19 research outputs found
Alteration of Bcl11b upon stimulation of both MAP Kinase- and Gsk3-dependent signaling pathways in double negative thymocytes
Get PDFBcl11b is a transcription factor critical for thymocyte development. We previously characterized the kinetic post-translational modifications (PTMs) of Bcl11b in double positive (DP) thymocytes during stimulation of the T cell receptor-activated MAP kinase pathway. However, the PTMs of Bcl11b in thymocytes from other developmental stages in the thymus, primarily double negative (DN) cells, have not been previously identified. We found that kinetic modifications of Bcl11b in DN cells are somewhat different than the patterns observed in DP cells. Distinct from DP thymocytes, phosphorylation and sumoylation of Bcl11b in DN cells were not oppositely regulated in response to activation of MAP kinase, even though hyper-phosphorylation of Bcl11b coincided with near complete desumoylation. Additionally, prolonged stimulation of the MAP kinase pathway in DN cells, unlike DP thymocytes, did not alter Bcl11b levels of sumoylation or ubiquitinylation, or stability. On the other hand, activation of Wnt/Gsk3-dependent signaling in DN cells resulted in composite dephosphorylation and sumoylation of Bcl11b. Moreover, stimulation of MAP kinase and/or Wnt signaling pathways differentially affects gene expression of some Bcl11b target and maturation- associated genes. Defining the signaling pathways and regulation of sequence-specific transcription factors (SSTFs) by PTMs at various stages of thymopoiesis may improve our understanding of leukemogenesis.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author
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Gene networks during cardiogenesis and skeletal myogenesis
No full textOrganismal development requires a precisely orchestrated transcriptional program to correctly deploy genetic information into the genome. This process requires sophisticated gene regulatory networks at multiple spatial and temporal levels from early embryonic development to adult physiological conditions. Molecular differences that define cell types are set up during the pattern formation phase of development. Selective gene expression provides molecular markers such as sequence specific DNA-binding transcription factors (SSTFs) to define cell types. Homeodomain transcription factors are essential for embryonic pattern formation and cell specification and therefore can affect several mechanistically distinct aspects of organ development. The Pitx2 homeobox gene is expressed in the lateral plate mesoderm and it is involved in cardiac and skeletal muscle development. Mutations of Pitx2 are associated with the human Axenfeld-Rieger syndrome. Pitx2 null mice die at embryonic day 13.5 and exhibit un-septated atria and outflow tract that leads to deformed valves and arrythmias. Pitx2 promotes the proliferation of the branchial arch mesoderm-derived cells and their remodeling process, the epithelial-mesenchymal transition, to form the outflow tract cushions by influencing the expression of SSTFs in the cardiac mesoderm. Pitx2 is expressed in skeletal muscle cells from their time as progenitors until they form mature muscle groups. In Pitx2 null mutant mice the skeletal muscles have been specified, the muscles were formed but their higher order assembly was disrupted. Pitx2 was a key player of the embryonic muscle progenitors as they transition to the fetal state. Embryonic muscle progenitors were able to delaminate from the dermomyotome and migrate to the forelimb pre-patterned anlagen but were not fast to transition to the fetal state, as their G1 phase was arrested and their motility was disrupted. We have developed a Pitx2 conditional genetic system that eliminates the early cause of death and allows us to investigate the role Pitx2 at later stages. The molecular mechanisms of muscle regeneration in adults share many characteristics with the myogenic programs that generate skeletal muscle during development. These studies will enhance the molecular understanding of myogenic development and generate muscle-impaired mouse lines for drug and regeneration studies
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The role of the homeodomain transcription factor Pitx2 in regulating skeletal muscle precursor migration and higher order muscle assembly
