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Biomedical subjects

C E Stewart

Publications and source records attributed to C E Stewart.

At least 37 records · Page 2Linked to original sources
PubMed2000

Characterisation of the IGF system in a primary adult human skeletal muscle cell model, and comparison of the effects of insulin and IGF-I on protein metabolism.

In an attempt to address the complex and clinically challenging question of the causes of muscle wasting in patients with cachexia, we have developed a primary adult human skeletal muscle cell model. The cultured cells were characterised by immunocytochemistry using antibodies to the myofibrillar protein constituents desmin and titin. Myotube formation was confirmed biochemically by a fourfold increase in the activity of the muscle-specific enzyme creatinine kinase, and myoblast withdrawal from the cell cycle, which is essential for terminal differentiation, was associated with progressive retinoblastoma protein dephosphorylation. Having successfully confirmed the phenotype of these adult human muscle cells, we assessed their interaction with the insulin-like growth factor (IGF) system. IGF-I is known to stimulate myoblast survival, proliferation and differentiation in cell lines, and, like insulin, is a potent anabolic agent in the regulation of protein metabolism. We have shown that IGF-I stimulated both replication and differentiation of myoblasts, whilst fibroblast growth factor-2 stimulated replication but inhibited differentiation. Examining the IGF system during the process of terminal differentiation, we found that both myoblasts and myotubes expressed insulin, IGF-I and insulin-IGF-I hybrid receptors, with the levels of all three receptor types increasing on differentiation. The cells also produced a wide range of IGF binding proteins (IGFBPs) including IGFBP-2, IGFBP-4 and abundant IGFBP-3, which has not been shown to be produced by any other skeletal muscle cell line examined to date. Both insulin and IGF-I had anabolic effects on myotube protein metabolism at physiological concentrations. Insulin was more potent than IGF-I: use of the IGF analogue long R(3)IGF-I demonstrated that the effects of exogenous IGF-I on protein metabolism were not affected by the high levels of endogenous IGFBP production. In summary, we have developed and characterised a clinically relevant in vitro model with which to address the aetiology of muscle wasting associated with chronic catabolic conditions, and we anticipate that future work will enable the development of novel, effective therapeutic interventions.

Adult↗
PubMed1999

Increased tyrosine kinase activity but not calcium mobilization is required for ceramide-induced apoptosis.

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro, potentially via activation of the IGF-I receptor (IGF-IR). We have previously shown that lower doses of the sphingolipid analogue C2-ceramide are required to induce apoptosis in IGF-IR-minus vs -positive murine fibroblasts, indicating a protective feedback loop in the latter and corroborating evidence that the IGF-IR functions as a survival receptor [1, 2]. Since, unexpectedly, C2-ceramide was capable of activating MAP kinase, phosphorylating the IGF-I receptor, and promoting entry into the G2 phase of the cell cycle, we wished to further determine the mechanisms involved. Using IGF-IR-positive fibroblasts we demonstrate here for the first time that ceramide is capable of activating a tyrosine kinase which acts at the level of the IGF-IR to increase cell death. We also demonstrate that in the presence of sodium orthovanadate, ceramide-induced death is increased, and the phosphorylation of a 75-kDa protein which associates with the IGF-I receptor is enhanced. Although the identity of this protein is not known, we speculate that it may link into the Raf kinase signaling pathway; indeed, inhibitors of MEKK reduce ceramide-induced apoptosis, thus substantiating this theory [1, 2]. Although calcium mobilization did cause apoptosis in these cells, it was not required as a mediator of ceramide-induced apoptosis. Finally, the potential hydrolysis of ceramide to sphingosine-1-phosphate was not the cause of increased MAP kinase activation, substantiating the role of an IGF-IR interacting tyrosine kinase, which may be involved in apoptosis.

Animals↗
PubMed1999

Increased, not decreased activation of the insulin-like growth factor (IGF) receptor signalling pathway during ceramide-induced apoptosis.

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro. The IGF-I receptor (IGF-IR) is believed to mediate protective effects of the IGFs against apoptosis. To determine whether ceramide-mediated induction of apoptosis involved a decreased survival effect of the IGF-IR, apoptosis was induced in IGF-I receptor positive (R+) and negative (R-) murine fibroblasts by incubation with increasing doses of the sphingolipid analogue, C2 ceramide. Lower ceramide doses were required to induce death in receptor negative compared with receptor positive fibroblasts (P< 0.05 at ceramide doses of 2 microM or greater), not only corroborating evidence that the IGF-I receptor functions as a survival receptor, but also suggesting that ceramide is not inducing apoptosis by suppressing a survival effect of the IGF-IR. Ceramide has been reported to induce death through suppression of MAP kinase, and activation of JUN kinase signalling; since our initial data suggested that ceramide had not affected an anti-apoptotic signalling event of the IGF-IR, we monitored the activation of these enzymes. To our surprise, in the presence of ceramide, not only was JUN kinase activity increased, but so too was MAP kinase. Inhibition of MAP kinase, using the MEKK inhibitor, PD98059, significantly reduced ceramide-induced cell death (P< 0. 001). Ceramide also enhanced IGF-induced tyrosine phosphorylation of the IGF-I receptor and activated PI-3 kinase. The cumulative effects of these events resulted in increased progression to the G2 phase of the cell cycle, arrest without subsequent mitosis, and apoptosis. These results indicate that ceramide is capable of eliciting apparently contradictory events within a single cell type, and suggest that in the presence of an IGF-IR, survival is enhanced because ceramide can activate PI-3 kinase, believed to be an anti-apoptotic enzyme.

