Biomedical subjects

D Fulton

Publications and source records attributed to D Fulton.

At least 19 recordsLinked to original sources

PubMed2002

Baculovirus expression and antigenic characterization of the capsid proteins of three Norwalk-like viruses.

Human caliciviruses (HuCVs) are antigenically diverse. The antigenic relationships among different HuCVs have been difficult to study because HuCVs cannot be passaged in the laboratory. In this study, we describe cloning, sequencing and expression of the viral capsid proteins of three HuCVs that were identified in outbreaks of acute gastroenteritis in Virginia in 1997-1998. Yields of the capsid proteins similar to previously expressed recombinant Norwalk virus were obtained using the baculovirus expression system. Recombinant VA97207 capsid protein (rVA97207) and rVA98387, but not rVA98115, formed virus-like particles (VLPs). All three recombinant capsid antigens detected seroresponses in patients involved in outbreaks of acute gastroenteritis associated with genetically homologous or related HuCVs. The antigenic relationships of the three strains were further characterized using hyperimmune antisera against the three capsid antigens as well as four previously characterized recombinant capsid antigens of Norwalk (rNV), Mexico (rMxV), Hawaii (rHV), and Grimsby viruses (rGrV). VA98387 shared 98% aa identity with GrV; rVA98387 was detected by antisera to GrV. VA98115 shared 87% aa identity with Desert Storm virus and 65% aa identity with prototype Norwalk virus (NV); rVA98115 reacted weakly with NV antisera. VA97207 shared 80% aa identity with Amsterdam and 75% aa identity with Leeds strains and rVA97207 was not detected by any of the heterologous antibodies. In conclusion, VA97207 and VA98115 may belong to CV antigenic types not previously expressed, while VA98387 is a GrV-like virus. Low levels of cross-reactive antibodies were detected between types. Further studies to characterize these antigens and to develop enzyme immune assays (EIAs) for these strains are in progress.

Animals↗

PubMed2001

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells.

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.

Adenoviridae↗

PubMed2001

Acidic hydrolysis as a mechanism for the cleavage of the Glu(298)-->Asp variant of human endothelial nitric-oxide synthase.

The 894G-->T polymorphism within exon 7 of the human endothelial nitric-oxide synthase (eNOS) gene codes for glutamate or aspartate, respectively, at residue 298 and has been associated with several diseases of cardiovascular origin. A recent report indicates that Asp(298)-eNOS (E298D) is cleaved intracellularly to 100- and 35-kDa fragments, suggesting a mechanism for reduced endothelial function. Here we have documented the precise cleavage site of the E298D variant as a unique aspartyl-prolyl (Asp(298)--Pro(299)) bond not seen in wild-type eNOS (Glu(298)). We show that E298D-eNOS, as isolated from cells and in vitro, is susceptible to acidic hydrolysis, and the 100-kDa fragment can be generated ex vivo by increasing temperature at low pH. Importantly, cleavage of E298D was eliminated using a sample buffer system designed to limit acidic hydrolysis of Asp--Pro bonds. These results argue against intracellular processing of E298D-eNOS and suggest that previously described fragmentation of E298D could be a product of sample preparation. We also found that eNOS turnover, NO production, and the susceptibility to cellular stress were not different in cells expressing WT versus E298D-eNOS. Finally, enzyme activities were identical for the respective recombinant enzymes. Thus, intracellular cleavage mechanisms are unlikely to account for associations between the exon 7 polymorphism and cardiovascular diseases.

Amino Acid Substitution↗

PubMed2001

Intrathecal treatment of neoplastic meningitis due to breast cancer with a slow-release formulation of cytarabine.

DepoCyte is a slow-release formulation of cytarabine designed for intrathecal administration. The goal of this multi-centre cohort study was to determine the safety and efficacy of DepoCyte for the intrathecal treatment of neoplastic meningitis due to breast cancer. DepoCyte 50 mg was injected once every 2 weeks for one month of induction therapy; responding patients were treated with an additional 3 months of consolidation therapy. All patients had metastatic breast cancer and a positive CSF cytology or neurologic findings characteristic of neoplastic meningitis. The median number of DepoCyte doses was 3, and 85% of patients completed the planned 1 month induction. Median follow up is currently 19 months. The primary endpoint was response, defined as conversion of the CSF cytology from positive to negative at all sites known to be positive, and the absence of neurologic progression at the time the cytologic conversion was documented. The response rate among the 43 evaluable patients was 28% (CI 95%: 14-41%); the intent-to-treat response rate was 21% (CI 95%: 12-34%). Median time to neurologic progression was 49 days (range 1-515(+)); median survival was 88 days (range 1-515(+)), and 1 year survival is projected to be 19%. The major adverse events were headache and arachnoiditis. When drug-related, these were largely of low grade, transient and reversible. Headache occurred on 11% of cycles; 90% were grade 1 or 2. Arachnoiditis occurred on 19% of cycles; 88% were grade 1 or 2. DepoCyte demonstrated activity in neoplastic meningitis due to breast cancer that is comparable to results reported with conventional intrathecal agents. However, this activity was achieved with one fourth as many intrathecal injections as typically required in conventional therapy. The every 2 week dose schedule is a major advantage for both patients and physicians.

