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W McDonald

Publications and source records attributed to W McDonald.

At least 19 recordsLinked to original sources
PubMed2001

Dynamics of C6 astrocytoma invasion into three-dimensional collagen gels.

OBJECTIVE: The dynamic mechanisms underlying the three-dimensional invasive paradigm of C6 astrocytoma cells has been assessed. METHODS: Spheroids of C6 astrocytoma cells were implanted into three-dimensional collagen type I gels (vitrogen 100) and individual C6 astrocytoma cell invasion monitored. Time-lapsed videomicroscopy was used to assess the dynamic components of cell invasion in three dimensions while scanning and transmission electron microscopy were used to assess matrix architecture and the static aspects of cell invasion. RESULTS: Videomicroscopy outlined an invasion paradigm continuum with repeating phases. A cell surface ruffling phase was followed by invadopodia extension and pull up phases. For some cells the collagen type I matrix extracellular matrix appeared to modify the ability of C6 cells to carry out their invasion paradigm. CONCLUSIONS: C6 astrocytoma cells invading a three-dimensional collagen type I matrix utilize a invasion paradigm made up of a number of phases. A better understanding of the dynamic invasion paradigms of malignant glial cells may be useful in the development of effective treatment strategies to prevent or modify malignant glioma invasion.

Animals↗
PubMed2001

The A20R protein is a stoichiometric component of the processive form of vaccinia virus DNA polymerase.

In vitro analysis of the catalytic DNA polymerase encoded by vaccinia virus has demonstrated that it is innately distributive, catalyzing the addition of <10 nucleotides per primer-template binding event in the presence of 8 mM MgCl(2) or 40 mM NaCl (W. F. McDonald and P. Traktman, J. Biol. Chem. 269:31190-31197, 1994). In contrast, cytoplasmic extracts isolated from vaccinia virus-infected cells contain a highly processive form of DNA polymerase, able to catalyze the replication of a 7-kb template per binding event under similar conditions. To study this holoenzyme, we were interested in purifying and characterizing the vaccinia virus processivity factor (VPF). Our previous studies indicated that VPF is expressed early after infection and has a native molecular mass of approximately 48 kDa (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). Using these criteria, we established a six-step chromatographic purification procedure, in which a prominent approximately 45-kDa band was found to copurify with processive polymerase activity. This species was identified as the product of the A20 gene. By use of recombinant viruses that direct the overexpression of A20 and/or the DNA polymerase, we verified the physical interaction between the two proteins in coimmunoprecipitation experiments. We also demonstrated that simultaneous overexpression of A20 and the DNA polymerase leads to a specific and robust increase in levels of processive polymerase activity. Taken together, we conclude that the A20 gene encodes a component of the processive DNA polymerase complex. Genetic data that further support this conclusion are presented in the accompanying report, which documents that temperature-sensitive mutants with lesions in the A20 gene have a DNA(-) phenotype that correlates with a deficit in processive polymerase activity (A. Punjabi et al, J. Virol. 75:12308-12318, 2001).

Amino Acid Sequence↗
PubMed2000

Prenatal and breast milk morphine exposure following maternal intrathecal morphine treatment.

A case of an infant who had morphine exposure during and following pregnancy while her mother was treated with intrathecal (i.t.) morphine is presented along with a review of the relevant literature. Successful maternal pain management was achieved during pregnancy and while breastfeeding. Minimal maternal serum and breast milk levels in the first 7 postpartum weeks were found. Also, infant sleep and arousal behavior and general development at 2 and 7 months were unremarkable, illustrating the possible safe and efficacious use of i.t. morphine during and following pregnancy. Further work is required to understand opioid pharmacology during breastfeeding, which will be used to develop an empirical approach to breastfeeding and morphine use.

Adult↗
PubMed2000

The role of coagulation abnormalities in the development of Perthes' disease.

Recent reports have suggested an association between Perthes' disease and an underlying thrombophilic or hypofibrinolytic tendency. In Northern Ireland there is a high incidence of Perthes' disease (11.7 per 100,000 or 1 in 607 children) in a stable paediatric population. We reviewed 139 children with Perthes' disease and compared them with a control group of 220 aged- and gender-matched healthy primary schoolchildren with similar racial and ethnic backgrounds. There were no significant deficiencies of antithrombotic factors protein C, protein S, antithrombin III or resistance to activated protein C. A total of 53 (38.1%) of the children with Perthes' disease had a prolonged activated partial thromboplastin time (>38) compared with 13 (5.9%) of the control group (p < 0.001). Our findings have shown that using standard assays, thrombophilia secondary to antithrombotic factor deficiency or resistance to activated protein does not appear to be an aetiological factor for Perthes' disease. The cause of the prolonged activated partial thromboplastin time, usually associated with a clotting factor deficiency, is under further investigation.