Animals↗
PubMed1997

Artifact from an electrosurgical ground pad.

Artifacts from medical intervention are frequently present in medicolegal autopsies and must be distinguished from injuries of forensic interest, particularly in deaths from trauma. In this case, burns resulting from incomplete skin contact of an electrosurgical ground pad during surgery for multiple gunshot wounds were initially confused with abrasions caused by impact of bullet fragments. Inspection of the decedent's clothing and of the medical records revealed the true etiology of this injury.

Adult↗
PubMed1996

Insulin-like growth factor-II is an autocrine survival factor for differentiating myoblasts.

Recent studies indicate that insulin-like growth factor-II (IGF-II) acts as an autocrine differentiation factor for skeletal myoblasts in culture. IGF-II mRNA and protein are induced as early events in muscle differentiation, and the rate and extent of IGF-II secretion correlate with both biochemical and morphological differentiation. Here we show that IGF-II also functions as an essential survival factor during the transition from proliferating to differentiating myoblasts. Stably transfected C2 muscle cell lines were established in which a mouse IGF-II cDNA was expressed in the antisense orientation relative to the constitutively active Moloney sarcoma virus promoter. IGF-II antisense cells proliferated normally in growth medium containing 20% serum but underwent rapid death when placed in low serum differentiation medium. Death was accompanied by characteristic markers of apoptosis with more than 90% of cells showing DNA fragmentation within 12-16 h. Myoblast death was prevented by IGF-I, des [1-3] IGF-I, IGF-II, and insulin with a dose potency consistent with activation of the IGF-I receptor; death also could be blocked by the protein synthesis inhibitor, cycloheximide. Exogenous IGFs additionally stimulated passage through a single cell cycle and subsequently induced terminal differentiation. Cell survival and cell cycle progression also were enhanced by fibroblast growth factor-2 and platelet-derived growth factor-bb, but these peptides did not promote differentiation. Our results define a novel system for studying apoptotic cell death and its prevention by growth factors, underscore the importance of IGF action in minimizing inappropriate cell death, and indicate that shared signal transduction pathways may mediate myoblast survival in vitro.

Animals↗
PubMed1996

Overexpression of insulin-like growth factor-II induces accelerated myoblast differentiation.

Previous studies have shown that exogenous insulin-like growth factors (IGFs) can stimulate the terminal differentiation of skeletal myoblasts in culture and have established a correlation between the rate and the extent of IGF-II secretion by muscle cell lines and the rate of biochemical and morphological differentiation. To investigate the hypothesis that autocrine secretion of IGF-II plays a critical role in stimulating spontaneous myogenic differentiation in vitro, we have established C2 muscle cell lines that stably express a mouse IGF-II cDNA under control of the strong, constitutively active Moloney sarcoma virus promoter, enabling us to study directly the effects of IGF-II overproduction. Similar to observations with other muscle cell lines, IGF-II overexpressing myoblasts proliferated normally in growth medium containing 20% fetal serum, but they underwent enhanced differentiation compared with controls when incubated in low-serum differentiation medium. Accelerated differentiation of IGF-II overexpressing C2 cells was preceded by the rapid induction of myogenin mRNA and protein expression (within 1 h, compared with 24-48 h in controls) and was accompanied by an enhanced proportion of the retinoblastoma protein in an underphosphrylated and potentially active form, by a marked increase in activity of the muscle-specific enzyme, creatine phosphokinase, by extensive myotube formation by 48 h, and by elevated secretion of IGF binding protein-5 when compared with controls. These results confirm a role for IGF-II as an autocrine/paracrine differentiation factor for skeletal myoblasts, and they define a model cell system that will be useful in determining the biochemical mechanisms of IGF action in cellular differentiation.

Animals↗
PubMed1996

Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism.