Adult↗

PubMed2001

Effect of radiation on cytokine and cytokine receptor messenger-RNA profiles in p53 wild and mutated human glioblastoma cell lines.

OBJECTIVE: Glioblastoma cells produce cytokines with proinflammatory or immunosuppressive properties, or both, which, in addition to altered p53 gene expression, have been shown to be associated with glioblastoma resistance to radiotherapy. The reported data concerning cytokines have been isolated and sometimes discordant, and a comprehensive profile analysis of cytokines and their corresponding receptors in irradiated glioblastomas has received limited attention. The object of this study was to test the hypothesis that radiation alone in clinically relevant doses would not significantly alter expression of endogenous cytokines and their receptors in human glioblastoma celll ines with wild-type and mutant p53. DESIGN AND METHOD: Culture specimens of 4 glioblastoma cell lines of different p53 gene expression (U87, U118, U251, U373) were irradiated with cobalt 60 at a dose of 10 Gy. After 48 hours, radiosensitivity was defined through a colony formation assay, cell cycle distribution was analyzed by flow cytometry, and cytokine and cytokine receptor messenger-RNA (mRNA) profiles were defined with an RNase protection assay. Different single doses of radiation at varying time intervals after culture were applied also to wild-type p53 cell lines. RESULTS: All cell lines were relatively radioresistant at lower doses of 1 and 2 Gy. Immunosuppressive cytokine and cytokine receptor mRNA of the Th2 (IL-13Ralpha, IL-4) and Th3 family (TGF-beta1, 2 and 3, TGF-betaRI and RII) were expressed. In contrast, only 2 proinflammatory Th1 cytokine receptor genes (IFN-gammaRa and IFN-gammaRbeta), but no significant Th1 cytokine gene expression, were detected. Even though the population examined included a large fraction of reproductively dead cells, cytokine and cytokine receptor mRNA profiles were not altered significantly by irradiation in all cell lines, regardless of the p53 status. CONCLUSION: These results suggest that cobalt irradiation alone at clinically relevant doses does not significantly alter the cytokine and cytokine receptor profiles in human glioblastoma cell lines.

Cobalt Radioisotopes↗

PubMed2001

Post-translational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough?

Endothelial nitric oxide synthase (eNOS) is important for cardiovascular homeostasis, vessel remodeling, and angiogenesis. Given the impact of endothelium- derived nitric oxide (NO) in vascular biology, much work in the past several years has focused on the control of NO synthesis by regulatory proteins that influence its function. Indeed calcium-activated calmodulin is important for regulation of NOS activity. Herein we discuss why other proteins, in addition to calmodulin, are necessary for eNOS regulation and summarize the biology of negative and positive regulators of eNOS function in vitro, in cells, and in blood vessels.

Animals↗

PubMed2000

Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells.

17beta-Estradiol (E(2)) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E(2)-stimulated endothelial NO release can occur without an increase in cytosolic Ca(2+). Here we demonstrate for the first time, to our knowledge, that E(2) rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E(2) treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182, 780. E(2) stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E(2)-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca(2+) levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E(2)-stimulated NO release. The membrane-impermeant E(2)BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca(2+) fluxes and describes an important pathway relevant to cardiovascular pathophysiology.

Adenoviridae↗

PubMed2000

Temporal events underlying arterial remodeling after chronic flow reduction in mice: correlation of structural changes with a deficit in basal nitric oxide synthesis.