Antithrombin III↗
PubMed1998

The importance of cell density in the interpretation of growth factor effects on collagenase IV activity release and extracellular matrix production from C6 astrocytoma cells.

We have examined the influence of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) on the release of collagenase type IV activity and the production of extracellular matrix (ECM) molecules using C6 astrocytoma cells in monolayer culture. Collagenase type IV activity was significantly increased in a dose dependent manner in the low cell density group by treatment with FGF-2 and VEGF but significantly decreased in a dose dependent fashion in the high cell density group. These results were corroborated using Western blot technique with an antibody to gelatinase A. Addition of exogenous laminin and fibronectin to the media decreased collagenase type IV activity in a dose dependent fashion with the minimum concentration of 0.1 microgram/ml. Laminin and fibronectin reached a concentration of 0.1 microgram/ml in only the high cell density group after treatment with the growth factors tested. These findings indicate that C6 astrocytoma cells appear to have two regulatory mechanisms for collagenase type IV activity which are dependent on cell density. In a low cell density, C6 astrocytoma cells respond to the dominant effect of FGF-2 and VEGF by increasing the release of collagenase IV activity. In a high cell density collagenase type IV activity is decreased due to it's down regulation by released ECM molecules in response to FGF-2 and VEGF. These regulatory mechanisms may be crucial to the understanding of the coordination of tumor-associated angiogenesis by malignant glial cells.

Analysis of Variance↗
PubMed1997

Low-dose oral type I interferons reduce early virus replication of murine cytomegalovirus in vivo.

Immunity to viral infections involves both innate and antigen-specific immune responses. The antiviral properties of interferons (IFNs) are part of the innate immune response. Low doses of type I IFNs (IFN-alpha and IFN-beta) administered daily (10 IU per mouse) by the oral route significantly reduced the early replication of murine cytomegalovirus (MCMV) in both the spleen and liver of MCMV-infected susceptible BALB/c mice. Significant inhibition of virus replication was observed for two different inoculum doses of virus (2 x 10(4) pfu per mouse [0.6 LD50] and 2 x 10(4.12) pfu per mouse [0.8 LD50]). Analysis of IFN retention, using [35S]-labeled IFN-alpha1 compared with the nonreceptor binding mutant IFN-alpha1 (R33M) administered orally to mice, revealed binding of wild-type IFN-alpha1 to several tissues. In particular, IFN was retained by tissues proximal to lymphoid regions, including the posterior nasal cavity, posterior tongue, small intestine, and rectum. These findings suggest that type I IFNs may inhibit MCMV replication by distal binding of the orally administered IFN to various tissues, which in turn augment the primary immune response to virus infection.

Administration, Oral↗
PubMed1997

Implantation of C6 astrocytoma spheroid into collagen type I gels: invasive, proliferative, and enzymatic characterizations.

A three-dimensional model has been developed in which C6 astrocytoma spheroids of defined sizes are embedded into collagen type I gels. The authors have monitored cell invasive behavior; obtained quantitative data on cell invasion, proliferation, and enzymatic activity; assessed cell-cell interactions by altering the spheroid size used; and studied cell-matrix interactions by modifying the matrix components. Their results show that C6 astrocytoma cells detach from the spheroid surface and invade the gel as single cells by means of a system that appears to be dependent on metalloprotease function. These invasive cells have a low proliferative index. Larger spheroids with central hypoxic microregions possess cells that invade the gel at faster rates; this could be correlated with the release of increased collagen type I degrading activity. Extracellular matrix proteins, such as laminin, fibronectin, and collagen type IV have no significant influence on invasive activity, whereas hyaluronic acid decreases and human central nervous system myelin increases invasion. New strategies directed at the treatment of malignant gliomas must take into account the subpopulation of malignant cells located long distances from the major tumor mass. The spheroid invasion model may provide specific insights into the behavior of these invasive cells.

Animals↗
PubMed1996

Release of collagen type IV degrading activity from C6 astrocytoma cells and cell density.

