Caring for people with end-stage dementia.

This article considers the role of palliative care in the management of patients with dementia. It aims to broaden the knowledge of nurses providing general care as well as specialist palliative and end of life care to patients with dementia in all settings. The article helps nurses to identify the characteristics of end-stage dementia and meet the associated challenges that this diagnosis poses. Nurses should then be in a better position to recognise and support patients and their families and ensure that palliative care is included in care planning for this group of patients.

Protein kinase B phosphorylates AHNAK and regulates its subcellular localization.

AHNAK is a ubiquitously expressed giant phosphoprotein that was initially identified as a gene product subject to transcriptional repression in neuroblastoma. AHNAK is predominantly nuclear in cells of nonepithelial origin, but is cytoplasmic or associated with plasma membrane in epithelial cells. In this study we show that the extranuclear localization of AHNAK in epithelial cells depends on the formation of cell-cell contacts. We show that AHNAK is a phosphorylation substrate of protein kinase B (PKB) in vitro and in vivo. Nuclear exclusion of AHNAK is mediated through a nuclear export signal (NES) in a manner that depends on the phosphorylation of serine 5535 of AHNAK by PKB, a process that also plays a major role in determining extranuclear localization of AHNAK. AHNAK is a new PKB substrate whose function, though unknown, is likely to be regulated by its localization, which is in turn regulated by PKB.

Activation of Raf as a result of recruitment to the plasma membrane.

The small guanine nucleotide binding protein Ras participates in a growth promoting signal transduction pathway. The mechanism by which interaction of Ras with the protein kinase Raf leads to activation of Raf was studied. Raf was targeted to the plasma membrane by addition of the COOH-terminal localization signals of K-ras. This modified form of Raf (RafCAAX) was activated to the same extent as Raf coexpressed with oncogenic mutant Ras. Plasma membrane localization rather than farnesylation or the presence of the additional COOH-terminal sequence accounted for the activation of RafCAAX. The activation of RafCAAX was completely independent of Ras; it was neither potentiated by oncogenic mutant Ras nor abrogated by dominant negative Ras. Raf, once recruited to the plasma membrane, was not anchored there by Ras; most activated Raf in cells was associated with plasma membrane cytoskeletal elements, not the lipid bilayer. Thus, Ras functions in the activation of Raf by recruiting Raf to the plasma membrane where a separate, Ras-independent, activation of Raf occurs.

Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B.

Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase. Chromatographic separation of brain cytosol revealed a kinase activity that phosphorylated and activated PKB only in the presence of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Phosphorylation occurred exclusively on threonine-308, a residue implicated in activation of PKB in vivo. PtdIns(3,4,5)P3 was determined to have a dual role: Its binding to the pleckstrin homology domain of PKB was required to allow phosphorylation by the upstream kinase and it directly activated the upstream kinase.

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.

Mechanisms of translational deregulation in human tumors and therapeutic intervention strategies.

Analysis of the recurrent genetic aberrations present in human tumors provides insight into how normal cells escape appropriate proliferation and survival cues. Commonly mutated genes encode proteins that monitor DNA damage (e.g., p53), proteins that regulate the cell cycle (such as Rb), and proteins that regulate signal transduction pathways (such as APC, PTEN and Ras). Analysis of the relevant targets and downstream events of these genes in normal and tumor cells will clearly highlight important pathways for tumorigenesis. However, more infrequent mutations are also informative in defining events critical for the process of tumorigenesis, and often delineate important pathways lying downstream of commonly mutated oncogenes and tumor suppressors. Together, these studies have led to the conclusion that deregulated protein synthesis plays an important role in human cancer. This review will discuss the evidence implicating mRNA translation as an important downstream consequence of signal transduction pathways initiated by mutated oncogenes and tumor suppressors, as well as additional genetic findings implicating the importance of global and specific translational control in human cancer. It will also discuss therapeutic strategies that take advantage of differences in translational regulation between normal and tumor cells.

Induction of NF-kappaB by the Akt/PKB kinase.