No full textCells of the ventrolateral dermomyotome delaminate and migrate into the limb buds where they give rise to all muscles of the limbs. The migratory cells proliferate and form myoblasts, which withdraw from the cell cycle to become terminally differentiated myocytes. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed specifically in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Microarrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors from Wild type and Pitx2 null mice. Changes in gene expression were observed in genes encoding cytoskeletal, adhesion and fusion proteins which play a role in cell motility and myoblast fusion. We observed decreased cellular motility, disrupted cytoskeleton organization and focal adhesion distribution, decreased fusion of mononucleated myoblasts into multinucleated myotubes and decreased proliferation in presence of Ptix2. These studies suggest that Pitx2 plays a critical role in regulating the timing of myoblast filling the limb anlagen which may have detrimental consequences for higher order muscle architecture
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Roles of homeodomain transcription factors during organogenesis
No full textThe spatial and temporal patterning of sequence specific transcription factors (SSTFs) contributes to cell type specification and organ formation during embryogenesis. Homeodomain transcription factors are evolutionally conserved among invertebrate and vertebrate animals. They are responsible for body segmentation and organogenesis. Lbx1 and Pitx2 both are homeodomain transcription factors contributing to SSTF pattern formation during multiple organ formations. We studied how homeodomain transcription factors regulate SSTF and non-SSTF genes in a population-specific manner using the Lbx1[superscript EGFP] and Pitx2[superscript LacZ] mouse models. We have studied the role of Lbx1 in dorsal horn interneuron specification and Pitx2 in forelimb muscle formation. The two top non-SSTF target genes, NPY and Chmp2b, of Lbx1 are studied for expression pattern and potential neuronal function in neural tube. The T box, Hox gene families and Pax genes were identified as Pitx2 target genes via microarray analysis and their expression pattern were analyzed in forelimb. The expression domains of signaling molecules were altered in absence of Pitx2, suggesting that Pitx2 played a general role in pattern formation in forelimb mesenchyme
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Transcriptional repression by CTIP2, a C2H2 zinc finger protein
No full textCTIP2, a novel C2H2 zinc finger protein, is a transcriptional repressor that functions by at least two mechanisms. CTIP2 interacts with and stimulates transcriptional repression mediated by COUP-TF family members. CTIP2 also represses transcription independently of COUP-TF proteins by direct, sequence-specific DNA binding activity. CTIP2 has been implicated in lymphoid malignancies and development of T lymphocytes and the central nervous system (CNS). However, very little is known concerning the molecular mechanism(s) by which CTIP2 functions in these processes. The goal of the studies described herein was to contribute towards a greater understanding of cellular functions of CTIP2 through the characterization of domains of CTIP2 required for transcriptional regulatory activity, identification of CTIP2 target genes, and elucidation of molecular mechanisms underlying the transcriptional repression mediated by CTIP2.
CTIP2 was found to repress transcription by recruiting at least three different histone deacetylases (HDAC) to the promoter template of target genes. The three HDACs that were identified as being involved in CTIP2-mediated transcriptional repression were SIRT1, HDAC1, and HDAC2. The latter two were found in the same complex, which we identified as the Nucleosome Remodeling and Deacetylation (NuRD) complex. The SIRT1 and NuRD complexes appeared to be differentially recruited to CTIP2 target genes as a function of promoter, and possibly cellular, context. CTIP2 was found to recruit the NuRD complex, but not the SIRT1 complex, to the promoter of the cyclin-dependent kinase inhibitor p57KIP2 gene, a new transcriptional target of CTIP2 in neuroblastoma cells.
By analyses of regions of CTIP2 required for functionality, we found that the C-terminal zinc finger (ZnF) 5-7 module conferred self-associative activity, which appeared to be obligatory for high-affinity DNA binding and transcriptional repression of the protein. In contrast, the centrally located ZnF3-4 module of CTIP2 may confer sequence-specific DNA binding activity.
