Math-Towers: Online Interactive Collaborative Mathematics Explorations & Problem-Solving for Students Ages 11 to 15 years (Grades 6 to 10)

Math-Towers (www.math-towers.ca) is an online resource for students in grades 6 to 10 that supports collaborative problem-solving and investigations. This paper presents the philosophical position motivating the development of Math-Towers and describes how the site presents and motivates the mathematical challenges and supports participants' exploration and collaboration.

Trauma and Family Therapy: Reflections on September 11 from Northern Ireland

This article draws on the author's experience of living and working in Belfast, Northern Ireland. Connections between the traumatic events of September 11 and the situation in the north of Ireland of ongoing civil conflict and violence are developed and aspects of therapeutic practice are described. Coping with the effects of trauma presents systemic therapists with multiple and complex challenges in whatever sociopolitical context they practice. A therapist stance that combines flexibility and openness of attitude with the creative use of therapeutic practices, including those from other modalities, will assist systemic therapists in rising to these challenges.

The Role of Thymidylate Synthase Induction in Modulating p53-regulated Gene Expression in Response to 5-Fluorouracil and Antifolates

Thymidylate synthase (TS) is a critical target for chemotherapeutic agents such as 5-fluorouracil (5-FU) and antifolates such as tomudex (TDX),multitargeted antifolate, and ZD9331. Using the MCF-7 breast cancer line, we have developed p53 wild-type (M7TS90) and null (M7TS90-E6) isogenic lines with inducible TS expression (approximately 6-fold induction compared with control after 48 h). In the M7TS90 line, inducible TS expression resulted in a moderate approximately 3-fold increase in 5-FU IC-50(72 h) dose and a dramatic >20-fold increase in the IC-50(72 h) doses of TDX, multitargeted antifolate, and ZD9331. S-phase cell cycle arrest and apoptosis induced by the antifolates were abrogated by TS induction. In contrast, cell cycle arrest and apoptosis induced by 5-FU was unaffected by TS expression levels. Inactivation of p53 significantly increased resistance to 5-FU and the antifolates with IC-50(72 h) doses for 5-FU and TDX of >100 and >10 microM, respectively, in the M7TS90-E6 cell line. Furthermore, p53 inactivation completely abrogated the cell cycle arrest and apoptosis induced by 5-FU. The antifolates induced S-phase arrest in the p53 null cell line; however, the induction of apoptosis by these agents was significantly reduced compared with p53 wild-type cells. Both inducible TS expression and the addition of exogenous thymidine (10 microM) blocked p53 and p21 induction by the antifolates but not by 5-FU in the M7TS90 cell line. Similarly, inducible TS expression and exogenous thymidine abrogated antifolate but not 5-FU-mediated up-regulation of Fas/CD95 in M7TS90 cells. Our results indicate that in M7TS90 cells, inducible TS expression modulates p53 and p53 target gene expression in response to TS-targeted antifolate therapies but not to 5-FU.

Functional comparison of the endothelial nitric oxide synthase Glu298Asp polymorphic variants in human endothelial cells

The G894T endothelial nitric oxide synthase (eNOS) polymorphism results in a Glu to Asp substitution at position 298. This position is located externally on the protein and as the regulation of eNOS is dependent on its subcellular localization and interaction with modulatory proteins, we aimed to address whether the substitution of Asp at 298 had any effect on these mechanisms. Initially, we developed a novel method to accurately determine molar quantities of each variant by expressing them as green fluorescent protein (GFP) fusion proteins and using recombinant adenoviruses to facilitate transient infection of human microvascular endothelial cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting of eNOS298Asp revealed a 135-kDa proteolytic fragment which was not present with eNOS298Glu. This proteolysis was prevented by using LDS buffer confirming that this differential cleavage is an artefact of sample preparation and unlikely to occur intracellularly. Nitric oxide was measured following stimulation with calcium ionophore or oestrogen in the presence of varying sepiapterin concentrations. GFP fluorescence was used to quantify the amount of fusion protein and calculate intracellular specific activity. There was no significant difference in intracellular specific activity between Glu298 and Asp298 eNOS in response to calcium ionophore or oestrogen. Tetrahydrobiopterin supplementation increased eNOS activity of both variants in an identical manner. The presence of the GFP also facilitated the visualization of the variants by confocal microscopy and demonstrated that both localized to the plasma membrane and the Golgi. These findings demonstrate that the Asp substitution at 298 does not have a major effect in modulating eNOS activity in vivo.

Truncated estrogen receptor alpha 46-kDa isoform in human endothelial cells: relationship to acute activation of nitric oxide synthase.

Background Estrogen acutely activates endothelial nitric oxide synthase (eNOS). However, the identity of the receptors involved in this rapid response remains unclear. Methods and Results We detected an estrogen receptor (ER) transcript in human endothelial cells that encodes a truncated 46-kDa ER (1a-hER-46). A corresponding 46-kDa ER protein was identified in endothelial cell lysates. Transfection of cDNAs encoding the full-length ER (ER-66) and 1a-hER-46 resulted in appropriately sized recombinant proteins identified by anti-ER antibodies. Confocal microscopy revealed that a proportion of both ER-66 and hER-46 was localized outside the nucleus and mediated specific cell-surface binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding studies. Both ER isoforms colocalized with eNOS and mediated acute activation of eNOS in response to estrogen stimulation. However, estrogen-stimulated transcriptional activation mediated by 1a-hER-46 was much less than with ER-66. Furthermore, 1a-hER-46 inhibited classical hER-66 mediated transcriptional activation in a dominant-negative fashion. Conclusions These findings suggest that expression of an alternatively spliced, truncated ER isoform in human endothelial cells confers a unique ability to mediate acute but not transcriptional responses to estrogen.