Type IV collagen is a major protein component of the vascular basement membrane and its degradation is crucial to the initiation of tumor-associated angiogenesis. The authors have investigated the influence of cell density on the release of collagen type IV degrading activity by C6 astrocytoma cells in monolayer culture. The release of collagen type IV degrading activity was assessed biochemically, immunocytochemically, and by Western blot analysis. The results demonstrate that increasing plating density and increasing cell density are associated with decreased collagen type IV degrading activity released per tumor cell. These findings indicate the existence of regulatory mechanisms dependent on cell-cell communication, which modulate release of collagen type IV degrading activity. The extrapolation of these results to the in vivo tumor microenvironment would suggest that individual and/or small groups of invading tumor cells, distant from the main tumor mass, would release substantial collagen type IV degrading activity, which may be crucial to their continued invasion and to angiogenesis.

Animals↗
PubMed1995

An extracellular proteasome-like structure from C6 astrocytoma cells with serine collagenase IV activity and metallo-dependent activity on alpha-casein and beta-insulin.

An extracellular proteasome-like (EP) structure has been isolated from serum-free media conditioned by C6 astrocytoma cells. EP has a native molecular mass of 1000 kDa and is composed of three subunits, two isoelectric variants at 70 kDa and one at 65 kDa. The extracellular proteasome degraded collagen IV, alpha-casein, beta-insulin, and certain synthetic peptide substrates. A 68-kDa type IV collagenase, identified as the activated form of gelatinase A, was also isolated from this medium. The type IV collagenase activity of the proteasome was sensitive to serine protease inhibitors, while the 68-kDa collagenase IV represented the matrix metalloprotease gelatinase A. The general protease activity of the proteasome was sensitive to metalloprotease inhibitors. Western blot analysis indicates a sequence relationship between the 68-kDa type IV collagenase and either one or both of the 70-kDa isoelectric variants of the proteasome; however, the two enzymes appear to be distinct functionally. Comparison with known proteasomes indicates that EP represents a novel proteasome. The complexity of degradative enzymes in the extracellular microenvironment implies that complete inhibition of tumor growth requires at least a combination of serine and metalloprotease inhibitors.

Amino Acid Sequence↗
PubMed1995

Degradation of collagen type IV by C6 astrocytoma cells.

The key event associated with the initiation of angiogenesis is the localized degradation of the vascular basement membrane. Because of its complex structure, any remodelling and/or modification of the basement membrane must involve the co-ordinated function of a number of different enzyme systems. Type IV collagen is a major protein component (60-90%) of the basement membrane and its degradation is crucial to the initiation of angiogenesis. This study has focused on the mechanisms by which C6 astrocytoma cells degrade human type IV collagen. C6 astrocytoma cells use components of two major degradative pathways to degrade collagen type IV. The major matrix metalloproteinase identified is the activated form (68-KDa) of gelatinase A (72-KDa matrix metalloproteinase) and a serine sensitive 1000-KDa collagenase type IV degrading activity which appears to have the characteristics of a novel extracellular proteasome.

Astrocytoma↗
PubMed1992

Serum proteolytic activity during the growth of C6 astrocytoma.

Tumor growth is dependent on the ability of neoplastic cells to induce angiogenesis. Blood-vessel remodeling requires the reconstruction of the nonfibrous proteins and type IV collagen components of the basement membrane. This study has assessed the influence of the growth of C6 astrocytoma cells in the rat spheroid implantation model on serum general protease and type IV collagenase activity. The results demonstrate that general protease activity increased in serum, reaching maximum values on Day 6 and Day 13 following spheroid implantation, and that type IV collagenase activity increased in serum, obtaining maximum values on Day 8 and Day 15. The measurement of serum proteolytic activity may be of value in the detection of recurrent tumors.

Animals↗
PubMed1992

Proteolytic activity during the growth of C6 astrocytoma in the murine spheroid implantation model.

General protease and collagenase IV activity are involved in the remodelling of the vascular basement membrane that occurs during tumor-induced angiogenesis. This study has assessed the level of these enzymes in tumor, peritumoral or contralateral cerebral cortex tissue during the growth of C6 astrocytoma in the rat spheroid implantation model. General proteolytic activity was increased in tumor tissue beginning on day 8 following spheroid implantation, then increased to a maximum value on day 11 and decreased to control values on day 18. A similar pattern was seen for collagenase IV activity but maximal activity occurred on day 13. The peritumor and tumor patterns of activity were similar. General protease activity was increased in the hemisphere contralateral to the tumor suggesting that the growth of C6 astrocytoma in rat brain was influencing biochemical events distant from the tumor. C6 astrocytoma cells orchestrate a cascade of proteolytic events which may play a crucial role in angiogenesis associated with tumor growth in the model system studied.

