No description available.

A two by six factorial design was used to investigate the effect of zinc and all-trans retinoic acid (RA) on the growth of two human breast cancer cell lines differing in their expression of metallothionein (MT) and of estrogen receptors; MCF7 cells express estrogen receptors, BT-20 cells do not. Cells were treated with zinc to induce MT then treated with six concentrations of RA. Cell proliferation, lipid peroxidation, MT protein, MT mRNA and glutathione concentrations were measured. / BT-20 cells expressed higher constitutive MT concentrations than MCF7 cells. MT was significantly induced by zinc treatment in BT-20 cells but not in MCF7 cells. Low RA concentrations stimulated growth proliferation but higher concentrations inhibited cell proliferation. High RA concentrations increased lipid peroxidation. There was a significant negative correlation between lipid peroxidation and cell proliferation. Growth inhibition and lipid peroxidation were reduced by zinc in BT-20 cells but not in MCF7 cells. Glutathione did not appear to be a significant factor. / Induction of metallothionein by zinc may modulate the growth inhibitory effects of all-trans retinoic acid in human breast cancer cells. One mechanism of growth inhibition may be through increased lipid peroxidation.

Checkpoint kinase 1 (Chk1) is a key player in the DNA damage response signalling pathway and the mode of Chk1 activation whereby it undergoes ATRdependent phosphorylation at Ser317 and Ser345 is well characterised. It has been suggested that phosphorylation at the ATR sites relieves the auto-inhibitory action conferred by the C-terminal negative regulatory domain on the catalytic core of Chk1. In this study, we show that Chk1 activity can also be stimulated by docking to an N-terminal region of the growth regulator p21waf1 and this docking domain is necessary for efficient Chk1-dependent phosphorylation of p21 at Ser146. In addition, Chk1 and p21 are shown to form a transient interaction by immunoprecipitation. Interestingly, although the isolated p21 docking domain can activate Chk1 in trans, a mutant where the C-terminal 70 amino acids are truncated is refractory to stimulation whereas mutation of the ATR phosphoacceptor sites does not affect docking dependent activation. Furthermore, when the amino acid sequence of the p21 docking domain was aligned with the sequence of Chk1, homology to the F region on the kinase domain was identified. Mutation of two conserved tryptophan residues within the homology region appears to release the C-terminus from intramolecular interactions rendering it susceptible to cleavage and refractory to allosteric stimulation. Furthermore, small peptides based on this region of Chk1, like the p21 docking domain, are able to activate Chk1 in trans and disrupt interaction between the N-terminal and Cterminal domains. Interestingly, peptide microarray showed that Chk1 stimulated by activating peptide is able to phosphorylate novel peptide substrates which are not observed with unstimulated Chk1. The data suggest that the last C-terminal 70 amino acids of Chk1 play an important role in auto-inhibition through interaction with the F region of the core catalytic domain. Binding to p21 is able to activate Chk1 by inhibiting the auto-inhibitory interaction independent of phosphorylation at the Ser317 and Ser345 sites. Furthermore, activating peptide is able to modulate Chk1 specificity towards other substrates.