The serine/threonine kinase Akt (also known as protein kinase B, PKB) is activated by numerous growth-factor and immune receptors through lipid products of phosphatidylinositol (PI) 3-kinase. Akt can couple to pathways that regulate glucose metabolism or cell survival [1]. Akt can also regulate several transcription factors, including E2F, CREB, and the Forkhead family member Daf-16 [2] [3] [4]. Here, we show that Akt can regulate signaling pathways that lead to induction of the NF-kappaB family of transcription factors in the Jurkat T-cell line. This induction occurs, at least in part, at the level of degradation of the NF-kappaB inhibitor IkappaB, and is specific for NF-kappaB, as other inducible transcription factors are not affected by Akt overexpression. Furthermore, the effect requires the kinase activity and pleckstrin homology (PH) domain of Akt. Also, Akt does not act alone to induce cytokine promoters and NF-kappaB reporters, because signals from other pathways are required to observe the effect. These studies uncover a previously unappreciated connection between Akt and NF-kappaB induction that could have implications for the control of T-cell growth and survival.

Protein kinase B (PKB/Akt) activity is elevated in glioblastoma cells due to mutation of the tumor suppressor PTEN/MMAC.

Glioblastomas are highly malignant tumors of the central nervous system that are resistant to radiation and chemotherapy [1]. We explored the role of the phosphatidylinositol (PI) 3-kinase signal transduction pathway in glioblastomas, as this pathway has been shown to inhibit apoptosis induced by cytokine withdrawal and the detachment of cells from the extracellular matrix [2]. Components of this pathway have been implicated in tumor development [3-6]. We show that glioblastoma cells, in contrast to primary human astrocytes, contain high endogenous protein kinase B (PKB/Akt) activity and high levels of PI 3,4,5-triphosphate (PI(3,4,5)P3) and PI(3,4)P2, the lipid products of PI 3-kinase. These glioblastoma cells express mutant forms of the putative 3' phospholipid phosphatase PTEN, also known as MMAC. Expression of wild-type PTEN derived from primary astrocytes, but not of mutant forms of PTEN, reduced the levels of 3' phosphoinositides and inhibited PKB/Akt activity. PTEN antagonized the activation of PKB/Akt by growth factors, by activated PI 3-kinase and by PI-dependent protein kinase-1 (PDK1), but did not antagonize the phospholipid-independent activation of PKB/Akt lacking the pleckstrin homology (PH) domain. These results suggest a role for PTEN in regulating the activity of the PI 3-kinase pathway in malignant human cells.

Inhibition of PDK-1 activity causes a reduction in cell proliferation and survival.

3-Phosphoinositide-dependent kinase-1 (PDK-1) was identified by its ability to phosphorylate and activate protein kinase B (PKB) in vitro [1,2] and can phosphorylate and activate additional protein kinases in the AGC family in vitro [3-6]. Its role in vivo has, however, only begun to be addressed. We used antisense oligonucleotides directed against PDK-1 expression to explore the role of PDK-1 in human glioblastoma cells (U87-MG), which express a mutant PTEN allele. Reduction in PDK-1 levels resulted in inhibition of PKB activity, and a reduction in phosphorylation on Thr308 and Ser473 of PKB. p70 S6 kinase (p70(S6K)) activity was also reduced. Cell proliferation was dramatically inhibited following treatment with PDK-1 antisense oligonucleotides, due to a combination of decreased cell doubling and an increase in apoptosis. This is in contrast to direct inhibition of phosphoinositide 3-OH kinase (PI 3-kinase), which results in G1 arrest with no effect on apoptosis. This study confirms both PKB and p70(S6K) as in vivo substrates for PDK-1. The effect of acute PDK-1 loss on cell proliferation and survival suggests the involvement of PI 3-kinase dependent and independent signaling events, and implicates PDK-1 as a potential therapeutic target for human neoplasms.

PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase.

