bluuebunny/crossref_metadata_2025 huggingface dataset:
The p110δ subunit of phosphoinositide 3-kinase is required for the lipopolysaccharide response of mouse B cells
Authors: Hebeis B.J. , Vigorito E. , Turner M.
Published: October 2004

PI3K (phosphoinositide 3-kinase) IA family members contain a regulatory subunit and a catalytic subunit. The p110δ catalytic subunit is expressed predominantly in haematopoietic cells. There, among other functions, it regulates antigen receptor-mediated responses. Using mice deficient in the p110δ subunit of PI3K, we investigated the role of this subunit in LPS (lipopolysaccharide)-induced B cell responses, which are mediated by Toll-like receptor 4 and RP105. After injection of DNP-LPS (where DNP stands for 2,4-dinitrophenol), p110δ−/− mice produced reduced levels of DNP-specific IgM and IgG when compared with wild-type mice. In vitro, the proliferation and up-regulation of surface activation markers such as CD86 and CD25 induced by LPS and an antibody against RP105 were decreased. We analysed the activation state of key components of the LPS pathway in B cells to determine whether there was a defect in signalling in p110δ−/− B cells. They showed normal extracellular-signal-regulated kinase phosphorylation, but anti-RP105-induced protein kinase B, IκB (inhibitor of nuclear factor κB) and c-Jun N-terminal kinase activation was severely reduced. This demonstrates that the p110δ subunit of PI3K is involved in the LPS response in B cells and may represent a link between the innate and the adaptive immune system.
PDE6 is an effector for the Wnt/Ca2+/cGMP-signalling pathway in development
Authors: Wang H. , Lee Y. , Malbon C.C.
Published: October 2004

Wnt signalling in development operates via members of the Frizzleds, G-protein-coupled receptors that bind specific Wnt ligands and mediate signalling via distinct pathways. The Wnt/Ca2+/cGMP pathway mediated by Frizzled-2 was discovered recently. Activation of this pathway leads to increased intracellular concentrations of Ca2+ and decreased intracellular concentrations of cGMP. The nature of the phosphodiesterase responsible for this Frizzled-2-mediated effect on cGMP levels was identified based on three separate criteria: (i) sensitivity to selective enzyme inhibitors, (ii) behaviour on chromatographic separation, and (ii) isolation by two-dimensional gels in tandem with direct mapping by MS of tryptic digests of the activity. On the basis of results from these three analyses, the cGMP-specific phosphodiesterase, PDE6, is demonstrated to be an effector for the Wnt/Ca2+/cGMP signalling pathway of development, which is mediated by Frizzled-2.
Plakoglobin expression and localization in zebrafish embryo development
Authors: Martin E.D. , Grealy M.
Published: October 2004

Plakoglobin (γ-catenin) and β-catenin are major components of the adherens junctions and can be localized to the nucleus by activation of the Wnt signalling pathway. In addition, plakoglobin is also found in desmosomes, a vertebrate-specific cell–cell adhesion structure. Plakoglobin expression and localization were examined at the protein level during zebrafish embryonic development by Western blotting and confocal microscopy. Plakoglobin was expressed throughout embryo development at the protein level. Western blotting revealed that embryonic plakoglobin protein content increased between 12- and 24-h post-fertilization (hpf). Confocal microscopy showed that at stages up to 12 hpf, plakoglobin and β-catenin were co-localized and expressed in both the nucleus and in cell–cell junctions. At 24- and 72-hpf, separate patterns were seen for plakoglobin and β-catenin. These data indicate that plakoglobin localization in the heart region shifts from adherens junctions to desmosomes during heart chamber development.
A molecular cell biology of lithium
Authors: Williams R. , Ryves W.J. , Dalton E.C. , Eickholt B. , Shaltiel G. , Agam G. , Harwood A.J.
Published: October 2004

Lithium (Li+), a mood stabilizer, has profound effects on cultured neurons, offering an opportunity to investigate its cellular biological effects. Here we consider the effect of Li+ and other psychotropic drugs on growth cone morphology and chemotaxis. Li+ inhibits GSK-3 (glycogen synthase kinase-3) at a therapeutically relevant concentration. Treated cells show a number of features that arise due to GSK-3 inhibition, such as altered microtubule dynamics, axonal branching and loss of semaphorin 3A-mediated growth cone collapse. Li+ also causes growth cones to spread; however, a similar effect is seen with two other mood stabilizers, valproic acid and carbamazepine, but without changes in microtubules or axon branching. This common effect of mood stabilizers is mediated by changes in inositol phosphate signalling, not GSK-3 activity. Given the presence of neurogenesis in the adult brain, we speculate that changes in growth cone behaviour could also occur during treatment of mental disorders.
Uncoupling insulin signalling by serine/threonine phosphorylation: a molecular basis for insulin resistance
Authors: Zick Y.
Published: October 2004