GTP Cyclohydrolase I Gene Transfer Augments Intracellular Tetrahydrobiopterin in Human Endothelial Cells: Effects on Nitric Oxide Synthase Activity and Dimerisation.

Objectives: Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS) activity. BH4 levels are regulated by de novo biosynthesis; the rate-limiting enzyme is GTP cyclohydrolase I (GTPCH). BH4 activates and promotes homodimerisation of purified eNOS protein, but the intracellular mechanisms underlying BH4-mediated eNOS regulation in endothelial cells remain less clear. We aimed to investigate the role of BH4 levels in intracellular eNOS regulation, by targeting the BH4 synthetic pathway as a novel strategy to modulate intracellular BH4 levels. Methods: We constructed a recombinant adenovirus, AdGCH, encoding human GTPCH. We infected human endothelial cells with AdGCH, investigated the changes in intracellular biopterin levels, and determined the effects on eNOS enzymatic activity, protein levels and dimerisation. Results: GTPCH gene transfer in EAhy926 endothelial cells increased BH4 >10-fold compared with controls (cells alone or control adenovirus infection), and greatly enhanced NO production in a dose-dependent, eNOS-specific manner. We found that eNOS was principally monomeric in control cells, whereas GTPCH gene transfer resulted in a striking increase in eNOS homodimerisation. Furthermore, the total amounts of both native eNOS protein and a recombinant eNOS–GFP fusion protein were significantly increased following GTPCH gene transfer. Conclusions: These findings suggest that GTPCH gene transfer is a valid approach to increase BH4 levels in human endothelial cells, and provide new evidence for the relative importance of different mechanisms underlying BH4-mediated eNOS regulation in intact human endothelial cells. Additionally, these observations suggest that GTPCH may be a rational target to augment endothelial BH4 and normalise eNOS activity in endothelial dysfunction states.

Relationship between G894T polymorphism (Glu 298 Asp variant) in endothelial nitric oxide synthase and nitric oxide mediated endothelial function in human atherosclerosis.

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), plays important roles in normal vascular homeostasis, and reduced endothelial NO bioactivity is an important feature of vascular disease states. The Glu298Asp (G894T) polymorphic variant of eNOS has been associated with vascular disease, but functional data are lacking. Accordingly, we examined the relationships between NO-mediated endothelial function, the presence of the eNOS Glu298Asp variant, and clinical risk factors for atherosclerosis. Endothelium-dependent vasorelaxations to different agonists were determined in human saphenous veins obtained from patients with coronary artery disease and identified risk factors (n = 104). Patients were genotyped for the eNOS G894T polymorphism. Nitric oxide-mediated endothelial vasorelaxations were highly variable between patients. Reduced vasorelaxations were associated with increased number of clinical risk factors for atherosclerosis (r = - 0.54, P

Performance analysis of an improved power-saving medium access protocol for IEEE 802.11 point coordination function WLAN

Wireless enabled portable devices must operate with the highest possible energy efficiency while still maintaining a minimum level and quality of service to meet the user's expectations. The authors analyse the performance of a new pointer-based medium access control protocol that was designed to significantly improve the energy efficiency of user terminals in wireless local area networks. The new protocol, pointer controlled slot allocation and resynchronisation protocol (PCSAR), is based on the existing IEEE 802.11 point coordination function (PCF) standard. PCSAR reduces energy consumption by removing the need for power saving stations to remain awake and listen to the channel. Using OPNET, simulations were performed under symmetric channel loading conditions to compare the performance of PCSAR with the infrastructure power saving mode of IEEE 802.11, PCF-PS. The simulation results demonstrate a significant improvement in energy efficiency without significant reduction in performance when using PCSAR. For a wireless network consisting of an access point and 8 stations in power saving mode, the energy saving was up to 31% while using PCSAR instead of PCF-PS, depending upon frame error rate and load. The results also show that PCSAR offers significantly reduced uplink access delay over PCF-PS while modestly improving uplink throughput.

Nitric Oxide Produced in Response to Engagement of beta 2 Integrins on Human Neutrophils Activates the Monomeric GTPases Rap1 and Rap2 and Promotes Adhesion

We found that engagement of beta 2 integrins on human neutrophils increased the levels of GTP-bound Rap1 and Rap2. Also, the activation of Rap1 was blocked by PP1, SU6656, LY294002, GF109203X, or BAPTA-AM, which indicates that the downstream signaling events in Rap1 activation involve Src tyrosine kinases, phosphoinositide 3-kinase, protein kinase C, and release of calcium. Surprisingly, the integrin-induced activation of Rap2 was not regulated by any of the signaling pathways mentioned above. However, we identified nitric oxide as the signaling molecule involved in beta 2 integrin-induced activation of Rap1 and Rap2. This was illustrated by the fact that engagement of beta 2 integrins increased the production of nitrite, a stable end-product of nitric oxide. Furthermore, pretreatment of neutrophils with N-monomethyl-L-arginine, or 1400W, which are inhibitors of inducible nitric-oxide synthase, blocked integrin-induced activation of Rap1 and Rap2. Similarly, Rp-8pCPT-cGMPS, an inhibitor of cGMP-dependent serine/threonine kinases, also blunted the integrin-induced activation of Rap GTPases. Also nitric oxide production and its downstream activation of cGMP-dependent serine/threonine kinases were essential for proper neutrophil adhesion by beta 2 integrins. Thus, we made the novel findings that beta 2 integrin engagement on human neutrophils triggers production of nitric oxide and its downstream signaling is essential for activation of Rap GTPases and neutrophil adhesion.