Publications by authors named "Wolfman A"

Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3(-/-) cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased.

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We demonstrate that both c-N-Ras and c-K(B)-Ras are constitutively associated with purified mitochondria. c-K(B)-Ras is associated with the mitochondrial outer membrane, and c-N-Ras is associated with both the outer membrane and inner mitochondrial compartments. The mitochondrial morphology is abnormal in both c-N-Ras negative and K-Ras negative cells.

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Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-alpha/beta). Although Tyk2(-/-) mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2(-/-) mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function.

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Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.

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K-Ras-negative fibroblasts are defective in their steady-state expression of MMP-2. This occurs through c-K(B)-Ras dependent regulation of basal levels of AKT activity. In this report, we have extended those studies to demonstrate that in the absence of K-Ras expression, PDGF-BB fails to induce significant AKT activation, although this was not the case in N-Ras-negative cells.

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We studied the PEMF power attenuation in tissues representative of clinical applications (blood and cortical bone) to determine the amount of power available for PEMF purported biological effects. The experimental system consisted of a pair of nearly circular, parallel and coaxial coils separated by a distance of one coil diameter. The power attenuation was measured using a small search coil connected to a digital oscilloscope.

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Murine pre-osteoblasts and fibroblast cell lines were used to determine the effect of pulsed electromagnetic field (PEMF) exposure on the production of autocrine growth factors and the activation of early signal transduction pathways. Exposure of pre-osteoblast cells to PEMF minimally increased the amount of secreted TGF-beta after 1 day, but had no significant effects thereafter. PEMF exposure of pre-osteoblast cells also had no effect on the amount of prostaglandin E(2) in the conditioned medium.

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23A2 myoblasts expressing GAP-resistant, constitutively active G12V:H-Ras (A2:G12V:H-Ras myoblasts) display a transformed morphology and do not undergo mitogen-deprivation-induced differentiation or the associated apoptosis. To determine the phenotype induced by F156L:H-Ras, a constitutively active mutant with enhanced nucleotide exchange activity rather than impaired GAP-stimulated GTPase activity, myoblast cell lines were established that stably express F156L:H-Ras at levels of H-Ras comparable to the A2:G12V:H-Ras myoblasts. These A2:F156L:H-Ras myoblast cell lines do not possess a transformed morphology, and while differentiation and apoptosis are impaired, these processes are not abrogated as in the A2:G12V:H-Ras myoblasts.

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We tested the hypothesis that exposure of a mouse preosteoblast cell line to pulsed electromagnetic fields (PEMF) would affect components of the extracellular matrix. We report that exposure of MC3T3-E1 cells to a single PEMF waveform significantly reduced the amount of mature, alpha1(I) collagen in the extracellular matrix (ECM) and the conditioned medium, without affecting the amount of total ECM protein. This decrease was not due to changes in the steady-state level of Col1A1 mRNA or to degradation of mature collagen.

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Longitudinal in vivo micro-computerized tomography (CT) imaging was used to monitor bone resorption in a rat fibula osteotomy model. Quantitative image post-processing techniques were developed for spatially aligning the longitudinal data sets. Nominal length and bone volume in the proximal and distal segments of the fibula after the osteotomy were measured, and quantitative comparisons of bone loss over a 13-week period post-surgery were made in five individual rats.

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Article Synopsis
  • The study tested whether pulsed electromagnetic field (PEMF) treatments can speed up the healing process for bone trauma using live rats as subjects.
  • Starting 5 days after surgery, the rats' injured limbs received daily PEMF treatment, which led to a significant increase in the rate and volume of bone callus formation compared to untreated limbs.
  • Results showed that PEMF treatment improved the strength and healing of bone more effectively than a different PEMF waveform, emphasizing the importance of specific treatment characteristics on biological outcomes.
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The effectiveness of non-invasive pulsed electromagnetic fields (PEMF) on stimulating bone formation in vivo to augment fracture healing is still controversial, largely because of technical ambiguities in data interpretation within several previous studies. To address this uncertainty, we implemented a rigorously controlled, blinded protocol using a bilateral, mid-diaphyseal fibular osteotomy model in aged rats that achieved a non-union status within 3-4 weeks post-surgery. Bilateral osteotomies allowed delivery of a PEMF treatment protocol on one hind limb, with the contralateral limb representing a within-animal sham-treatment.