Insulin resistance refers to a decreased capacity of circulating insulin to regulate nutrient metabolism. Recent studies reveal that agents that induce insulin resistance exploit phosphorylation-based negative feedback control mechanisms otherwise utilized by insulin itself to uncouple the insulin receptor from its downstream effectors and thereby terminate insulin signal transduction. This article focuses on the Ser/Thr protein kinases which phosphorylate insulin receptor substrates and the major Ser sites that are phosphorylated, as key elements in the uncoupling of insulin signalling and the induction of an insulin resistance state.
Analysis of insulin signalling by RNAi-based gene silencing
Authors: Zhou Q.L. , Park J.G. , Jiang Z.Y. , Holik J.J. , Mitra P. , Semiz S. , Guilherme A. , Powelka A.M. , Tang X. , Virbasius J. , Czech M.P.
Published: October 2004

Using siRNA-mediated gene silencing in cultured adipocytes, we have dissected the insulin-signalling pathway leading to translocation of GLUT4 glucose transporters to the plasma membrane. RNAi (RNA interference)-based depletion of components in the putative TC10 pathway (CAP, CrkII and c-Cbl plus Cbl-b) or the phospholipase Cγ pathway failed to diminish insulin signalling to GLUT4. Within the phosphoinositide 3-kinase pathway, loss of the 5′-phosphatidylinositol 3,4,5-trisphosphate phosphatase SHIP2 was also without effect, whereas depletion of the 3′-phosphatase PTEN significantly enhanced insulin action. Downstream of phosphatidylinositol 3,4,5-trisphosphate and PDK1, silencing the genes encoding the protein kinases Akt1/PKBα, or CISK(SGK3) or protein kinases Cλ/ζ had little or no effect, but loss of Akt2/PKBβ significantly attenuated GLUT4 regulation by insulin. These results show that Akt2/PKBβ is the key downstream intermediate within the phosphoinositide 3-kinase pathway linked to insulin action on GLUT4 in cultured adipocytes, whereas PTEN is a potent negative regulator of this pathway.
Physical and functional interaction between integrins and hERG potassium channels
Authors: Arcangeli A. , Becchetti A. , Cherubini A. , Crociani O. , Defilippi P. , Guasti L. , Hofmann G. , Pillozzi S. , Olivotto M. , Wanke E.
Published: October 2004

Integrins are adhesion receptors capable of transmitting intracellular signals that regulate many different cellular functions. Among integrin-mediated signals, the activation of ion channels can be included. We demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) potassium channels occurs in both human neuroblastoma and leukaemia cells after the activation of the β1 integrin subunit. This activation is apparently a determining factor inducing neurite extension and osteoclastic differentiation in both the cell types. More recently, we provided evidences that β1 integrins and hERG channels co-precipitate in both the cell types. Preliminary results suggest that a macromolecular signalling complex indeed occurs between integrins and the hERG1 protein and that hERG channel activity can modulate integrin downstream signalling.
Crumbs homologue 1 in polarity and blindness
Authors: Meuleman J. , van de Pavert S.A. , Wijnholds J.
Published: October 2004

Several retinal dystrophies, including retinitis pigmentosa type 12 and Leber congenital amaurosis, are caused by a large variety of mutations in the CRB1 (Crumbs homologue 1) gene. This discovery led to an increased focus on the function of CRB1 and the Drosophila homologue Crumbs. In the present study, we review the current knowledge on Crumbs and its vertebrate homologues, their function in cell polarity and their pathogenicity in retinal degeneration.
Cytoskeletal proteins talin and vinculin in integrin-mediated adhesion
Authors: Critchley D.R.
Published: October 2004

The cytoskeletal proteins talin and vinculin form part of a macromolecular complex on the cytoplasmic face of integrin-mediated cellular junctions with the extracellular matrix. Recent genetic, biochemical and structural data show that talin is essential for the assembly of such junctions, whereas vinculin appears to be important in regulating adhesion dynamics and cell migration.
The integrity of cholesterol-enriched microdomains is essential for the constitutive high activity of protein kinase B in tumour cells
Authors: Elhyany S. , Assa-Kunik E. , Tsory S. , Muller T. , Fedida S. , Segal S. , Fishman D.
Published: October 2004

A deregulated activity of PKB/Akt (where PKB stands for protein kinase B) renders tumour cells resistant to a variety of apoptosis-inducing stimuli. Elucidation of the mechanisms responsible for this deregulation is of prime importance for the development of novel anti-cancer drugs. Results of the present study demonstrate that the constitutive activity of PKB/Akt in B16BL6 melanoma cells depends on the integrity of cholesterol-enriched membrane microdomains, since the exposure of cells to cholesterol-depleting agents decreases the phosphorylation of this enzyme, with no change in its total protein level. Inhibitors of Hsp90 (heat-shock protein 90) decreased phosphorylation of PKB/Akt with a similar pattern. Dephosphorylation of the enzyme, as a consequence of raft disintegration, could be precluded by inhibition of serine/threonine (but not tyrosine) phosphatases. Our results imply that destabilization of lipid rafts seemingly affects the association of Hsp90 with the respective serine/threonine phosphatases, thereby increasing the accessibility to PKB/Akt to deactivating phosphatases. We have found recently that reconstituted expression of H-2K class I glycoproteins in class I-deficient B16BL6 cells also decreases the phosphorylation of PKB/Akt. Therefore it is possible that raft-associated regulation of this important enzyme involves both H-2K glycoproteins and Hsp90.
Hetero-oligomers of α2A-adrenergic and μ-opioid receptors do not lead to transactivation of G-proteins or altered endocytosis profiles
Authors: Zhang Y.Q. , Limbird L.E.
Published: October 2004