The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process stimulated by IGFs. We stably transfected C2 cells with IGFBP-5 cDNAs under control of a constitutively active promoter. Compared with vector-transfected control cells, C2 myoblasts expressing the IGFBP-5 transgene in the sense orientation exhibit increased IGFBP-5 levels in the extracellular matrix during proliferation, and subsequently fail to differentiate normally, as assessed by both morphological and biochemical criteria. Compared to controls, IGFBP-5 sense myoblasts show enhanced survival in low serum medium, remaining viable for at least four weeks in culture. By contrast, myoblasts expressing the IGFBP-5 antisense transcript differentiate prematurely and more extensively than control cells. The inhibition of myogenic differentiation by high level expression of IGFBP-5 could be overcome by exogenous IGFs, with des (1-3) IGF-I, an analogue with decreased affinity for IGFBP-5 but normal affinity for the IGF-I receptor, showing the highest potency. These results are consistent with a model in which IGFBP-5 blocks IGF-stimulated myogenesis, and indicate that sequestration of IGFs in the extracellular matrix could be a possible mechanism of action. Our observations also suggest that IGFBP-5 normally inhibits muscle differentiation, and imply a role for IGFBP-5 in regulating IGF action during myogenic development in vivo.

Animals↗
PubMed1996

Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors.

The insulin-like growth factors (IGFs), IGF-I and IGF-II, comprise a conserved pair of secreted proteins with diverse effects on growth, development, and metabolism. Insulin-like growth factor action is initiated upon binding to cell-surface receptors and is modulated through interactions with secreted IGF binding proteins (IGFBPs). The last decade has seen an explosion of new information about the physiological roles of the IGFs. In this review, we critically examine this information from biochemical, cell biological, and molecular genetic perspectives. We discuss the structures and functions of the two IGF receptors, outline the actions of the six IGFBPs, and summarize and interpret recent studies highlighting essential roles for components of the IGF system in the growth and development of the embryo and fetus, in tissue differentiation, in cell survival and proliferation, and in cancer. These results are discussed in the context of new opportunities for understanding the mechanisms of IGF action in multiple biological processes.

Animals↗
PubMed1994

Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality.

Murine embryos that inherit a nonfunctional insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor (Igf2r) gene from their fathers are viable and develop normally into adults. However, the majority of mice inheriting the same mutated allele from their mothers die around birth, as a consequence of major cardiac abnormalities. These mice do not express IGF2R in their tissues, are 25-30% larger than their normal siblings, have elevated levels of circulating IGF2 and IGF-binding proteins, and exhibit a slight kink in their tails. These results show that Igf2r is paternally imprinted and reveal that the receptor is crucial for regulating normal fetal growth, circulating levels of IGF2, and heart development.

Alleles↗
PubMed1993

Potentiation of insulin-like growth factor-I (IGF-I) activity by an antibody: supportive evidence for enhancement of IGF-I bioavailability in vivo by IGF binding proteins.

The effects of ovine polyclonal antibodies raised against human recombinant IGF-I were investigated in GH-deficient rodents in vivo both in the presence and absence of exogenous IGF-I. Dwarf mice (negligible endogenous serum IGF-I) treated with anti-IGF-I serum which had been pre-incubated with IGF-I exhibited a significantly greater rate of daily weight gain than did mice treated with the same dose of IGF-I alone (P < 0.001) or even a 2.5-fold higher dose (P < 0.001). Similar increases in whole body weight gain were observed in dwarf rats, with a concomitant increase in dissected muscle weight. Serum IGF-I concentrations were greater in all animals treated with the complex of anti-IGF-I antibodies and IGF-I than in those administered only IGF-I. Size exclusion chromatography of dwarf rat serum indicated the presence of high mol wt material (> 160 kDa) capable of binding 125I-IGF-I in the anti-IGF-I treated rats. We suggest that this particular polyclonal antibody is behaving in a similar manner to an enhancing IGF binding protein, maintaining a reservoir of bioactive IGF-I. Since the antibody has a slightly lower affinity (2 x 10(8) liters/M) than that of the type 1 receptor, these data provide tentative indirect evidence to support the hypothesis that the recently discovered mechanisms which apparently decrease the affinities of several IGFBPs may indeed result in increased IGF-I bioavailability.

Animals↗
PubMed1985

Chemical skin burns.

Copious lavage with water is the cornerstone of treatment for most types of chemical burns. Particularly dangerous are burns from hydrofluoric acid, which may act as both an alkali and an acid in its effects on the tissues. There are specific antidotes for certain types of chemical burns. For phenol burns, polyethylene glycol solutions are better solvents than water.

Acids↗
PubMed1984

Temporal bone trauma: high-resolution computed tomographic evaluation.

High-resolution computed tomography (CT) was used to examine 12 patients with temporal bone trauma. The CT findings were correlated with the clinical signs and symptoms (hearing loss, facial nerve dysfunction) and with the operative findings in the 5 patients who underwent surgery. CT was able to demonstrate ossicular chain disruption or fractures causing the clinical findings in the majority of cases. The CT findings affected patient care by alerting the otologist to the abnormalities likely to be encountered during surgery.

Adult↗