To define the cellular events of vascular remodeling in mice, we measured blood flow and analyzed the morphology of remodeled vessels at defined points after a flow-reducing remodeling stimulus for 3, 7, 14, and 35 days. Acute ligation of the left external carotid artery reduced blood flow in the left common carotid artery (LC) compared with sham and contralateral right common carotid arteries (RCs). In morphometric analyses, the decrease in diameter in LCs was reversible by vasodilator perfusion 3 days after ligation, whereas ligation for 7 days or greater resulted in a permanent diameter reduction. Coincident with structural remodeling at day 7 was an increase in cell death in remodeled LCs. Functionally, rings from remodeled LCs contracted to prostaglandin F(2alpha) and relaxed to acetylcholine in a manner identical to that of control arteries. However, remodeled LCs were hypersensitive to the nitrovasodilator sodium nitroprusside (at day 7) and exhibited a marked reduction in basal NO synthesis at 7 and 14 days after ligation. The impairment of endothelial NO synthase function was likely due to post-translational mechanisms, given that endothelial NO synthase mRNA and protein levels did not change in remodeled LCs. These data define the ontogeny of flow-triggered luminal remodeling in adult mice and suggest that endothelial dysfunction occurs during reorganization of the vessel wall.

Animals↗

PubMed2000

Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt.

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.

Actins↗

PubMed2000

Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway.

A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.

Angiopoietin-1↗

PubMed2000

Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation.

Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) production constitutively, in the absence of agonist challenge. Here, we examine the potential mechanism of how aspartate at 1179 increases eNOS activity using purified proteins. Examination of NO production and cytochrome c reduction resulted in no substantial changes in the K(m)/EC(50) for L-arginine, calmodulin, and calcium, whereas there was a 2-fold increase in the rate of NO production for S1179D and a 2-4-fold increase in reductase activity (based on cytochrome c reduction). The observed increase in activity for both assays of NOS function indicates that a faster rate of electron flux through the reductase domain is likely the rate-limiting step in NO formation from eNOS. In addition, S1179D eNOS did show an increased resistance to inactivation by EGTA compared with wild type eNOS. These results suggest that a negative charge imposed at serine 1179, either by phosphorylation or by replacement with aspartate, increases eNOS catalytic activity by increasing electron flux at the reductase domain and by reducing calmodulin dissociation from activated eNOS when calcium levels are low.

Animals↗

PubMed2000

NCIC-CTG phase II study of gemcitabine in patients with malignant glioma (IND.94).

PURPOSE: We conducted a phase II multicentre study of gemcitabine in patients with anaplastic astrocytoma and glioblastoma multiforme at first relapse. PATIENTS AND METHODS: Patients with anaplastic astrocytoma or glioblastoma multiforme receiving a stable dose of steroids and ECOG performance status < or = 3 were eligible for this study at the time of first relapse. One adjuvant chemotherapy regimen was permissible. Patients received gemcitabine 1000 mg/m2 i.v. weekly x 3, repeated on a four-weekly cycle. RESULTS: Of 20 patients enrolled, 15 were evaluable for response, 19 for non-hematological toxicity and 18 for hematological toxicity. Seven patients had anaplastic astrocytoma (AA) and twelve glioblastoma multiforme (GBM). Age ranged from 28-71 years (median 50). Fifteen patients discontinued therapy due to disease progression. The median number of cycles administered was 1 (range 1-11); only two patients received more than three cycles. Hematologic toxicity was acceptable and no grade 4 toxicity was seen. One patient developed Pneumocystis pneumonia and eventual pulmonary embolism; one died of gastric hemorrhage related to steroid therapy. No objective responses were seen. Nine patients had stable disease (median duration 2.7 months, range 0.9-11.2). CONCLUSIONS: Gemcitabine given in this dose and schedule seems well tolerated but is not active in patients with recurrent high-grade gliomas.

Adult↗

PubMed2000

The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals.

Recent studies suggest that statins can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. We show here that statins rapidly activate the protein kinase Akt/PKB in endothelial cells. Accordingly, simvastatin enhanced phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS), inhibited apoptosis and accelerated vascular structure formation in vitro in an Akt-dependent manner. Similar to vascular endothelial growth factor (VEGF) treatment, both simvastatin administration and enhanced Akt signaling in the endothelium promoted angiogenesis in ischemic limbs of normocholesterolemic rabbits. Therefore, activation of Akt represents a mechanism that can account for some of the beneficial side effects of statins, including the promotion of new blood vessel growth.

Animals↗

PubMed2000

A phase I trial of 1,3-bis(2-chloroethyl)-1-nitrosourea plus temozolomide: a North American Brain Tumor Consortium study.

