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S R Pennington

Publications and source records attributed to S R Pennington.

At least 19 recordsLinked to original sources
PubMed2001

Arrays for protein expression profiling: towards a viable alternative to two-dimensional gel electrophoresis?

Two-dimensional gel electrophoresis (2-DE) is used as a platform method for the measurement of protein expression patterns within cells, tissues or organisms. This approach can support expression profiling of several thousand proteins in multiple samples and as such it is currently unrivaled as a tool for the analysis of protein expression, which is a key component of the rapidly expanding field of proteomics. However, 2-DE has a number of significant limitations and as a consequence, alternative approaches for the measurement of expression of proteins within complex samples are actively being explored. Here we review some existing and emerging methods for protein expression analysis. In particular, we review a range of technologies that might be integrated to support the development of 'arrays' or 'chips' for rapid, high-throughput analysis of protein expression in a manner analogous to the current use of DNA arrays for mRNA expression analysis. We conclude that such separation-independent platforms may ultimately supersede two-dimensional (2-D) gel-based analyses for global protein expression analysis but that before this the technologies might provide important new platforms for diagnostic and prognostic monitoring of diseases.

Databases, Protein↗
PubMed2001

Regulation of growth factor induced gene expression by calcium signalling: integrated mRNA and protein expression analysis.

There is considerable indirect evidence that growth factor induced changes in the intracellular concentration of calcium play an important role in the regulation of the mammalian cell cycle. However, the precise mechanism by which this may be achieved remains unclear. Here we show that SKF-96365, an inhibitor of growth factor induced capacitative calcium entry (CCE), inhibits cell cycle progression by preventing entry into S phase. SKF-96365 changes the temporal profile of growth factor induced calcium signalling and recent studies have shown that alterations in the temporal and spatial patterns of calcium signalling can differentially regulate gene expression. We have therefore sought to examine the effect of inhibition of CCE on growth factor induced gene expression during G1. To achieve this we have initiated a combined transcriptomic and proteomic approach to measure CCE regulated gene expression using cDNA arrays and two-dimensional polyacrylamide gel electrophoresis, respectively. The initial results of this on-going analysis are reported here. They reveal that inhibition of CCE influences the expression of 29 genes at the mRNA level and 22 genes at the protein level. We report the identification of the mRNAs whose expression is altered by inhibition of CCE and describe the potential functional significance of some of these changes. The value of integrating a transcriptomic and two-dimensional gel electrophoresis based proteomic approach to studies of gene expression is discussed.

3T3 Cells↗
PubMed2000

Mastoparan transiently permeabilizes Swiss 3T3 cells and induces c-fos proto-oncogene expression. Role of calcium and G protein activation.

Mastoparan, a widely used tetradecapeptide activator of Gi/Go G proteins, has been reported to be a potent co-mitogen for Swiss 3T3 fibroblasts. However, we have previously shown that the peptide promotes the release of lactate dehydrogenase from Swiss 3T3 cells and evokes only a modest and delayed increase in DNA. We suggested that the ability of the peptide to permeabilise these cells may account for its mitogenic action. Here we show that mastoparan caused a rapid release of fluorescein from cells which had been pre-incubated with fluorescein diacetate, indicating that the peptide increases membrane permeability to small molecules. Furthermore, the release of lactate dehydrogenase evoked by mastoparan was lost after prolonged (24 h) incubation of cells with the peptide. Together, these data indicate that mastoparan-induced cell permeabilisation is both rapid and transient. We have also shown that mastoparan increased c-fos mRNA accumulation and that this response was not influenced by pertussis toxin or indomethacin. Although mastoparan increased the intracellular calcium concentration, the removal of extracellular calcium had no effect on mastoparan stimulated c-fos mRNA accumulation. These data show that mastoparan-induced c-fos mRNA accumulation is not mediated by activation of a G protein and subsequent activation of phospholipase D nor by a non-selective increase in calcium influx. The data have significance for the interpretation of studies in which mastoparan is, or has been, used as an activator of Gi/Go.

3T3 Cells↗
PubMed2000

Changes in p21(Cip1) and p27(Kip1) expression are not required for cell cycle entry and progression to S phase in Swiss 3T3 cells.

The cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), play an important role in the regulation of progression through G(1) to S phase in mammalian cells. Here we report that confluent 3T3 cells expressed p21(Cip1) and p27(Kip1) predominantly in the nucleus, and the level of both proteins declined as the cells entered the cell cycle and progressed through G(1) in response to serum growth factors. However, when confluent cells were serum starved prior to treatment, no downregulation of p21(Cip1) or p27(Kip1) expression was observed. Notably, serum starvation did not significantly influence the capacity of the cells to progress to the S phase. It was observed that serum starvation reduced cell density. Further, when cells were plated at a range of different densities, starved of serum to render them quiescent and then subsequently treated with serum, a reduction in p21(Cip1) and p27(Kip1) expression was observed in cells plated at high density but not in those at low density. Again, the extent and timing of progression to S phase was not influenced by cell density. To establish the potential role of cell:cell contact in the observed density-dependent regulation of p21(Cip1) and p27(Kip1) expression, cells were plated onto micorarrays of adhesive islands that prevented individual cells from making any contact with other cells. Under these conditions serum growth factors induced p21(Cip1) and p27(Kip1) downregulation, and hence, there is no requirement for cell:cell contact. Together, these data indicate that there are conditions under which 3T3 cells can progress to the S phase without downregulation of p21(Cip1) and p27(Kip1). The significance of these observations and mechanisms by which density-dependent regulation of p21(Cip1) and p27(Kip1) expression may occur are discussed.

3T3 Cells↗
PubMed1998

Attachment staining: a novel method for flow cytometric quantitation of protein expression in limited numbers of adherent cells.

Measurement of protein expression in adherent cells in culture by flow cytometry, although potentially a very powerful method, has so far found limited application. Where the method has been applied successfully, cells have been resuspended for analysis either prior to experimental treatment or before immunostaining. Such approaches have several limitations and do not permit analysis of limited numbers of cells. Here we describe a method of attachment staining which allows protein expression in a few thousand adherent cells to be analyzed by flow cytometry. The method has been successfully used for the measurement of growth factor-induced c-Fos protein expression and measurement of c-Fos expression in samples of < 3,000 adherent fibroblasts.

3T3 Cells↗
PubMed1997

Changes in nuclear protein composition in response to chronic electrical stimulation of skeletal muscle.

The adaptive response of skeletal muscle to increased functional demand involves phenotypic changes that affect contractile properties, energy metabolism and calcium kinetics. Some of these changes are known to be initiated at a pre-translational level, but the underlying regulatory mechanisms have not yet been identified. In this study we used chronic electrical stimulation (10 Hz, continuous) to initiate fast-to-slow muscle fibre-type transformation, and two-dimensional electrophoresis (2-DE) to assess changes in nuclear protein composition after 24 and 72 h. We report an early and sustained increase in the level of a 3 kDa protein in stimulated fast muscle (n = 6). The presence of the same protein in control slow muscle is consistent with a possible functional role in the determination of the slow phenotype.

Animals↗
PubMed1996

Inhibition of mitogen-induced DNA synthesis by bafilomycin A1 in Swiss 3T3 fibroblasts.

Quiescent cells (in G0) can be stimulated to enter the cell cycle and proceed to DNA synthesis in S-phase by a wide range of growth factors and mitogens. Activation of cell-surface growth factor receptors with intrinsic protein tyrosine kinase activity initiates autophosphorylation of the receptors and subsequent activation of signal transduction cascades. After activation the receptors undergo ligand-induced internalization to endosomes, which become acidified by the action of a vacuolar H(+)-ATPase (V-ATPase). The extent to which vesicular acidification plays a role in mitogenic signalling by receptors with intrinsic tyrosine kinase activity remains unknown. Here we have shown that bafilomycin A1, a specific inhibitor of V-ATPase, inhibits endosome acidification and mitogen-induced DNA synthesis in Swiss 3T3 fibroblasts. Addition of bafilomycin A1 at successively later times during G1 progressively decreased the inhibition of DNA synthesis such that no inhibition was observed when bafilomycin A1 was added at the onset of S-phase. Bafilomycin A1 also induced a dramatic but reversible change in the morphology of Swiss 3T3 cells. However, the rapid activation of c-fos mRNA accumulation by epidermal growth factor and insulin was unaffected by bafilomycin A1. Together, the results suggest that activation of the V-ATPase plays an important role in the mitogenic signalling pathways that occur during the G1 phase of the cell cycle but is not required for the initial epidermal growth factor and insulin-evoked signalling events that lead to c-fos mRNA expression.

3T3 Cells↗
PubMed1996

Mastoparan causes cell permeabilisation and delayed activation of DNA synthesis in Swiss 3T3 fibroblasts.

Mastoparan, a tetradecapeptide from wasp venom, preferentially activates the heterotrimeric G proteins, Go and Gi by promoting GDP/GTP exchange. The peptide was originally identified as a potent secretagogue and has since been shown to promote DNA synthesis in Swiss 3T3 fibroblasts. Here, we have shown that mastoparan (10-20 microM), either alone or in combination with the co-mitogen insulin, had no effect on DNA synthesis when incubated with cells for 24 h. However, in the presence of insulin, the peptide evoked a small increase in DNA synthesis after incubation for 40 h. Thus, unlike other mitogens, mastoparan caused a delayed activation of DNA synthesis. At concentrations of mastoparan (15-17.5 microM) which promoted DNA synthesis, the peptide caused a rapid release of lactate dehydrogenase from the cells. These data suggest that the mitogenic action of mastoparan occurs by a mechanism distinct from that of physiological mitogens.