BACKGROUND: Phosphoinositide 3-kinases (PI 3-kinases) are thought to play an important role in coordinating the responses elicited by a variety of growth factors, oncogene products and inflammatory stimuli. These responses include activation of membrane ruffling, chemotaxis, glucose transport, superoxide production, neurite outgrowth and pp70 S6 kinase. Some of these responses are also known to be regulated by Rac, a small GTP-binding protein related to Ras. Neither the transducing elements upstream of Rac, nor those downstream of PI 3-kinase, have been defined. RESULTS: We show here that platelet-derived growth factor (PDGF) can stimulate an increase in the level of GTP-Rac by at least two distinct mechanisms: firstly, by increased guanine nucleotide exchange; and secondly, by inhibition of a Rac GTPase activity. The first of these mechanisms is essential for the activation of Rac, and we show that it is dependent upon PDGR-stimulated synthesis of phosphatidylinositol (3,4,5)-trisphosphate. CONCLUSIONS: These results suggest that Rac activation lies downstream of PI 3-kinase activation on a PDGF-stimulated signalling pathway. Furthermore, as Rac has been implicated in at least two diverse cellular responses that are also though to require activation of PI 3-kinase--a reorganization of the actin cytoskeleton known as membrane ruffling and the neutrophil oxidative burst--these results suggest that Rac may be a major effector protein for the PI 3-kinase signalling pathway in many cell types.

5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells.

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G(1) in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27(KIP1). Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

p27Kip1 is required for PTEN-induced G1 growth arrest.

The tumor suppressor PTEN is one of the most commonly inactivated genes in human cancer. Glioblastoma multiforme cells harboring mutant PTEN have abnormally high levels of 3' phosphoinositides and elevated protein kinase B activity. Expression of wild-type PTEN in glioma cells, containing endogenous mutant PTEN, reduces 3' phosphoinositides levels, inhibits PKB activity, and induces G1 cell cycle arrest. We investigated the mechanism of the PTEN-induced growth arrest in glioma cell lines. Expression of PTEN is associated with increased expression of p27Kip1, decreased expression of cyclins A and D3, inhibition of cdk2 activity, and dephosphorylation of pRb. Inactivation of p53, by the human papilloma virus E6 oncoprotein, does not prevent PTEN-induced G1 arrest, implying that p53 is not required for G1 arrest. In contrast, p27Kip1 antisense oligonucleotides abrogated the growth arrest induced by PTEN. Furthermore, blocking p27Kip1 expression prevented the PTEN-induced reduction of cyclin-dependent kinase 2 activity, indicating that p27Kip1 functions upstream of cyclin-dependent kinase 2 in the PTEN regulatory cascade. These results implicate p27Kip1 as a critical mediator of PTEN-induced G1 arrest.

Adenoviral transgene expression of MMAC/PTEN in human glioma cells inhibits Akt activation and induces anoikis.

The MMAC/PTEN tumor suppressor gene encodes for a phosphatase that recently has been shown to have phosphotidylinositol phosphatase activity, implicating its possible involvement in phosphatidylinositol 3'-kinase-mediated signaling. To investigate possible alterations in growth factor-mediated signal transduction, an adenovirus containing MMAC/PTEN, Ad-MMAC, previously shown to inhibit growth and tumorigenicity in glioma cells, was used to acutely express the transgene. Human glioma cells infected with Ad-MMAC but not with control adenoviruses exhibited an inhibition of phosphorylation of both activating residues of Akt, Ser-473, and Thr-308, along with Akt's serine/threonine kinase activity, without significantly altering Akt expression. The effects of functional MMAC/PTEN expression were relatively specific, because members of several other growth factor-mediated signaling pathways showed no altered responses. The presence of MMAC/PTEN also inhibited phosphorylation of BAD, although no evidence of apoptosis in the in situ treated cells was observed. However, U251 glioma cells infected with Ad-MMAC were induced to undergo anoikis at a significantly higher rate than U251 cels treated with control viruses or mock infected with media. These results demonstrate that the acute administration of MMAC/PTEN results in the inhibition of Akt-mediated signaling, growth inhibition, and anoikis, implying that loss of MMAC/PTEN increases cellular proliferation and significantly augments a cell's survival potential during cellular processes that are associated with malignancy.

Akt activation by growth factors is a multiple-step process: the role of the PH domain.