Animals↗
PubMed1991

General protease and collagenase (IV) activity in C6 astrocytoma cells, C6 spheroids and implanted C6 spheroids.

Tumor growth is dependent on the ability of neoplastic cells to induce angiogenesis. Remodelling of blood vessels requires reconstruction of the collagen (type IV) and non-fibrous protein components of basement membrane. This study assessed the general protease and collagenase (IV) activities of C6 astrocytoma cells in monolayer and spheroid culture and C6 astrocytoma spheroids growing in vivo. Extracellular release of non-specific proteases and collagenase IV was maximal during early exponential cell growth. Increased spheroid size resulted in enhanced extracellular activity of both enzyme groups assessed. The size of the implanted spheroid influenced the activity measured in vivo. General proteolytic activity was significantly greater in tumor tissue at all spheroid sizes while only the implantation of 750 microns spheroids resulted in significantly increased collagenase type IV activity. The growth of C6 astrocytoma cells in monolayer and spheroid culture in vitro and in vivo is associated with distinct alterations in intracellular and extracellular activity of the proteolytic enzymes assessed. Increased extracellular release of these enzymes may play important roles in tumor-associated angiogenesis, tumor invasiveness, tumor induced hemorrhage and tumor-associated edema.

Animals↗
PubMed1991

Effect of an inhibitor of lipid peroxidation, U78517F, on C6 astrocytoma growth.

A potent in vitro inhibitor of lipid peroxidation, U78517F, has been employed to assess its influence on C6 astrocytoma growth in monolayer culture and tumor growth and protein extravasation in the rat C6 astrocytoma spheroid implantation model. Results demonstrate that 1 microM concentration of U78517F inhibits cell division, but is not tumoricidal to C6 astrocytoma cells in monolayer culture. Concentrations of 5 microM and above significantly decreased astrocytoma cell viability. Following spheroid implantation, rats were treated with one of these U78517F regimes: 12 mg/kg/day for 13 days post-implantation, 4 mg/kg/day for 13 days post-implantation or 12 mg/kg/day commencing seven days post-implantation until 13 days post-implantation. Tumor wet and dry weights were lower in all treatment groups, but these decreases were not statistically significant. Protein extravasation as measured by Evans blue extravasation was not significantly reduced by any treatment regime used. It is concluded that U78517F inhibits C6 astrocytoma growth in monolayer culture at and above 5 microM concentrations, but the treatment regimes utilized did not significantly decrease tumor growth or permeability in the C6 astrocytoma spheroid implantation model.

Animals↗
PubMed1989

Subcellular localization of superoxide dismutases, glutathione peroxidase and catalase in developing rat cerebral cortex.

The levels of copper- and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activity have been assessed in a nuclear fraction (NF), a mitochondrial fraction (MF) and a non-mitochondrial, non-nuclear fraction (NMNNF) isolated from developing rat cerebral cortex. The NF showed increasing CuZn and MnSOD activities and static, low activities of GSH-Px and CAT during development. The MF had increased levels of MnSOD and GSH-Px activities and a rapid decrease in CAT activity associated with development. Histochemical methods have localized greater CAT activity in mitochondria isolated from 2-day-old rat brain when compared to 77-day-old animals. Development was associated with increasing CuZnSOD activity, a decrease in CAT activity and, after an initial fall at 19 days, increasing GSH-Px activity in the NMNNF. Measurable activity of MnSOD were found in the NMNNF and appeared to be static during the time period assessed. A distinct ontogenetic pattern of oxidative enzyme activities and subcellular locations is associated with development in rat cerebral cortex.

Age Factors↗
PubMed1987

Distribution of superoxide dismutase, glutathione peroxidase and catalase in developing rat brain.

This study was carried out to assess the developmental pattern of copper- and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), glutathione peroxidase (GSH-Px) and catalase (CAT) activity in rat brain. The enzymes studied were assayed in different brain regions (cerebral cortex, striatum, cerebellum and brainstem) and enzyme values were corrected for erythrocyte contamination. The cerebral ontogenetic pattern of these enzymes is characterized by increasing CuZnSOD activity, a progressive decrease in CAT activity and, after an initial 10-day fall, increasing GSH-Px activity. The activity of MnSOD appeared to be quite stable up to 40 weeks of age. Similar and comparable changes were seen in all brain regions studied.

Animals↗