The North American Brain Tumor Consortium conducted a phase I trial of the combination 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide. Eligibility included a patient with a cancer type that was considered refractory to standard therapy. Prior nitrosourea treatments were not permitted. There were parallel dose escalations in two treatment schedules. Forty-five patients were enrolled during an 18-month period. The maximum tolerated doses (MTDs) when temozolomide followed BCNU (Arm A) were temozolomide at 550 mg/m2/p.o. and BCNU at 150 mg/m2/i.v.), whereas the MTD when temozolomide preceded BCNU (Arm B) was temozolomide at 400 mg/m2/p.o. and BCNU at 100 mg/m2/i.v. Toxicity was predominantly hematologic, although there were three instances of pulmonary toxicity, which in one case could have represented potentiation of nitrosourea-induced pulmonary fibrosis. The half-life of temozolomide was 1.86 (+/-0.31) h. There was a moderate relationship between dose and peak concentration and a strong relationship between dose and plasma concentration time curve. Pharmacokinetic parameters of temozolomide were unaffected by the treatment schedule, so the difference in MTD between the schedules is likely due to a biologic rather than a pharmacokinetic sequence interaction. There were 9 partial responses among 43 patients evaluable for response, including 5 of 25 with a histologic diagnosis of glioblastoma. The recommended dose and schedule for phase II trials of this regimen are BCNU 150 mg/m2/i.v. followed in 2 h by temozolomide 550 mg/m2/p.o. repeated every 6 weeks. We are also recommending screening and periodic pulmonary function testing during treatment to assess the possible potentiation of nitrosourea-induced pulmonary fibrosis.

Antineoplastic Agents, Alkylating↗

PubMed2000

Optic nerve sheath fenestration for a reversible optic neuropathy in radiation oncology.

To the authors' knowledge, there is a paucity of published accounts of management of radiation-induced optic neuropathy (RION) by optic nerve sheath fenestration (ONSF) in the conventional medical literature. With higher doses of radiation being given by using conformal techniques, more radiation-induced optic neuritis and neuropathy will be identified. We report here the successful use of ONSF to restore vision to three consecutive patients with pending anterior RION, and the importance of early identification and intervention in these potentially reversible cases.

Adult↗

PubMed2000

Quantification of eNOS mRNA in the canine cardiac vasculature by competitive PCR.

The goal of the present study was to develop a competitive PCR assay to measure changes in the expression of endothelial nitric oxide synthase (eNOS) mRNA levels throughout the canine vascular tree. A partial sequence of canine eNOS cDNA (1.86 kb), inducible NOS (1.95 kb), and neuronal NOS (1.16 kb) was cultured from canine aortic endothelial cells, LPS-treated canine splenic vein endothelial cells, and from canine left ventricle, respectively. Competitor eNOS cDNA (eNOS-C) was constructed via recombinant PCR. Thus, with the use of a standard curve competitive PCR with eNOS-C, the amount of eNOS mRNA in 500 ng of total RNA was greatest in the circumflex > right coronary artery > left anterior descending coronary artery > aorta. The isolation of coronary microvessels from the left ventricle was associated with an enrichment of endothelial cell markers such as eNOS, von Willebrand factor, and caveolin-1, an observation supported by the detection of up to 15-fold higher levels of eNOS mRNA in coronary microvessels relative to the larger arteries. The ability to quantify changes in eNOS mRNA levels throughout the canine vasculature should provide greater insight into the molecular mechanisms of how this gene is regulated in physiological and pathophysiological states.

Animals↗

PubMed2000

Acute modulation of endothelial Akt/PKB activity alters nitric oxide-dependent vasomotor activity in vivo.

The serine/threonine protein kinase Akt (protein kinase B) phosphorylates endothelial cell nitric oxide synthase (eNOS) and enhances its ability to generate nitric oxide (NO). Because NO is an important regulator of vasomotor tone, we investigated whether Akt can regulate endothelium-dependent vasomotion in vivo using a rabbit femoral artery model of gene transfer. The endothelium of isolated femoral arteries was infected with replication-defective adenoviral constructs expressing beta-galactosidase, constitutively-active Akt (myr-Akt), or dominant-negative Akt (dn-Akt). Femoral arteries transduced with myr-Akt showed a significant increase in resting diameter and blood flow, as assessed by angiography and Doppler flow measurements, respectively. L-NAME, an eNOS inhibitor, blocked myr-Akt-mediated vasodilatation. In contrast, endothelium-dependent vasodilatation in response to acetylcholine was attenuated in vessels transduced with dn-Akt, although these vessels showed normal responses to nitroglycerin, an endothelium-independent vasodilator. Similarly, relaxation of murine aorta ex vivo in response to acetylcholine, but not nitroglycerin, was inhibited by transduction of dn-Akt to the endothelium. These data provide evidence that Akt functions as key regulator of vasomotor tone in vivo.

Acetylcholine↗

PubMed1999

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation, but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

Animals↗