The protein kinase encoded by the Akt proto-oncogene is activated by phospholipid binding, membrane translocation and phosphorylation. To address the relative roles of these mechanisms of Akt activation, we have employed a combination of genetic and pharmacological approaches. Transient transfection of NIH3T3 cells with wild-type Akt, pleckstrin homology (PH) domain mutants, generated on the basis of a PH domain structural model, and phosphorylation site Akt mutants provided evidence for a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by constitutive phosphorylation of threonine 450 (T450) and perhaps serine 124 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translocation step; and (3) a PI3-K-dependent step, characterized by phosphorylation at T308 and S473, that occurs in the cell membrane and is required for activation. When forced to translocate to the membrane, wild-type Akt and PH domain Akt mutants that are defective in the first step become constitutively active, suggesting that the purpose of this step is to prepare the protein for membrane translocation. Both growth factor stimulation and forced membrane translocation, however, failed to activate a T308A mutant. This, combined with the finding that T308D/S473D double mutant is constitutively active, suggests that the purpose of the three-step process of Akt activation is the phosphorylation of the protein at T308 and S473. The proposed model provides a framework for a comprehensive understanding of the temporal and spatial requirements for Akt activation by growth factors.

Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins.

MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser86 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Ser86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.

The role of genetic abnormalities of PTEN and the phosphatidylinositol 3-kinase pathway in breast and ovarian tumorigenesis, prognosis, and therapy.

Breast and ovarian cancers exhibit similar epidemiologic, genotypic, and phenotypic characteristics. Phosphatidylinositol 3-kinase (PI3K) and the PTEN tumor suppressor gene product phosphorylate and dephosphorylate the same 3' site in the inositol ring of membrane phosphatidylinositols. Germ-line mutations in the PTEN tumor suppressor gene are causative of Cowden's breast cancer predisposition syndrome, and PTEN is frequently mutated in sporadic breast cancers. In contrast, amplification of multiple components of the PI3K pathway is a hallmark of serous epithelial ovarian cancers. The resultant activation of the PI3K pathway in both breast and ovarian cancers contributes to cell-cycle progression, decreased apoptosis, and increased metastatic capabilities. Strikingly, both ovarian and breast cancer cells are selectively sensitive to pharmacologic and genetic manipulation of the PI3K pathway, making molecular therapeutics targeting this pathway particularly attractive approaches for these cancers.

Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro.

The c-Raf-1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine kinase and G-protein-linked receptors. c-Raf-1 binds to the proto-oncogene Ras in a GTP-dependent manner, but the exact mechanism of activation of c-Raf-1 by Ras is still unclear. We have established a system to study the activation of c-Raf-1 in vitro. This involves mixing membranes from cells expressing oncogenic H-RasG12V, with cytosol from cells expressing epitope-tagged full-length wild-type c-Raf-1. This results in a fraction of the c-Raf-1 binding to the membranes and a concomitant 10- to 20-fold increase in specific activity. Ras was the only component in these membranes required for activation, as purified recombinant farnesylated K-Ras.GTP, but not non-farnesylated K-Ras.GTP or farnesylated K-Ras.GDP, was able to activate c-Raf-1 to the same degree as intact H-RasG12V membranes. The most potent activation occurred under conditions in which phosphorylation was prohibited. Under phosphorylation-permissive conditions, activation of c-Raf-1 by Ras was substantially inhibited. Consistent with the results from other groups, we find that the activation of c-Raf-1 by Src in vivo occurs concomitant with tyrosine phosphorylation on c-Raf-1, and in vitro, activation of c-Raf-1 by Src requires the presence of ATP. Therefore we propose that activation of c-Raf-1 by Ras or by Src occurs through different mechanisms.

Travel sickness in patients attending a geriatric day hospital.

A survey of 172 patients attending a geriatric day hospital revealed that 33 patients (19%) suffered travel sickness at some time. Sixteen (49%) of these patients also had a past history of travel sickness, while only 10 (7%) of those who were not sick gave a positive past history. The use of emergency vehicles for transporting patients may have increased the level of travel sickness. Seven (4%) of the patients had defaulted or considered defaulting on attendance because of the fear of travel sickness. Patients commenced on drug therapy for sickness responded well and experienced few side-effects and no interference with rehabilitation.

GPs and contracting for secondary health care.

Publication of the NHS White Paper Working for Patients and subsequent legislation has introduced the requirement for district health authorities to assess the health and health care needs of their resident populations. Sam Ramaiah and colleagues describe their experience in South Tees of working with GPs to fulfil this objective.