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Biomedical subjects

L Stephens

Publications and source records attributed to L Stephens.

At least 19 recordsLinked to original sources
PubMed2001

The crystal structure of the PX domain from p40(phox) bound to phosphatidylinositol 3-phosphate.

More than 50 human proteins with a wide range of functions have a 120 residue phosphoinositide binding module known as the PX domain. The 1.7 A X-ray crystal structure of the PX domain from the p40(phox) subunit of NADPH oxidase bound to PtdIns(3)P shows that the PX domain embraces the 3-phosphate on one side of a water-filled, positively charged pocket and reveals how 3-phosphoinositide specificity is achieved. A chronic granulomatous disease (CGD)-associated mutation in the p47(phox) PX domain that abrogates PtdIns(3)P binding maps to a conserved Arg that does not directly interact with the phosphoinositide but instead appears to stabilize a critical lipid binding loop. The SH3 domain present in the full-length protein does not affect soluble PtdIns(3)P binding to the p40(phox) PX domain.

Amino Acid Sequence↗
PubMed2001

Red cell antibodies in patients with homozygous sickle cell disease: a comparison of patients in Jamaica and the United Kingdom.

The transfusion history and frequency of red cell antibodies in patients with homozygous sickle cell (SS) disease have been compared in 190 subjects from the Jamaican cohort study and 37 patients attending a sickle cell clinic in Manchester, England. The proportion of patients transfused did not differ between the groups although the number of units transfused and the frequency of red cell antibodies were significantly greater in the Manchester group. Immune antibodies occurred in three Jamaicans (2.6% of those transfused) and 16 UK subjects (76% of those transfused). Multiple antibodies occurred in 10 (63%) UK subjects but in no Jamaicans. Indications for transfusion also differed between the groups, Jamaican patients typically receiving 1-2 units for acute anaemia or acute chest syndrome, whereas UK patients frequently had multiple transfusions in preoperative exchange or prophylaxis programmes. The greater red cell alloimmunization among UK patients probably reflects both the greater use of transfusion and the disparity between donor and recipient populations in the UK.

Adolescent↗
PubMed2000

Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma.

Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.

Animals↗
PubMed2000

Colorectal carcinomas in mice lacking the catalytic subunit of PI(3)Kgamma.

Phosphoinositide-3-OH kinases (PI(3)Ks) constitute a family of evolutionarily conserved lipid kinases that regulate a vast array of fundamental cellular responses, including proliferation, transformation, differentiation and protection from apoptosis. PI(3)K-mediated activation of the cell survival kinase PKB/Akt, and negative regulation of PI(3)K signalling by the tumour suppressor PTEN (refs 3, 4) are key regulatory events in tumorigenesis. Thus, a model has arisen that PI(3)Ks promote development of cancers. Here we report that genetic inactivation of the p110gamma catalytic subunit of PI(3)Kgamma (ref. 8) leads to development of invasive colorectal adenocarcinomas in mice. In humans, p110gamma protein expression is lost in primary colorectal adenocarcinomas from patients and in colon cancer cell lines. Overexpression of wild-type or kinase-dead p110gamma in human colon cancer cells with mutations of the tumour suppressors APC and p53, or the oncogenes beta-catenin and Ki-ras, suppressed tumorigenesis. Thus, loss of p110gamma in mice leads to spontaneous, malignant epithelial tumours in the colorectum and p110gamma can block the growth of human colon cancer cells.

Adenocarcinoma↗
PubMed2000

Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine.

The specific phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kgamma bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.

Adenosine Triphosphate↗
PubMed1999

Structural insights into phosphoinositide 3-kinase catalysis and signalling.

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that function both as signal transducers downstream of cell-surface receptors and in constitutive intracellular membrane and protein trafficking pathways. All PI3Ks are dual-specificity enzymes with a lipid kinase activity which phosphorylates phosphoinositides at the 3-hydroxyl, and a protein kinase activity. The products of PI3K-catalysed reactions, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), PtdIns(3,4)P2 and PtdIns(3)P, are second messengers in a variety of signal transduction pathways, including those essential to cell proliferation, adhesion, survival, cytoskeletal rearrangement and vesicle trafficking. Here we report the 2.2 A X-ray crystallographic structure of the catalytic subunit of PI3Kgamma, the class I enzyme that is activated by heterotrimeric G-protein betagamma subunits and Ras. PI3Kgamma has a modular organization centred around a helical-domain spine, with C2 and catalytic domains positioned to interact with phospholipid membranes, and a Ras-binding domain placed against the catalytic domain where it could drive allosteric activation of the enzyme.

Amino Acid Sequence↗
PubMed1998

Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B.

Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid products phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3] and PtdIns(3,4)P2 in the signaling pathways used by a wide variety of growth factors, antigens, and inflammatory stimuli. PKB is a direct target of these lipids, but this regulation is complex. The lipids can bind to the pleckstrin homologous domain of PKB, causing its translocation to the membrane, and also enable upstream, Thr308-directed kinases to phosphorylate and activate PKB. Four isoforms of these PKB kinases were purified from sheep brain. They bound PtdIns(3,4,5)P3 and associated with lipid vesicles containing it. These kinases contain an NH2-terminal catalytic domain and a COOH-terminal pleckstrin homologous domain, and their heterologous expression augments receptor activation of PKB, which suggests they are the primary signal transducers that enable PtdIns(3,4,5)P3 or PtdIns- (3,4)P2 to activate PKB and hence to control signaling pathways regulating cell survival, glucose uptake, and glycogen metabolism.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1998

Increased platelet transfusion requirement is associated with multiple organ dysfunctions in patients undergoing hematopoietic stem cell transplantation.

Organ dysfunction following hematopoietic stem cell transplantation (HSCT) may be a manifestation of a systemic inflammatory response. We speculate that part of the platelet transfusion requirement in HSCT patients results from this systemic inflammatory response, and increased transfusion requirement would be associated with, or precede, organ dysfunction. We studied 199 adults undergoing autologous (n=173) or allogeneic (n=26) HSCT. Patients with CNS (P=0.008) or pulmonary (P=0.002) dysfunction, or with VOD (P=0.05) received a higher mean number of platelet transfusions per week than patients who did not have these dysfunctions. Furthermore, a higher number of platelet transfusions during any 1 week period was significantly associated with development of pulmonary (P=0.0002) or renal (P < 0.0001) dysfunction in the following week. This predictive value was strongest early in the HSCT course, but remained significant over all 4 weeks. In multivariate analysis the number of platelet transfusions during the previous week was independently predictive for development of pulmonary dysfunction in week 2 (P=0.01) and week 3 (P=0.055). We believe that occurrence of increased platelet transfusion requirement prior to onset of dysfunction is consistent with the concept that an antecedent inflammatory response results in both platelet consumption and various organ dysfunctions. Increased platelet transfusion requirement may act as an early marker of subsequent organ dysfunction. Additionally, there may be a direct role of platelets in the development and progression of organ dysfunction in HSCT patients.

Adolescent↗
PubMed1997

Factors predicting morbidity following hematopoietic stem cell transplantation.

Circulating anticoagulants protein C (PC) and antithrombin III (AT) are markers of, and possibly involved in the pathogenesis of, significant organ dysfunction, in patients undergoing autologous peripheral blood stem cell (PSBC) or autologous bone marrow (BM) transplantation. The effect of the stem cell source, the use of hematopoietic growth factors (GFs), and the specific preparative regimen on the incidence of organ system dysfunction or on post-transplant levels of circulating anticoagulants has not been well studied. We analyzed 205 patients in an attempt to correlate organ dysfunction and AT and PC deficiencies with these transplant-specific factors (78 BMT with GM-CSF after transplant, 95 PBSCT without GM-CSF after transplant, and 32 PBSCT with GM-CSF after transplant). Patients transplanted with PBSC had a lower incidence of pulmonary dysfunction (20 vs 40%, P = 0.006) and liver dysfunction (4 vs 13%, P = 0.05) than patients receiving BM. The use of GF after transplant did not influence the development of subsequent organ dysfunction. In multivariate analysis, the stem cell source was again predictive of pulmonary dysfunction. In contrast, although patients transplanted with PBSC also had a lower incidence of PC deficiency (50 vs 81%, P < 0.01) and AT deficiency (20 vs 54%, P < 0.01) as compared with patients receiving BM, use of GM-CSF after transplant was a more significant risk factor for the development of anticoagulant deficiency (PBSC with GF vs PBSC without GF: PC deficiency 50 vs 78%, P = 0.007; AT deficiency 20 vs 47%, P = 0.005). In the multivariate analysis GM-CSF use was the only significant risk factor for development of anticoagulant deficiency. Since the clinical significance of anticoagulant deficiency has been well shown, further studies examining these effects of hematopoietic GFs appear warranted.

Antithrombin III Deficiency↗
PubMed1996

A heterotrimeric GTPase-regulated isoform of PI3K and the regulation of its potential effectors.

We have purified two forms of phosphoinositide 3-kinase (PI3K) that are activated by heterotrimeric G-protein beta gamma-subunits. These novel isoforms of PI3K are structurally distinct to those forms of PI3K which have already been cloned. They are both heterodimers made up of a p120 and a p101 and a p117 and a p101 protein. The p101 species in both heterodimers are identical and show no substantial homology with any other proteins or DNA sequences. The p117 and p120 are highly related. The p101 and p120 species have been cloned from a pig neutrophil mRNA library. The p120 has similarities with other known PI3K catalytic subunits. They may be responsible for conferring cells with the capacity to produce phosphatidylinositol (3,4,5)-trisphosphate in response to activation of G-protein-coupled receptors. Activation of both the monomeric G-protein rac and PI3K(s) have been implicated in receptor-stimulated membrane-ruffling. We have observed that agonist-stimulated guanine nucleotide exchange on rac can be inhibited by a variety of PI3K inhibitors. This suggests PI3K may lie upstream of rac in receptor-driven pathways regulating cell movement.

