human prostatic adenocarcinoma cell lines.

Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines.

Adenocarcinoma↗
PubMed1992

Biochemical, cytogenetic, and morphological characteristics of human primary and metastatic prostate cancer cell lines.

Metastatic properties of human prostatic cancer cell lines (ND-1 and DU-145) were examined using various biochemical techniques. DU-145 cells had a higher metastatic potential than ND-1 cells. Cytogenetic analysis by G-banding demonstrated an aneuploid karyotype with considerable structural rearrangement. ND-1 cells had a modal chromosome number range lower than DU-145 cells (45-66, compared to 54-62). Ploidy analysis revealed that DU-145 cells showed hyperdiploidy with a greater amount of proliferation than the majority of ND-1 cells. Electron microscopic studies revealed little change in the cell morphology of either line. DU-145 cells had lower phosphatidyl choline levels and higher sphingomyelin levels than ND-1. DU-145 cells had much lower arachidonic acid levels than ND-1 cells. SDS-polyacrylamide gel electrophoresis revealed protein differences between the two cell lines. This study demonstrates for the first time that lipids, proteins and cytogenetic parameters differ in human primary and secondary prostate cancer cell lines.

Adenocarcinoma↗
PubMed1990

Free radical inhibition and serial chemiluminescence in evolving experimental pancreatitis.

Oxygen free radical activity and inhibition were examined in experimental pancreatitis. Twenty-five rats were randomized to five groups: controls received intravenous saline, to simulate pancreatitis one group received intravenous caerulein (5 micrograms kg-1 h-1), and three groups received sodium taurocholate via the pancreatic duct (0.2 ml, 5 per cent), either alone, following allopurinol or immediately before superoxide dismutase. Chemiluminescence (a phenomenon based on the emission of light during chemical reactions and which is dependent on oxygen free radical activity) was used as an index of oxygen free radical activity and was measured in tissue samples at 5-min intervals following induction of pancreatitis. The control mean(s.e.m.) serum amylase level 1 h after induction of pancreatitis was 635(13) units. It was significantly elevated in caerulein-induced pancreatitis, 1833(118) units (P less than 0.05) and exceeded 3000 units in all taurocholate-infused animals. Mean(s.e.m.) chemiluminescence ranged from 44 (8) mV 100 mg-1 at time zero to 404(113) mV 100 mg-1 at 1 h in controls. In caerulein-induced pancreatitis mean(s.e.m.) chemiluminescence peaked at 20 min (1399(239) mV 100 mg-1, P less than 0.02) and in taurocholate-induced pancreatitis at 15 min (2316(95) mV 100 mg-1, P less than 0.004). Superoxide dismutase significantly reduced chemiluminescence and hyperamylasaemia in taurocholate groups. Increasing oxygen free radical activity paralleled evolving pancreatitis. Superoxide dismutase may have a therapeutic role in pancreatitis.

Allopurinol↗