Complexes of α2A-ARs (α2A-adrenergic receptors) and MORs (μ-opioid receptors), probably hetero-oligomers, were detected by co-immunoisolation after extraction from HEK-293 cells (human embryonic kidney 293 cells). Functional communication between these receptors is revealed by α2A-AR activation of a pertussis toxin-insensitive Giα subunit (termed as Gi1) when fused with the MOR and evaluated in membranes from pertussis toxin-treated cells. However, the α2A-AR does not require transactivation through MOR, since quantitatively indistinguishable results were observed in cells co-expressing α2A-AR and a fusion protein of Gi1 with the first transmembrane span of MOR (myc–MOR-TM1). Functional cross-talk among these α2A-AR–MOR complexes does not occur for internalization profiles; incubation with adrenaline (epinephrine) leads to endocytosis of α2A-AR but not MOR, while incubation with DAMGO ([D-Ala,NMe-Phe,Gly-ol]enkephalin) leads to endocytosis of MOR but not α2A-AR in cells co-expressing both the receptors. Hence, α2A-AR and MOR hetero-oligomers, although they occur, do not have an obligatory functional influence on one another in the paradigms studied.
AKAP (A-kinase anchoring protein) domains: beads of structure–function on the necklace of G-protein signalling
Authors: Malbon C.C. , Tao J. , Shumay E. , Wang H.-Y.
Published: October 2004

AKAPs (A-kinase anchoring proteins) are members of a diverse family of scaffold proteins that minimally possess a characteristic binding domain for the RI/RII regulatory subunit of protein kinase A and play critical roles in establishing spatial constraints for multivalent signalling assemblies. Especially for G-protein-coupled receptors, the AKAPs provide an organizing centre about which various protein kinases and phosphatases can be assembled to create solid-state signalling devices that can signal, be modulated and trafficked within the cell. The structure of AKAP250 (also known as gravin or AKAP12), based on analyses of milligram quantities of recombinant protein expressed in Escherichia coli, suggests that the AKAP is probably an unordered scaffold, acting as a necklace on which ‘jewels’ of structure–function (e.g. the RII-binding domain) that provide docking sites on which signalling components can be assembled. Recent results suggest that AKAP250 provides not only a ‘tool box’ for assembling signalling elements, but may indeed provide a basis for spatial constraint observed for many signalling paradigms. The spatial dimension of the integration of cell signalling will probably reflect many functions performed by members of the AKAP family.
Amylin receptors: molecular composition and pharmacology
Authors: Hay D.L. , Christopoulos G. , Christopoulos A. , Sexton P.M.
Published: October 2004

Several receptors which bind the hormone AMY (amylin) with high affinity have now been identified. The minimum binding unit is composed of the CT (calcitonin) receptor at its core, plus a RAMP (receptor activity modifying protein). The receptors have been named AMY1(a), AMY2(a) and AMY3(a) in accordance with the association of the CT receptor (CT(a)) with RAMP1, RAMP2 and RAMP3 respectively. The challenge is now to determine the localization and pharmacological nature of each of these receptors. Recent attempts to achieve these aims will be briefly discussed.
Determining calmodulin binding to metabotropic glutamate receptors with distinct protein-interaction methods
Authors: Lidwell K. , Dillon J. , Sihota A. , O'Connor V. , Pilkington B.
Published: October 2004

mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that modulate synaptic transmission. The eight mammalian mGluRs form three groups based on sequence and functional similarities: group I (1 and 5), group II (2 and 3) and group III (4, 6–8) mGluRs. In the present study, we used a Y2H (yeast two hybrid) screen to identify proteins that interact with the C-terminal intracellular tail of mGluR3. Prominent among the candidate receptor interacting proteins was calmodulin, a Ca2+ sensor known to bind identifiable sequences in group I and III mGluRs. The Y2H method was used to investigate calmodulin binding to mGluRs but failed to confirm the documented interaction with group III mGluRs. Furthermore, subsequent biochemical analysis showed that calmodulin does not interact with group II mGluRs. This illustrates that certain Ca2+-dependent interactions are not recapitulated in yeast. Moreover, it highlights the necessity for supporting biochemical data to substantiate interactions identified with Y2H methods.
Interactions between G-protein-coupled receptors and periplakin: a selective means to regulate G-protein activation
Authors: Milligan G. , Murdoch H. , Kellett E. , White J.H. , Feng G.-J.
Published: October 2004

A substantial number of G-protein-coupled receptor-interacting proteins have been identified initially by the use of yeast two-hybrid screens. Using the C-terminal tail of both opioid receptors and the melanin concentrating hormone receptor-1 as bait, the actin and intermediate filament-binding protein periplakin was isolated. In each case, the site of interaction is within helix VIII of the receptor and periplakin limits agonist-mediated G-protein activation potentially by competing with G-protein for this region of the receptor.
Allosteric modulators of metabotropic glutamate receptors: lessons learnt from mGlu1, mGlu2 and mGlu5 potentiators and antagonists
Authors: Johnson M.P. , Nisenbaum E.S. , Large T.H. , Emkey R. , Baez M. , Kingston A.E.
Published: October 2004