Bovine fibroblasts and epithelial cells were isolated from surgically biopsied mammary tissue. Characterization of population doubling time, cytoskeletal intermediate filaments, cryopreservation survival, and viability were performed on all fibroblast and epithelial cells. Several clonal fibroblast cell lines were cotransfected with a plasmid bearing the SV-40 Large-T-antigen, and the pSV-2 neo plasmid. Transfected cells were subsequently selected with G418 sulfate and cloned. / MAC-T cells and non-clonal primary bovine mammary epithelial cells proliferated in response to IGF-I, insulin, serum and serum albumin. MAC-T cells did not proliferate when cultured in EGF, estrogen, progesterone, estrogen+progesterone, growth hormone, prolactin, and only modest proliferation was obtained after TGF-$ alpha$ treatment. Subsequent experiments used serum, insulin or IGF-I (and its analogues) to stimulate cellular proliferation. Serum albumin was not added to serum-free media preparations since it stimulated cellular proliferation. / TGF-$ beta$ receptors were characterized in MAC-T cells and normal fibroblasts. Affinity labelling studies revealed that MAC-T and MF-2 cells contained type I, II, and III autoregulatable receptors. Fibroblast proliferation, was inhibited 50% by TGF-$ beta$. TGF-$ beta$ inhibited MAC-T cellular proliferation at concentrations among the lowest ever reported, ED$ sb{ rm 50}$ = 4 pm. TGF-$ beta$ was not cytotoxic at concentrations 1000-fold higher. / Retinoic acid (RA) also inhibited proliferation of MAC-T cells. Inhibition of proliferation did not occur when cells were growth stimulated by IGF-I analogues that do not bind IGFBPs. Unlike TGF-$ beta$, RA treatment increased IGFBP-2 and decreased IGFBP-3 protein expression by cells into media and on the cell's membrane. RA was cytotoxic at concentrations 10-fold higher than ED$ sb{100}$. / Fibroblasts and epithelial cells expressed the gap junction (GJ) protein, connexin43, with transformed fibroblasts expressing significantly less connexin43. Perinuclear and cell surface connexin43 was immunodetected in epithelial and fibroblasts cells. TGF-$ beta$, RA or cAMP, increased connexin43 protein expression, especially phosphorylated species. Only cAMP noticeably altered immunolocalization patterns of connexin43, causing a shift from perinuclear pools to the cell surface. None of the growth inhibitors affected GJ communication as measured by dye transfer. Therefore, mammary epithelial cells are growth inhibited by TGF-$ beta$ and RA by distinct mechanisms and both growth inhibitors significantly enhance the gap junction protein, connexin43, without increasing GJ communication.

The signal transducer and activator of transcription (STAT) and protein inhibitor of STAT(PIAS) system represent an elegant regulatory mechanism of transcriptional control IN mammalian cytokine signalling. Abnormal activation of the system is associated with immune disorders and a large group of diverse tumours. PIAS3 is a multiple domain protein with distinct functions involved in regulation of cytokine-mediated gene activation pathways.Its over-expression significantly inhibits cell growth and renders cancer cells more sensitive to drugs. The objective of this study was to structurally and biochemically characterise the function of the PIAS3 protein using in silico, in vivo and in vitro analysis approaches.The conservation pattern of the PIAS protein family and critical conserved residues in the PINIT (Proline, Isoleucine, Asparagine, Isoleucine, Tyrosine) domain were identified. The PINIT domain model was generated based on the PINIT domain structure of yeast PIAS3 homologue Siz1 and structural determinants in the PIAS3-STAT3 interaction were evaluated.Guided by the in silico findings, in vivo analysis of the localisation of the PIAS3, mutantderivatives of PIAS3 (PIAS3-L97A, PIAS3-R99N, PIAS3-R99Q), PINIT and acidic domain was conducted. PIAS3 was completely localised in the nucleus while PIAS3 mutants appeared to exhibit diffuse cytoplasmic distribution. The PINIT domain was predominantly localised in the nucleus with some apparent perinuclear staining while the acidic domain exhibited a predominantly perinuclear staining pattern. Further analysis of the PINIT domain and the effect of the mutants on PIAS3-STAT3 interaction were assessed by in vitro analysis. Guided by in silico analysis, the PINIT domain and mutant derivatives of PINIT domain (PINIT-L97A, PINIT-R99N, and PINIT-R99Q) were heterologously expressed in Escherichia coli and subsequently purified using a combination of immobilized metal affinity and size exclusion based chromatography. The size and structural elements of the PINIT domain and its mutants were characterised. The 23 kDa PINIT domain was found to exist as a monomer in solution and its secondary structure was shown to consist of 66 % β-sheets by fourier transformed infrared spectroscopy consistent with the generated homology model.Using surface plasmonresonance spectroscopy (SPR) the PINIT domain was shown to bind to STAT3 in a specific concentration dependent manner. Recombinant PINIT-L97A,PINITR99N and PINIT-R99Q mutants, which exhibited similar structural integrity to the wildtype, were found to abrogate binding to STAT3. These findings suggest that these residues form part of a potential binding surface for stat3. In conclusion, this study has provided evidence that the PINIT domain is an important determinant of PIAS3 interaction with STAT3 and that the interaction is mediated by defined conserved residues directly involved in the PINITSTAT3 interaction.

Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.

The termina1 enzyme in the linoleoyl-CoA desaturase enzyme complex, delta-6-desaturase was implied in the control of cell proliferation in cancer cells. One of the aims of this study was to isolate the terminal enzyme. It was decided that in order to isolate this enzyme it was first necessary to isolate the entire complex and then to enzymatically solubilise the first two components of the complex i e cytochrome b5 reductase and cytochrome b5 from the complex resulting in a pure delta-6-desaturase . The first two components were isolated and purified using simplified and easily reproducible methodologies which could be utilised in the final purification of delta-6- desaturase. The entire enzyme complex, linoleoyl-CoA desaturase was also isolated in a pure form and this pure complex was used to attempt to isolate delta-6-desaturase. The terminal enzyme was isolated with some cytochrome b5 still bound to it. The methods used had proven to be successful and with some modifications should yield a pure enzyme. Zinc and GLA were known to play a role in the inhibition of cancer cell proliferation and zinc was hypothesised to inhibit cell growth by stimulating the activity of the linoleoyl-CoA desaturase enzyme complex which is involved in the regulation of cell proliferation. GLA is the product of the reaction that this enzyme complex catalyses and GLA has been shown to inhibit cancer ce ll growth. The effect of GLA on cell growth and linoleoyl-CoA desaturase activity was thus investigated. Results showed that both zinc and GLA inhibited cell growth and that the combined addition of zinc and GLA generally resulted in the inhibition of cell growth and the activation of linoleoyl-CoA desaturase activity in the BL-6 cells while having a less pronounced effect on the LLCMK cells. The results of this study support the hypothesis that zinc may be a cofactor of linoleoyl-CoA desaturase.

No description available.

Managing turf for high aesthetic value is costly. Such management usually involves mowing, disease prevention, insect control, and weed control. Mowing is the most expensive practice on golf courses and annual bluegrass (Poa annua L) is the most challenging weed problem in professional turf. The plant growth regulators trinexapac-ethyl and paclobutrazol are commonly used in VA for these two costly and challenging jobs. Prohexadione calcium (PC) is an experimental chemical that inhibits the same enzyme (3ß-hydroxyalase) as trinexapac-ethyl and may selectively suppress annual bluegrass. Experiments were conducted at the Virginia Tech Turfgrass Research Center and Glade Road Research Facility to determine the PC rate required to reduce clipping biomass of four turfgrass species as effectively as trinexapac-ethyl. Prohexadione calcium reduced clipping biomass of bermudagrass (Cynodon dactylon (L.) Pers.), Kentucky bluegrass (Poa pratenis L.), perennial ryegrass (Lolium perenne L.), and zoysiagrass (Zoysia japonica Steud.) equivalent to trinexapac-ethyl at 0.70, 0.22, 0.60, and 0.27 kg a.i./ha -1, respectively. Further experiments conducted at three locations across Virginia determined that PC was comparable to paclobutrazol for annual bluegrass suppression. Since turfgrass response to PC was different between annual bluegrass, Kentucky bluegrass, and perennial ryegrass, 14C labeled PC was used to assess absorption, translocation, and metabolism of PC between annual and Kentucky bluegrass, creeping bentgrass (Agrostis stolonifera L.), and perennial ryegrass. Annual and Kentucky bluegrass absorbed more PC than creeping bentgrass or perennial ryegrass and partially explained the selectivity between these species. Translocation and metabolism of PC did not differ between species. Our final objective launched experiments characterizing possible resistance to acetolactate synthase (ALS) inhibiting herbicides in annual bluegrass. Several selective herbicides for annual bluegrass control inhibit ALS. Since many weeds have developed resistance to ALS-inhibiting herbicides, the ALS gene in annual bluegrass was sequenced and derived amino acid sequences were at least 87% similar to other previously sequenced grass species. This sequencing data will be used in future experiments to predict the likelihood of ALS resistance in annual bluegrass. / Ph. D.