3T3 Cells↗
PubMed1995

Analysis of growth factor stimulated nucleocytoplasmic protein transport using two-dimensional gel electrophoresis.

The rapid activation of gene expression by growth factors frequently involves the nucleocytoplasmic redistribution of proteins which regulate the transcription of discrete sets of genes. In particular, it has become apparent that protein kinases and transcription factors including protein kinase A, mitogen activated protein kinase, protein kinase C, NF-kappa B and Stat91, can rapidly migrate from the cytosol to the nucleus in response to a wide variety of extracellular stimuli. Furthermore, it is assumed that these proteins represent important components of the signal transduction pathways which operate between the plasma membrane and the nucleus. Here two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to detect proteins which undergo rapid growth factor stimulated nucleocytoplasmic redistribution. Epidermal growth factor and insulin influenced the association of 15 individual protein spots with the nucleus. Estimates of the abundance of these proteins revealed that they represented approximately 1/1000 to 1/5000 of total nuclear protein. To permit identification by microsequencing, five of the protein spots have been recovered from multiple 2-D PAGE gels and concentrated by one-dimensional elution-concentration gel electrophoresis. The data obtained demonstrate that 2-D PAGE can be used to: (i) analyse putative regulators of growth factor activated gene transcription, and (ii) purify sufficient amounts of these proteins to facilitate identification by microsequence analysis.

3T3 Cells↗
PubMed1995

Cell cycle-dependent morphological changes in the actin cytoskeleton induced by agents which elevate cyclic AMP.

Agents which increase the intracellular concentration of cyclic adenosine-5'-monophosphate, induce a highly arborised morphology in a proportion of sub-confluent Swiss 3T3 fibroblasts. During this process the organisation of actin filaments progressively changes from a characteristic stress fibre pattern to leave a network of actin filaments within each and every arborisation. Despite this massive reorganisation of the actin cytoskeleton no changes are observed in the extent of polymerisation of actin during arborisation. The proportion of cells in asynchronous cultures undergoing arborisation at maximal concentrations of agents reaches a maximum of 30%; suggesting that the effect might be mediated only in cells during a restricted period of the cell cycle. More than 80% of serum-starved cells responded to these agents between 1 and 8 hours after readdition of serum, but not at other times, suggesting that the arborisation response can occur only in the G1 phase of the cell cycle.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Measurement of intracellular Ca2+ in single aequorin-injected and suspensions of fura-2-loaded ROS 17/2.8 cells and normal human osteoblasts. Effect of parathyroid hormone.

It is known that parathyroid hormone (PTH) activates the cyclic AMP (cAMP) signalling pathway in osteoblasts. In recent years it has been suggested that an elevation of the intracellular free Ca2+ concentration ([Ca2+]i) may also be involved in the regulation of osteoblast function by PTH. However, this remains controversial. Here we investigated the effect of PTH on the [Ca2+]i of ROS 17/2.8 cells and normal human osteoblasts. The [Ca2+]i was measured in single aequorin-injected cells and in suspensions of cells loaded with fura-2. Human PTH-(1-38)-peptide (1-300 nM) had no effect on the [Ca2+]i in single aequorin-injected ROS 17/2.8 cells (n = 17) measured at various times after injection (1-20 h), or in suspensions of fura-2-loaded ROS 17/2.8 cells (n = 9). Ionomycin (1 microM) increased the [Ca2+]i in fura-2-loaded and single aequorin-injected ROS 17/2.8 cells by 285 +/- 60 nM (n = 9) and 312 +/- 99 nM (n = 6) respectively, indicating that both methods detect changes in [Ca2+]i with equal sensitivity. In contrast, human PTH-(1-38) (10-100 nM) markedly stimulated cAMP accumulation in ROS 17/2.8 cells. In single aequorin-injected normal human osteoblasts there was no change in the [Ca2+]i in response to 100 nM human PTH-(1-38) or 100 nM bovine PTH-(1-84) (n = 18). In contrast, in suspensions of normal human osteoblasts loaded with fura-2, an increase in [Ca2+]i in response to human PTH-(1-38) (100 nM) was found (60 +/- 28 nM; n = 6). Considerable variation in the magnitude of the response was observed between individual preparations and donors. These data indicate that PTH activates cAMP accumulation without affecting [Ca2+]i in ROS 17/2.8 cells and that PTH causes a rise in [Ca2+]i only in a small subset of normal human osteoblasts. We suggest that the Ca2+ response to PTH in osteoblasts is limited by the state of differentiation of the cells, and may be due either to the presence of a distinct Ca2(+)-mobilizing receptor or to a cAMP-mediated Ca2+ response.

Aequorin↗