Although relatively few G-protein-coupled receptors are Class C, in recent years, this small family of receptors has become a focal point for the discovery of new and exciting allosteric modulators. The mGlu (metabotropic glutamate) receptors are illustrative in the discovery of both positive and/or negative allosteric modulators with unique pharmacological properties. For instance, allosteric modulators of the mGlu2 receptor act as potentiators of glutamate responses in clonal expression systems and in native tissue assays. These potentiators act to increase the affinity of orthosteric agonists for the mGlu2 receptor and shift potency curves for the agonist to the left. In electrophysiological experiments, the potentiators show a unique activation-state-dependent presynaptic inhibition of glutamate release and significantly enhance the receptor-mediated increase in G-protein binding, as seen with autoradiography. Similarly, potentiators of mGlu5 have been described, as well as allosteric antagonists or inverse agonists of mGlu1 and mGlu5. Binding and activity of the modulators have recently indicated that positive and negative allosteric sites can be, but are not necessarily, overlapping. Compared with orthosteric ligands, these modulators display a unique degree of subtype selectivity within the highly conserved mGlu family of receptors and can have very distinct pharmacological properties, such as neuronal frequency-dependent activity. This short review describes some of the unique features of these mGlu1, mGlu2 and mGlu5 allosteric modulators.
Proteomic analysis of chromatin-modifying complexes in Saccharomyces cerevisiae identifies novel subunits
Authors: Lee K.K. , Prochasson P. , Florens L. , Swanson S.K. , Washburn M.P. , Workman J.L.
Published: October 2004

Epigenetics is the alteration of phenotype without affecting the genotype. An underlying molecular mechanism of epigenetics is the changes of chromatin structure by covalent histone modifications and nucleosome reorganization. In the yeast, Saccharomyces cerevisiae, two of the most well-studied macromolecular complexes that perform these epigenetic changes are the ATP-dependent Swi/Snf chromatin-remodelling complex and the SAGA histone acetyltransferase complex. To understand fully the mechanism by which these large protein complexes perform their functions in the cell, it is crucial that all the subunits of these complexes are identified. In an attempt to identify new subunits associated with SAGA and Swi/Snf, we used tandem affinity purification, followed by a multidimensional protein identification technology to analyse the subunit composition. Our analysis identified two novel proteins, one associated with SAGA, YPL047W (Sgf11), and another associated with Swi/Snf, Rtt102.
Zebularine: a new drug for epigenetic therapy
Authors: Yoo C.B. , Cheng J.C. , Jones P.A.
Published: October 2004

Regulatory genes are often hypermethylated at their promoter 5′ regions and silenced in cancer. Epigenetic therapy with DNA methylation inhibitors have been shown to result in the demethylation and reactivation of these genes. Zebularine is a recently discovered mechanism-based inhibitor of DNA methylation, and has received much attention for its potential in clinical use. Further studies exploring the effectiveness of zebularine in a variety of settings could allow the development of novel therapies for cancer.
Control of gene expression by CpG island methylation in normal cells
Authors: Strathdee G. , Sim A. , Brown R.
Published: October 2004

The role of DNA methylation in the control of mammalian gene expression has been the subject of intensive research in recent years, partly due to the critical role of CpG island methylation in the inactivation of tumour suppressor genes during the development of cancer. However, this research has also helped elucidate the role that DNA methylation plays in normal cells. At present, it is also clear that DNA methylation forms an important part of the normal cell-regulatory processes that govern gene transcription. Methylation, targeted at CpG islands, is an important part of the mechanisms that govern X-chromosome inactivation; it is also essential for the maintenance of imprinted genes and, at least in some cases, is critical in determining the cell-type-specific expression patterns of genes. Study of these examples will be important in identifying the mechanisms that control targeting of DNA methylation and how these processes are disrupted during disease pathogenesis.
CpG-island methylation and epigenetic control of resistance to chemotherapy
Authors: Teodoridis J.M. , Strathdee G. , Plumb J.A. , Brown R.
Published: October 2004

Aberrant methylation of CpG islands (CpG-rich regions of DNA associated with the promoters of many genes) is associated with transcriptional inactivation of genes involved in tumour development. Genes involved in key DNA damage response pathways, such as cell-cycle control, apoptosis signalling and DNA repair can frequently become epigenetically silenced and methylated in tumours. This may lead to differences in intrinsic sensitivity of tumours to chemotherapy, depending on the specific function of the gene inactivated. Furthermore, chemotherapy itself may exert a selective pressure on epigenetically silenced drug sensitivity genes present in subpopulations of cells, leading to acquired chemoresistance. Clinical trials of epigenetic therapies are now in progress, and epigenetic profiling using DNA methylation will provide guidance on optimization of the use of these therapies with conventional chemotherapy, as well as helping to identify patient populations who may particularly benefit from such approaches.
HMGN proteins play roles in DNA repair and gene expression in mammalian cells
Authors: West K.L.
Published: October 2004

HMGN (high-mobility-group N) family members are vertebrate proteins that unfold chromatin and promote transcription and replication of chromatin templates in vitro. However, their precise roles in vivo have been elusive until recently. This paper summarizes recent advances from studies of Hmgn1 knockout mice and genetically engineered cell lines that are beginning to reveal the diverse roles that HMGN proteins play in DNA repair and transcription within mammalian cells.
Complex regulation and nuclear localization of JRK protein
Authors: Waldron R. , Moore T.
Published: October 2004