The results described herein provide a framework for understanding the mechanisms underlying the transcriptional regulatory activity of CTIP2, which may contribute to a better understanding of molecular and cellular basis for the activity of CTIP2 in vivo
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Selective oxidation of biological matrices for subsequent peroxidase and peroxidase-based quantitative analyses
No full textHydrogen peroxide quantification is of broad interest due to the common use of hydrogen peroxide as an oxidizing agent in industrial processing and laboratory research. Hydrogen peroxide assays are also of general importance for biological studies aimed at understanding the role of in situ generated reactive oxygen species. In the latter scenario particularly, assays amenable to high throughput processing are needed. Peroxidase-based methods are appropriate for such applications due to the high selectivity and sensitivity of enzyme catalyzed reactions. A problem commonly encountered when using peroxidase-based methods to quantify the level of hydrogen peroxide in biological samples is assay interference due the presence of assay-modifying endogenous compounds. This type of interference has limited the applicability of peroxidase/chromophore linked assays which are commonly used elsewhere for high throughput screening (e.g., the glucose oxidase/peroxidase assay for glucose quantification). Potential mechanisms of assay interference include enzyme inhibition/inactivation, substrate competition and product modification. In the present study we addressed the different mechanisms of interference, especially product (oxidized reporter molecule) modification, using the following system: horseradish peroxidase, 2,2' azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and a hydrogen peroxide-containing garlic paste extract (GPE). Methods using ABTS as an appropriate reporter molecule to circumvent the interference are based on removal
of confounding compounds, particularly referred to as natural antioxidants, prior to initiating the assay. Because confounding compounds interfere with the peroxidase-based assay by converting peroxidase-catalyzed ABTS oxidation product, ABTS•⁺ back to ABTS, prepared ABTS•⁺ was used to selectively oxidize, thus inactivate confounding compounds that would cause confounding in this assay. A calibration curve generated by using ABTS•⁺ treated GPE sample was not significantly different (p>0.05) from the curve obtained in the model buffer system. In contrast to a flat baseline generated by original GPE sample, the effectiveness of ABTS•⁺ treatment in hydrogen peroxide quantification in the presence of interference was proved. This assay allows one to simply determine the amount of hydrogen peroxide in a product in situ and thus avoids the need for sophisticated separation techniques. The limitation of the method is that the treatment required for removal of confounding compounds takes on the order of minutes and thus the method has the possibility of underestimating the hydrogen peroxide content in systems where such concentrations are changing on the seconds to minutes time scale.
The other focus of this project was a modified assay that eliminates a source of underestimation of peroxidase activity in plant extracts. Natural reducing agents endogenous to plant materials, such as phenolic compounds and ascorbic acid, may interfere with traditional peroxidase assays by reducing the oxidized product generated in the peroxidase reaction; in such assays the oxidized product is typically the reporter molecule that is monitored for enzyme quantification. The action of such reducing compounds results in an apparent lag in product development, which is interpreted as a lower enzyme activity. In such cases the time course of product production may appear sigmoidal. In some cases, these compounds may be sufficiently active as to completely obscure the rate of the reaction. This study describes a relatively simple way to alleviate complications from these compounds. The method is based on using ABTS as the reporter substrate. The oxidized product of the reaction is ABTS•⁺, which can be followed spectrophotometrically due to its relatively high molar absorptivity in the visible region. It is herein shown that one can selectively remove complicating
endogenous reducing compounds by treating the enzyme preparation with the oxidized product itself, ABTS•⁺, prior to initiating the assay. This approach is highly selective for those compounds likely to interfere with peroxidase quantification via reaction product reduction. The presented method is herein shown to remove lag phases associated with different plant extracts and, thus, more accurately reflect total peroxidase activity. The improved assay is relatively simple and should be applicable to a range of biological systems
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Some potential mechanisms for finely-tuned regulation of phospholipase C-β isozymes : studies of dimerization and phosphatidylinositol 3,4,5-trisphosphate activation
Get PDFPhospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled
signaling pathways. Prior research suggested that some isoforms of PLC-β may exist
and function as dimers, but little is known about dimerization of PLC-β. Data from coimmunoprecipitation
assays of differentially-tagged PLC-β constructs and sizeexclusion
chromatography of native PLC-β support homodimerization of PLC-β3 and
PLC-β1 isozymes, but not heterodimerization of these isozymes. Size-exclusion
chromatography data also suggest that PLC-β3 and PLC-β1 form higher affinity
homodimers than PLC-β2. Evidence supportive of limited PLC-β monomerhomodimer
equilibrium appears at 100 nM and lower. Further assessment of
homodimerization status by co-immunoprecipitation assays with differentially-tagged
PLC-β3 fragments demonstrated that at least two subdomains of PLC-β3 are involved
in dimer formation, one in the catalytic X and Y domains, and the other in the G
protein-regulated carboxy-terminal domain. Additionally, microscopic fluorescence
resonance energy transfer assays provide evidence consistent with the existence of
PLC-β homodimers in a whole cell context.
Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) has been proposed as a second
messenger that affects a variety of cellular responses. Previously, we had shown that
PLC-β1 and PLC-β3 bound immobilized PIP₃. In this study, PIP₃ was found to
potentiate Ca²⁺-stimulated PLC-β activities using an in vitro reconstitution assay.
LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 minutes agoniststimulated
total IP accumulation. Both LY294002 and wortmannin inhibited 90
seconds agonist-stimulated IP₃ accumulation in intact cells. Moreover, transfected
p110CAAX, a constitutively activated PI 3-Kinase catalytic subunit, increased 90
seconds oxytocin-stimulated IP₃ accumulation. Receptor-ligand binding assays
indicated that LY294002 did not affect G protein-coupled receptors directly,
suggesting a physiological role for PIP₃ in directly potentiating PLC-β activity. When
co-expressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly
localized to the plasma membrane. Conversely, a greater proportion of PLC-β3
associated with cytosolic fraction following H9c2 cells treatment with LY294002.
Additional observations suggest that the C-tail domain of PLC-β1 and β3, not the PH
or catalytic XY domain, is important for membrane association
Rx for Joy: Boosting Wellbeing and Fulfillment in Oncology Pharmacist Work
No full textThe October 2024 Better U Survey revealed that only 27% of oncology pharmacists felt energized and showed no signs of burnout, which is significantly lower than the University of Utah\u27s clinical team average of 45%. This project\u27s goal is to increase this rate by 5% by the Fall 2025 survey. To address this, using the IHI framework, we conducted What Matters to You? (WMTY) conversations with oncology pharmacists from three distinct groups and collected "bright spots‚" and "stops" using sticky notes. Feedback highlighted daily improvements, participative management, and camaraderie as key factors contributing to workplace challenges. We identified different needs across groups and established the foundation for projects targeting the disease state groups. Bone Marrow Transplant: Improved coordination of outside labs; Hematology: Improved coordination of oral chemotherapy refills; Medical Oncology: Organizing team potlucks. Our success can be attributed to early pharmacy management support, preparing teams with a project pitch, customizing interventions to specific teams, maintaining anonymity, and gaining provider and nursing leadership support. To ensure lasting improvements, we will ensure our chosen projects are executed by checking in regularly with the teams. We also will continue annual WMTY conversations to reassess team needs and continue participation in joy-in-work training. Citation: Perlo J, Balik B, Swensen S, Kabcenell A, Landsman J, Feeley D. IHI Framework for Improving Joy in Work. Cambridge, MA: Institute for Healthcare Improvement, 2017
An advanced pharmacy practice experience in Melbourne, Australia: practical guidance for global experiences
No full textThe Methylerythritol Phosphate Pathway Contributes to Carotenoid But Not Phytol Biosynthesis in <i>Euglena </i><i>g</i><i>racilis</i>
No full textThe biosynthesis of diadinoxanthin and β-carotene in Euglena gracilis was examined using [1-13C]-d-glucose and [5,5-2H2]-1-deoxy-d-xylulose. In contrast to previous studies on isoprenoid biosynthesis in E.
gracilis, the results demonstrate a role for the methylerythritol phosphate (MEP) pathway, along with
the mevalonate pathway, in carotenoid biosynthesis. Interestingly, the MEP pathway is not involved in
the biosynthesis of phytol, a result not previously observed for other chloroplast-containing organisms