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Fibroblasts constitutively express matrix metalloproteinase 2 (MMP-2), which specifically cleaves type IV collagen, a major structural component of basement membranes. The level of MMP-2 expression was not altered by serum withdrawal, suggesting that MMP-2 expression is regulated by a series of steady-state conditions that impinge on the MMP-2 promoter. Expression of a dominant-negative Ras protein significantly inhibited MMP-2 transcription, thereby suggesting a role for steady-state Ras function in the regulation of MMP-2 expression.

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Electromagnetic field visualization is important in multidisciplinary research on the molecular basis of therapeutic effects of pulsed electromagnetic fields (PEMF). We have compared classic PEMF representations by two-dimensional field lines and field magnitude contour plots with a field representation using three-dimensional field isosurfaces. Field simulations were performed for a clinically approved Spinal-Stim Lite system (Orthofix Inc.

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Cellular N-Ras provides a steady-state antiapoptotic signal, at least partially through the regulation of phosphorylated Akt and Bad levels. Fibroblasts lacking c-N-Ras expression are highly sensitive to the induction of apoptosis by a variety of agents. Reduction of pBad and pAkt levels using a phosphatidylinositol 3-kinase inhibitor was not sufficient to sensitize the control cell population to the high level of apoptosis observed in the N-Ras knockout cell lines, suggesting that c-N-Ras provides at least one other antiapoptotic signal.

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The proteins that compose the Ras family of small guanosine triphosphatases share a remarkably high degree of sequence similarity, yet recent evidence indicates that they may have unique biological properties. How is it that similar proteins carry out different jobs in the cell? Wolfman addresses this question by surveying recent reports that indicate that different biological roles may be born out of distinct subcellular localizations of the Ras proteins. It appears that the small differences in their amino acid sequences and their different posttranslational modifications may be all that is necessary to direct various Ras proteins to different sites.

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Cells expressing oncogenic Ras proteins transmit a complex set of signals that ultimately result in constitutive activation of signaling molecules, culminating in unregulated cellular function. Although the role of oncogenic Ras in a variety of cellular responses including transformation, cell survival, differentiation, and migration is well documented, the direct Ras/effector interactions that contribute to the different Ras biological end points have not been as clearly defined. Observations by other groups in which Ras-dependent transformation can be blocked by expression of either dominant negative forms of Phosphatidylinositol (PI) 3-kinase or PTEN, a 3-phosphoinositide-specific phosphatase, support an essential role for PI 3-kinase and its lipid products in the transformation process.

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Activation of MAP kinase leads to the activation of p53-dependent pathways, and vice-versa. Although the amount of p53 protein increases in response to MAP kinase-dependent signaling, the basis of this increase is not yet fully understood. We have isolated the mutant cell line AP14, defective in p53 expression, from human HT1080 fibrosarcoma cells, which have an activated ras allele.

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Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module.

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We report that c-N-Ras possesses an isoform-specific, functional role in cell survival under steady-state conditions. This function includes protection from programmed cell death by serum deprivation or upon treatment with apoptosis-inducing agents. The data demonstrate that c-N-Ras may play a functional role in the regulation of steady-state phosphorylated Akt and serine 136-phosphorylated Bad (Ser(136)-pBad).

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Amplification of several chromosomal regions have been observed in human breast carcinomas. One such region, 8p11, is amplified in 10-15% of tumor samples. Although the FGFR1 gene is located close to this region, and is often included within the amplicon, the observation that tumors exhibiting 8p11 amplification do not always overexpress FGFR1 suggests that another gene located close to FGFR1 is involved in the tumorigenic process.

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C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serum-free medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A.

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Both insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) are key modulators of skeletal myoblast differentiation. The critical signaling pathways used by either IGF-1 or FGF-2 to inhibit differentiation have not been determined. In this study, we show that both IGF-1 and FGF-2 inhibit the differentiation of 23A2 myoblasts and that both stimulate signaling through mitogen-activated protein kinase (MAPK) kinase (MEK) to MAPK roughly 8-fold in 23A2 myoblasts.

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The Ras GTPases function as molecular switches, regulating a multiplicity of biological events. However the contribution, if any, of a specific c-Ras isoform (Ha-, N-, or Ki-ras A or B) in the regulation of a given biological or biochemical process, is unknown. Murine C3H1OT1/2 fibroblasts transformed with activated (G12V)Ha-ras or (Q61K)N-ras proliferate in serum-free media and have constitutive MAPK activity.

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