The mouse jerky gene and its human orthologue, JRK/JH8, encode a putative DNA-binding protein with homology to the CENP-B (centromere-binding protein B). Disruption of the mouse jerky gene by transgene insertion causes generalized recurrent seizures reminiscent of human idiopathic generalized epilepsy. In addition (and similar to a cenp-b null mouse) jerky null mice exhibit postnatal weight loss and reduced fertility. Using fluorescence confocal microscopy, the cellular localization of a JRK–GFP fusion (where GFP stands for green fluorescent protein) was investigated in HeLa cells. JRK–GFP has a dynamic expression pattern in the interphase nucleus, localizing in a small number of punctate nuclear foci and in the nucleolus. The JRK–GFP foci number changes during the cell cycle, but a distinct pattern of three JRK–GFP foci is observed at G2. The endogenous protein behaves in a similar manner to the GFP-fusion protein. JRK–GFP was found to co-localize with CREST antigens (which recognize the centromere-binding proteins, CENP-A, -B and -C) through S and G2 phases of interphase and co-localized completely with a subset of PML nuclear bodies at G2. We speculate that JRK protein associates with a specific chromosomal centromeric locus in G2, where it associates fully with PML bodies. Research is underway to identify this locus.
Roles of the U5 snRNP in spliceosome dynamics and catalysis
Authors: Turner I.A. , Norman C.M. , Churcher M.J. , Newman A.J.
Published: October 2004

Most protein-coding genes in eukaryotes are interrupted by non-coding intervening sequences (introns), which must be precisely removed from primary gene transcripts (pre-mRNAs) before translation of the message into protein. Intron removal by pre-mRNA splicing occurs in the nucleus and is catalysed by complex ribonucleoprotein machines called spliceosomes. These molecular machines consist of several small nuclear RNA molecules and their associated proteins [together termed snRNP (small nuclear ribonucleoprotein) particles], plus multiple accessory factors. Of particular interest are the U2, U5 and U6 snRNPs, which play crucial roles in the catalytic steps of splicing. In the present review, we summarize our current understanding of the role played by the protein components of the U5 snRNP in pre-mRNA splicing, which include some of the largest and most highly conserved nuclear proteins.
Regulation of NF-κB by atypical activators and tumour suppressors
Authors: Perkins N.D.
Published: October 2004

The RelA(p65) NF-κB (nuclear factor κB) subunit is typically thought of as being antiapoptotic and tumour-promoting. However, in our laboratory, we have discovered that RelA can inhibit, rather than induce, antiapoptotic gene expression when activated by certain chemotherapeutic drugs, UV light or through the action of the ARF tumour suppressor. These observations explain why RelA can sometimes facilitate rather than inhibit apoptosis and also exhibits tumour-suppressor characteristics in vivo. A better understanding of these processes and an ability to analyse RelA function in tumours could lead to improved cancer diagnosis, choice of therapy and, ultimately, development of new drugs.
Factor inhibiting hypoxia-inducible factor (FIH) and other asparaginyl hydroxylases
Authors: Lancaster D.E. , McDonough M.A. , Schofield C.J.
Published: October 2004

FIH (Factor inhibiting hypoxia-inducible factor), an asparaginyl β-hydroxylase belonging to the super-family of 2-oxoglutarate and Fe(II)-dependent dioxygenases, catalyses hydroxylation of Asn-803 of hypoxia-inducible factor, a transcription factor that regulates the mammalian hypoxic response. Only one other asparaginyl β-hydroxylase, which catalyses hydroxylation of both aspartyl and asparaginyl residues in EGF (epidermal growth factor)-like domains, has been characterized. In the light of recent crystal structures of FIH, we compare FIH with the EGFH (EGF β-hydroxylase) and putative asparagine/asparaginyl hydroxylases. Sequence analyses imply that EGFH does not contain the HXD/E iron-binding motif characteristic of most of the 2-oxoglutarate oxygenases.
Silence is green
Authors: Herr A.J.
Published: October 2004

Small RNAs serve as the specificity determinant for a collection of regulatory mechanisms known as RNA silencing. Plants use these mechanisms to control the expression of endogenous genes and to suppress unwanted foreign nucleic acids. Several gene families implicated in silencing have undergone expansion and evidence exists for multiple RNA silencing pathways. Recent progress in defining the components of a number of these pathways is examined here.
Understanding the roles of RecQ helicases in the maintenance of genome integrity and suppression of tumorigenesis
Authors: Mankouri H.W. , Hickson I.D.
Published: October 2004

RecQ helicases are evolutionarily conserved enzymes required for the maintenance of genome stability. Mutations in three of the five known human RecQ helicase genes cause distinct clinical disorders that are characterized by genome instability and cancer predisposition. Recent studies have begun to reveal the cellular roles of RecQ helicases and how these enzymes may prevent tumorigenesis at the molecular level.
Poly(ADP-ribose) polymerase inhibitors as potential chemotherapeutic agents
Authors: Bryant H.E. , Helleday T.
Published: October 2004

