Biomedical subjects

John Siekierka

Publications and source records attributed to John Siekierka.

6 recordsLinked to original sources

PubMed2006

Inhibitors of unactivated p38 MAP kinase.

Inhibition of the p38 map kinase pathway has been shown to be beneficial in the treatment of inflammatory diseases. The first class of potent p38 kinase inhibitors was the pyridinylimidazole compounds from SKB. Since then several pyridinylimidazole-based compounds have been shown to inhibit activated p38 kinase in vitro and in vivo. We have developed a novel series of pyridinylimidazole-based compounds, which potently inhibit the p38 pathway by binding to unactivated p38 kinase and only weakly inhibiting activated p38 kinase activity in vitro.

Animals↗

PubMed2006

A multiplexed proteomics approach to differentiate neurite outgrowth patterns.

We report here a method for proteomics pattern discovery by utilizing a self-organizing map approach to analyze data obtained from a novel multiplex iTRAQ proteomics method. Through the application of this technique, we were able to delineate the early molecular events preceding dorsal root ganglia neurite outgrowth induced by either nerve growth factor (NGF) or an immunophilin ligand, JNJ460. Following pattern analysis we discovered that each neurotrophic agent promoted mostly distinct increases in protein expression with few overlapping patterns. In the NGF-treated group, proteins possessing "biosynthesis function" (p < 0.002) and "ribosome localization" (p < 0.0003) were increased, while proteins promoting "organogenesis" (p < 0.004) and related "signal transduction" (p < 0.008) functions were notably increased in the JNJ460-treated group. This study suggests that the properties of neurite outgrowth triggered by NGF and JNJ460 can be distinguished at the proteome level. Multiplexed proteomics analysis, along with pattern discovery bioinformatics tools, has the capability to differentiate subtle neuroproteomics patterns.

Animals↗

PubMed2006

Effects of drug combinations on smooth muscle cell proliferation: an isobolographic analysis.

Although sirolimus is a potent inhibitor of vascular smooth muscle cell (VSMC) proliferation and is effective at preventing restenosis in the majority of clinical revascularization procedures employing sirolimus-eluting stents, some VSMC may escape the antiproliferative effects of sirolimus. The present study examines the effects of combining sirolimus with other known cell cycle-specific antiproliferative agents (cladribine, topotecan or etoposide) on cultured coronary artery VSMC proliferation and utilizes a novel isobolographic approach to determine whether sirolimus/antiproliferative agent combinations produce subadditive, additive or supraadditive potentiation of antiproliferative activity. All agents were found to inhibit coronary artery VSMC proliferation in a dose-dependent manner. Cladribine was found to potentiate the antiproliferative activity of sirolimus in either an additive or supraadditive manner, depending upon the cladribine concentration. Topotecan potentiated the sirolimus antiproliferative activity by simple additivity while etoposide yielded subadditive potentiation. The present results demonstrate the utility of isobolographic analysis for identifying and optimizing antiproliferative drug combinations.

Algorithms↗

PubMed2005

Drug-eluting stents: sirolimus and paclitaxel differentially affect cultured cells and injured arteries.

Sirolimus and paclitaxel eluted from stents inhibit cell proliferation and other cellular processes by dramatically different mechanisms. In this study, the effects of sirolimus and paclitaxel on cultured human coronary artery smooth muscle and endothelial cell function or cell cycle changes in balloon-injured arteries were directly compared. Both sirolimus and paclitaxel inhibited smooth muscle and endothelial cell proliferation. However, only paclitaxel inhibited smooth muscle and endothelial cell migration at low (nM) concentrations. Sirolimus arrested smooth muscle and endothelial cells in the G0/G1 phase of the cell cycle without inducing apoptosis while paclitaxel produced apoptosis in both cell types at low nanomolar concentrations. Although both agents blocked neointimal formation, sirolimus applied locally to injured rat carotid arteries increased the percentage of cycling vascular cells in G0/G1 without inducing apoptosis while paclitaxel increased the percentage of cycling cells in S and G2/M phases while inducing apoptosis. These results suggest that sirolimus reduces neointimal hyperplasia through a cytostatic mechanism while paclitaxel produces apoptotic cell death.

Animals↗

PubMed2004

A novel role for the immunophilin FKBP52 in copper transport.

FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes. Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood. To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library. We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone. Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux. The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506. Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper. Overexpression of FKBP52 increased rapid copper efflux in (64)Cu-loaded cells, as did the overexpression of WD transporter. Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity.

Animals↗

PubMed2002

Opposing roles of serine/threonine kinases MEKK1 and LOK in regulating the CD28 responsive element in T-cells.

T-cell activation requires signals from both the T-cell receptor (TcR) and other co-stimulatory molecules such as CD28. TcR- and CD28-mediated signals are integrated during T-cell activation resulting in the expression of cytokine genes such as interleukin-2 (IL-2). An enhancer element (CD28RE) of the IL-2 gene specifically responsive to CD28 signals has been previously identified and characterized. This response element and an adjacent Activated Protein-1 (nuclear factor-interleukin-2B) site together (RE/AP1) were shown to complex with c-rel, AP-1 and other factors. However, details of the signal transduction pathways leading from CD28 to the composite response element remain poorly understood. We present data showing that overexpression of the serine threonine kinase, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase-1 (MEKK1), but not nuclear factor-kappa B inducing kinase, or MAP kinase/ERK kinase-1 (MEK1), can significantly increase the level of CD28RE/AP1-driven luciferase (Luc) reporter gene expression in Jurkat E6-1 cells. A MEKK1 dominant negative mutant blocked such activation induced by stimulation with Raji B cells and the superantigen staphylococcus enterotoxin E (SEE), as well as via CD3/CD28. Mutations in either site of the RE/AP1 element abolished MEKK1-induced Luc expression. Calcineurin inhibitors, CsA and FK520, or inhibitors of p38 kinase (SB 203580), or MEK1 (PD 098059), did not affect MEKK1-induced reporter activation. These results directly implicate MEKK1 in the CD28 signalling pathway that activates the CD28 response element. Co-expression of the lymphocyte-oriented kinase (LOK) kinase attenuated Raji/SEE-induced IL-2 production in Jurkat cells, as well as MEKK1 and Raji/SEE-induced reporter gene activation. These data suggest that MEKK1 and LOK may have opposing roles in regulating the CD28RE/AP1 element.

CD28 Antigens↗