La plupart des organes et organismes ont une forme remarquablement reproductible, malgré une très grande variabilité de forme et de croissance au niveau cellulaire. Des signaux supracellular, gradient de morphogène ou contraintes mécaniques, pourraient coordonner le comportement des cellules et via de multiples boucles de rétroaction canaliser les formes finales. Des progrès récents en imagerie du vivant, micromécanique et modélisation, permettent aujourd’hui d’analyser la relation entre la variabilité cellulaire, la communication intercellulaire et la forme globale d’un organe de façon quantitative. Nous avons choisi de travailler sur le sépale abaxial chez Arabidopsis thaliana, car sa forme est reproductible et il est facilement accessible pour l’imagerie. Nous avons choisi de nous concentrer sur l’analyse des microtubules corticaux : ils s’alignent le long des tensions maximales dans les tissus et, en guidant le dépôt des microfibrilles de cellulose, ils renforcent l’anisotropie mécanique des parois dans la direction des contraintes maximales. Nous avons observé un alignement supracellulaire des microtubules à la pointe du sépale et nous avons pu corréler ce comportement à un patron de tensions causé par un différentiel de croissance dans le sépale. En utilisant des approches micromécaniques et des mutants affectés dans la dynamique des microtubules, nous avons confirmé que les microtubules étaient capables de s’aligner en fonction des contraintes mécaniques, la forme finale du sépale dépendant de la force du rétrocontrôle. Nous proposons donc que cette réponse déclenche un arrêt de croissance de la pointe du sépale jusqu’à sa base et limite ainsi l'expansion des sépales. Plus localement, nous avons également analysé la contribution des conflits mécaniques entre cellules voisines, soit en utilisant le différentiel de croissance naturel autour d’un trichome, soit en générant des mosaïques artificielles avec le système cre-lox. Nos résultats suggèrent une contribution de l'hétérogénéité de croissance dans la forme finale des sépales, encore une fois par l'intermédiaire de la réponse des microtubules aux contraintes mécaniques. Ces résultats nous permettent donc d’élaborer un scénario dans lequel une rétroaction mécanique, locale et globale, sur les microtubules contrôle la forme finale du sépale. / Most organs and organisms have remarkably consistent final shapes, yet at the cellular level, growth and shape can be highly variable. Surpacellular signals, e.g. morphogen gradients or force fields, may coordinate cell behavior, involving multiple feedback loops, to yield such reproducible shapes. Because of the recent progress in live-imaging techniques, micromechanics and modeling, the relation between cellular noise, cell-cell communication and global shape is now amenable to quantitative analysis. We chose to work on the abaxial sepal, as it displays consistent shapes and is easily accessible for live imaging. We focus our analysis on cortical microtubules: they align along maximal tensile stress directions in plant tissues, and as they guide the deposition of cellulose microfibrils, the main load-bearing component in plant cell walls, they largely determine the mechanical anisotropy of cell walls, providing mechanical strength in the direction of maximal stress. We identified a supracellular alignment of microtubules at the tip of the sepal and we could match this pattern with predicted growth-derived tensile stress patterns. Using micromechanical approaches and mutants impaired in microtubule dynamics, we confirm that microtubules in the sepal can align along maximal tension directions, the final sepal shape depending on the strength of the feedback. We thus propose that this response triggers a wave of growth arrest from the tip of the sepal and thus restricts the expansion of the sepal. More locally, we also analyzed the contribution of mechanical conflicts between adjacent cells that grow at different rates, using the naturally occurring fast growing trichome cells as well as cre-lox induced artificial growth mosaics. Our results support a contribution of growth heterogeneity in final sepal shape, again via the microtubule response to mechanical forces. Altogether, this provides a scenario in which global and local mechanical feedback on microtubules channels the sepal final shape.