PARP [poly(ADP-ribose) polymerase] activity is up-regulated by binding to DNA strand breaks and its association with DNA repair is well documented. Many anticancer therapies work by inducing breaks in DNA, if unrepaired these can lead to cell death. As PARP promotes DNA repair there is a strong rational to suggest that its inhibition may increase the efficiency of certain cytotoxic treatments. This review discusses the advances made in PARP inhibitor design and the mechanism by which they enhance anti-tumour therapies.
Monitoring base excision repair proteins on damaged DNA using human cell extracts
Authors: Parsons J.L. , Dianov G.L.
Published: October 2004

BER (base excision repair) is a major pathway for the removal of simple lesions in DNA including base damage and base loss (abasic site). We have developed an assay, using formaldehyde cross-linking during repair in human cell extracts, to observe BER proteins involved in the repair of damaged DNA. This approach allows visualization of repair proteins on damaged DNA during BER in human cell extracts and provides a detailed view of the molecular events leading to repair.
Dissection of the signalling mechanisms underlying FcγRIIB-mediated apoptosis of mature B-cells
Authors: Carter N.A. , Harnett M.M.
Published: October 2004

The low-affinity receptor for IgG, FcγRIIB, negatively regulates BCR (B-cell antigen receptor)-mediated proliferative signalling and thus plays an important role in feedback inhibition of the humoral immune response. Whereas crosslinking of BCR on mature B-cells results in proliferation, co-ligation of FcγRIIB results in growth arrest and apoptosis. We have now investigated the signals underlying FcγRIIB-driven apoptosis and found this to be dependent on disruption of mitochondrial potential (Δψ), involve the pro-apoptotic Bcl-2 family members, Bid and Bad, and be caspase-independent.
Mitogenic stimulation of transcription by RNA polymerase III
Authors: Mauger E. , Scott P.H.
Published: October 2004

Regulation of protein synthesis is an important aspect of growth control. RNA polymerase (pol) III plays a key role in this process by catalysing production of tRNA and 5 S rRNA. Growth factors trigger a rapid increase in pol III activity and this is essential for cell proliferation. The transcription factor TFIIIB plays a key role in controlling pol III activity and is a target for regulation by a number of mechanisms. This review will focus on how TFIIIB is targeted by these proteins in response to mitogen stimulation.
Oxidative stress in colon tissue induced by vitamin E depletion
Authors: Drew J.E. , Mercer D.K. , Mayer C. , Farquharson A.J. , Morrice P.C. , Arthur J.R. , Duthie G.G.
Published: October 2004

Inflammatory disorders of the bowel and colon cancer are associated with elevated indices of oxidative stress. Analogous elevations in markers of oxidative stress and loss of cell-membrane integrity are also observed in the colons of rats deficient in vitamin E (D-α-tocopherol), the major lipid-soluble antioxidant in biological systems. The causal relationship between colon pathologies associated with oxidative stress and dietary deficiency in antioxidant vitamins such as vitamin E is still uncertain. Investigation of potential mechanisms by which lack of dietary vitamin E may lead to clinically relevant pathological changes in colon tissue was conducted using gene expression profiling strategies on vitamin E-sufficient and -deficient rats. Morphological changes and increased indices of lipid peroxidation were linked to vitamin E deficiency. These changes in colon tissue are potentially important in disease pathogenesis of the colon linked with oxidative stress or other direct consequences of inadequate levels of vitamin E.
Dietary carotenoids, connexins and cancer: what is the connection?
Authors: Bertram J.S.
Published: October 2004

Carotenoids and retinoids are chemically related; indeed a major source of vitamin A in humans occurs through enzymic cleavage of β-carotene. However, most dietary carotenoids cannot be converted into retinoids. Retinoids have demonstrated cancer-preventive activities in humans and experimental models; however, their toxicity has precluded wide-scale clinical use. In contrast, carotenoids are essentially non-toxic and their cancer-preventive activities, although strongly supported by epidemiological studies, have only been satisfactorily demonstrated in experimental systems. We have shown that in an experimental cell culture system consisting of carcinogen-treated 10T1/2 cells, both retinoids and all dietary carotenoids examined can reversibly inhibit neoplastic transformation in the post-initiation phase of carcinogenesis. This activity strongly correlates with their ability to increase gap junctional intercellular communication by up-regulating the expression of the gene CX43 (connexin43). Connexins comprise the structural unit of gap junctions, organelles which allow direct transfer of signals, nutrients and waste products between contacting cells. CX43 is the most widely expressed member of the gap junction family of genes, and we have demonstrated that its expression is strongly down-regulated in human cancers and in several premalignant conditions. When several human tumour cell lines were genetically engineered to conditionally express CX43 under the influence of a tetracycline promoter, their neoplastic phenotype was strongly attenuated. Specifically, induced cells were inhibited from growing in an anchorage-independent manner and, additionally, growth as xenografts in immunocompromised animals was also strongly attenuated. Growth inhibition in suspension was associated both with increased G1 cell-cycle arrest and with increased apoptosis. We propose a model whereby junctional communication allows the transfer of growth inhibitory signals from normal to neoplastic cells and that retinoids and carotenoids, by increasing signal transfer, act to prevent cancer.
Triacylglycerol-rich lipoprotein–gene interactions in endothelial cells
Authors: Williams C.M. , Maitin V. , Jackson K.G.
Published: October 2004