Enzyme Activation↗
PubMed1996

Poly (DL-lactide-co-glycolide) microspheres as carriers for peptide vaccines.

Peptides carrying an immunodominant T-helper cell epitope delineated from the rabies virus nucleoprotein either alone or in combination with a linear B-cell epitope from the same protein were incorporated into three different formulations of poly(DL-lactide-co-glycolide) (PLG) which were distinct in their composition, and consequently in their peptide release rates. In vitro peptides incorporated into any of the PLG formulations stimulated a peptide-specific T-cell line. Upon subcutaneous immunization of mice, the PLG formulation that showed the fastest peptide release rate induced the best immune response. This immune response was in magnitude comparable or even superior to that induced by peptide emulsified in complete Freund's adjuvant.

Amino Acid Sequence↗
PubMed1996

Prolonged deficiency of protein C following hematopoietic stem cell transplantation.

Deficiencies in circulating anticoagulant protein C (PC) occur in patients undergoing hematopoietic stem cell transplantation. These deficiencies may predispose to thrombotic and other complications, and may contribute to the morbidity of transplantation. In most patients, PC reaches a nadir 14 days after the preparative regimen and then begins to increase toward normal. However, low PC has been seen months after transplantation. Neither the frequency of, nor risk factors for, this prolonged deficiency are known. We examined 71 adults undergoing stem cell transplantation and found low PC antigen and activity in 21% and 20% of patients, respectively. Low PC at day 100 correlated strongly with low PC pre-chemotherapy (PC antigen, r = 0.69, P < 0.001; PC activity, r = 0.59, P < 0.001). The incidence of deficiency of PC at day 100 was lower in patients undergoing peripheral stem cell transplantation compared with patients undergoing autologous BMT (12.5% vs 35%; P = 0.05), although several significant confounding variables exist. We conclude that deficiencies in protein C persist at least 100 days after stem cell transplantation in nearly one quarter of patients undergoing this procedure. Therefore, patients undergoing stem cell transplantation may be at prolonged risk of thrombotic and other complications. Further studies to determine the risk of prolonged deficiency based on stem cell source need to be performed.

Adult↗
PubMed1995

Activation of the small GTP-binding proteins rho and rac by growth factor receptors.

The small GTP-binding proteins, rho and rac, control signal transduction pathways that link growth factor receptors to the activation of actin polymerization. In Swiss 3T3 cells, rho proteins mediate the lysophosphatidic acid and bombesin-induced formation of focal adhesions and actin stress fibres, whilst rac proteins are required for the platelet-derived growth factor-, insulin-, bombesin- and phorbol ester (phorbol 12-myristate 13-acetate)-stimulated actin polymerization at the plasma membrane that results in membrane ruffling. To investigate the role of p85/p110 phosphatidylinositol 3-kinase in the rho and rac signalling pathways, we have used a potent inhibitor of this activity, wortmannin. Wortmannin has no effect on focal adhesion or actin stress fibre formation induced by lysophosphatidic acid, bombesin or microinjected recombinant rho protein. In contrast, it totally inhibits plasma membrane edge-ruffling induced by platelet-derived growth factor and insulin though not by bombesin, phorbol ester or microinjected recombinant rac protein. We conclude that phosphatidylinositol 3,4,5 trisphosphate mediates activation of rac by the platelet-derived growth factor and insulin receptors. The effects of lysophosphatidic acid on the Swiss 3T3 actin cytoskeleton can be blocked by the tyrosine kinase inhibitor, tyrphostin. Since tyrphostin does not inhibit the effects of microinjected rho protein, we conclude that lysophosphatidic acid activation of rho is mediated by a tyrosine kinase.

3T3 Cells↗
PubMed1994

1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells.

Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits. This interaction takes place via the phosphorylated YMXM motifs of IRS-1 and the Src homology region 2 (SH2) domains of p85. In this study, the stable overexpression in a Chinese hamster ovary (CHO) cell line of a mutant p85 alpha (delta p85) protein, which lacks a binding site for p110, disrupted the complex formation between IRS-1 and the catalytic subunit of PI 3-kinase in intact cells during insulin stimulation. Activation of insulin receptor kinase and the tyrosine phosphorylation of IRS-1 remained unaffected. In this cell line, both insulin-stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate and the insulin-stimulated glucose uptake due to the translocation of GLUT1 glucose transporters were markedly impaired, whereas neither phorbol 12-myristate 13-acetate-stimulated glucose uptake nor the insulin-stimulated activation of RAS was impaired. These results suggest that PI 3-kinase is required for glucose transport in insulin signaling in CHO cells.

Animals↗