Lipoproteins such as LDL (low-density lipoprotein) and oxidized LDL have potentially adverse effects on endothelial cells due to their ability to activate pro-inflammatory pathways regulated via the transcription factor NF-κB (nuclear factor κB). Triacylglycerol-rich lipoproteins (the chylomicrons, very-low-density lipoprotein and their respective remnant particles) have also been implicated in the induction of a pro-inflammatory phenotype and up-regulation of adhesion molecule expression. Although early studies supported the proposal that LPL (lipoprotein lipase)-mediated hydrolysis of TRLs (triglyceride-rich lipoproteins) at the endothelium could activate the NFκB pathway, more recent studies provide evidence of pro- and anti-inflammatory responses when cells are exposed to fatty acids or TRL particles. A large number of genes are up- and down-regulated when cells are exposed to TRL, with the net effect reflecting receptor- and nonreceptor-mediated pathways that are activated or inhibited depending on fatty acid type, the lipid and apolipoprotein composition of the TRL and the presence or absence of LPL. Early concepts of TRL particles as essentially pro-inflammatory stimuli to the endothelium provide an overly simplistic view of their impact on the vascular compartment.
Haem oxygenase-1: a target for dietary antioxidants
Authors: Ogborne R.M. , Rushworth S.A. , Charalambos C.A. , O'Connell M.A.
Published: October 2004

HO-1 (haem oxygenase-1) is a stress-response enzyme involved in the catabolism of haem. In animal models, it plays a key protective role in vascular disease. HO-1 has anti-inflammatory effects in macrophages and is induced by a range of stimuli, including antioxidants, in various cell types. As dietary antioxidants are considered to be beneficial in vascular disease, their protective effects may occur through induction of HO-1. Emerging evidence suggests that a range of dietary and other naturally occurring antioxidants stimulate HO-1 expression in various cell types, although regulation by these compounds has not been investigated in detail. These studies suggest that HO-1 may be a target for dietary therapy in vascular disease.
Oxidant regulation of the bivalent cation transporter Nramp1
Authors: Yeung I.Y.L. , Phillips E. , Mann D.A. , Barton C.H.
Published: October 2004

Nramp1 (murine natural resistance-associated macrophage protein 1 gene)/Slc11a1 (solute carrier family 11 member a1 gene) encodes a bivalent-metal/iron transporter that is expressed within late endosomes/lysosomes of macrophages. A functionally null Nramp1 allele that exhibits impaired bivalent cation transport enables excessive growth of intracellular pathogens. Iron is important for many cellular activities, including defence against pathogens; however, redox-active/free iron can participate in Fenton chemistry that generates reactive oxygen species. Using Raw264.7 cells, non-functional for Nramp1, and stable Nramp1 transfectants, we have examined the effects of impaired bivalent cation transport on macrophage function using glutathione depletion as OS (oxidant stress). Our results demonstrate that OS itself is a signal for increasing Nramp1 transcription and that Nramp1 expression protects against OS. We suggest that OS-mediated protection by Nramp1 function may arise from direct removal of redox-active bivalent cations from a cytosolic pool. We show that OS transcriptional responses are probably mediated by the Sp1 transcription factor.
Investigating mitochondrial radical production using targeted probes
Authors: Murphy M.P.
Published: October 2004

A range of mitochondria-targeted probe molecules that comprise a lipophilic cation covalently attached to an active moiety have been developed. The lipophilic cation causes the accumulation of these molecules into mitochondria, driven by the mitochondrial membrane potential. To date, we have targeted antioxidants, spin traps, thiol reagents and DNA-alkylating compounds to mitochondria. The selective mitochondrial localization of these compounds enables us to investigate several aspects of the production of reactive oxygen species by mitochondria.
Reactive sulphur species in oxidative signal transduction
Authors: Jacob C. , Lancaster J.R. , Giles G.I.
Published: October 2004

Intense interest has been generated by the discovery that reactive oxygen species can function as intracellular second messengers. Reactive oxygen species have been implicated in diverse cellular processes, including growth factor signal transduction, gene expression and apoptosis. Additionally, there is evidence for proteins that are regulated by redox environment through the reversible oxidation of their cysteine residues. However, the direct reaction of reactive oxygen species with cysteine at physiological concentrations is generally a slow process, suggesting that intermediates are required to convey efficiently the oxidative stimulus. Here, we discuss the evidence that DSOs (disulphide-S-oxides) are formed from glutathione under oxidizing conditions and specifically modulate the redox status of thiols, indicating the existence of specialized cellular oxidative pathways. DSO inactivated glyceraldehyde 3-phosphate and alcohol dehydrogenases and released zinc from metallothionein and a zinc finger domain. In contrast, equivalent concentrations of H2O2 showed minimal effect. The antioxidants ascorbate, NADH, trolox and melatonin were unable to quench DSO-induced oxidation. These findings support the paradigm of oxidative signal transduction and provide a general pathway whereby reactive oxygen species can convert thiols into disulphides.
Direct interactions between NMDA and D1 receptors: a tale of tails
Authors: Lee F.J.S. , Liu F.
Published: October 2004

Considerable evidence has accumulated describing a complex interaction between the dopaminergic and glutamatergic pathways. Efforts to describe the mechanisms underlying this complex interaction have implicated a functional interaction between dopamine and glutamate receptors. Classically, the interaction between D1 and NMDA (N-methyl-D-aspartate) receptors has been proposed to involve the activation of second-messenger signalling cascades after receptor stimulation. However, in recent years, another paradigm has emerged which involves the direct interaction between D1 and NMDA receptors. The physical association between D1 and NMDA receptors is unique in that two different regions of the D1 C-terminus are able to couple specifically and physically with two different NMDA subunits. The selective modulation of multiple NMDA receptor-mediated functions by direct interactions with D1 receptors may form a new avenue to identify specific targets for therapeutics to modulate NMDA receptor-governed synaptic plasticity, neuronal development and disease states.
Evidence to support a spectrum of active states for the glucagon receptor
Authors: Strudwick N. , Bhogal N. , Evans N.A. , Blaney F.E. , Findlay J.B.C.
Published: October 2004

The ternary complex model suggests that G-protein-coupled receptors resonate between inactive (R) and active (R*) forms. Physiologically, R sites ordinarily predominate with a few R* sites giving rise to basal activity. Agonists recognize, stabilize and increase the R* population, thus altering intracellular activity. There is evidence to suggest the possibility of a spectrum of conformations between R and R*. Our aim is to study the consequences of putative GR (glucagon receptor)-activating mutations using glucagon and partial agonist des-His1-[Glu9]glucagon amide (glucagon-NH2). Alanine substitution in TM (transmembrane) helix 2 of Arg173 or of His177 detrimentally affected glucagon and glucagon-NH2 response maxima. TM2 receptor mutant, Phe181-Ala, displayed reduced maximum cAMP accumulation in response to glucagon-NH2. Thr353-Cys (TM6) and Glu406-Ala (TM7) receptors demonstrated constitutive activity and enhanced EC50 values for glucagon-NH2; Arg346-Ala (TM6) and Asn404-Ala (TM7) receptors were activated by sub-fmol glucagon concentrations, yet were not constitutively active and demonstrated wild-type receptor-like EC50 values for glucagon-NH2. Unlike Arg346-Ala receptors, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors demonstrated improved EC50 values for glucagon, whereas their maximal responses to and their affinity for glucagon were comparable with the wild-type receptor. In contrast, despite slightly reduced glucagon-NH2 affinity, Arg346-Ala, Thr353-Cys, Asn404-Ala and Glu406-Ala receptors displayed glucagon-NH2 response maxima that exceeded those seen for wild-type receptors. Interestingly, we observed biphasic glucagon-mediated signalling responses. Our results are consistent with the concept of different agonists promoting the formation of distinct active states from partially active R*low to fully active R*high forms.
Mass spectroscopic analysis of phosphatidylinositol synthesis using 6-deuteriated-myo-inositol: comparison of the molecular specificities and acyl remodelling mechanisms in mouse tissues and cultured cells
Authors: Postle A.D. , Dombrowsky H. , Clarke H. , Pynn C.J. , Koster G. , Hunt A.N.
Published: October 2004

Mammalian cell PtdIns (phosphatidylinositol) in vivo is enriched in the sn-1-stearoyl 2-arachidonoyl species, the physiological precursor of phosphatidylinositol 4,5-bisphosphate. Mechanisms regulating this specificity are unclear but are typically lost for cells in culture. We used ESI-MS (tandem electrospray ionization-mass spectrometry) to determine the molecular species of PtdIns synthesized by mouse tissues in vivo compared with cultured cells in vitro. After incorporation of deuteriated myo-d6-inositol over 3 h, endogenous and newly synthesized PtdIns and lysoPtdIns species were quantified from precursor scans of m/z 241− and m/z 247− respectively. PtdIns was synthesized as a wide range of species irrespective of the final membrane composition. Analyses of isotope enrichments argued against acyl remodelling as the major regulatory mechanism: composition of the lysoPtdIns pool under all conditions reflected that of either endogenous or newly synthesized PtdIns and was always at equilibrium. The kinetics of PtdIns synthesis, together with the prolonged time scale required for achieving final equilibrium compositions suggest that selective transport between membranes and/or hydrolysis of selected molecular species are the most probable mechanisms regulating compositions of PtdIns and, ultimately, phosphatidylinositol 4,5-bisphosphate.
Lipidomic analysis of the molecular specificity of a cholinephosphotransferase in situ
Authors: Hunt A.N. , Fenn H.C. , Clark G.T. , Wright M.M. , Postle A.D. , McMaster C.R.
Published: October 2004

Dynamic lipidomics using ESI–MS (tandem electrospray ionization mass spectrometry) of 9-deuterated choline (choline-d9) incorporation into mammalian cell PtdCho (phosphatidylcholine) permits assessment of the molecular specificity of synthesis. Bulk cell PtdCho synthesis occurs in spatially distinct locations, using separate CPTs (1,2 diacylglycerol CDP:choline cholinephosphotransferases). We assessed whether in vitro molecular selectivity of DAG (diacylglycerol) incorporation between CPTs is manifest in situ, by monitoring choline-d9 incorporation into PtdCho and lyso-PtdCho molecular species over 3 h in control cells and in CHO-K1 cells overexpressing hCEPT1. Compared with controls, the basal molecular species composition of hCEPT1 overexpressors was significantly enriched in arachidonate. This was not due to net accretion of cellular PtdCho arguing against effects of inadequate unsaturated PtdCho degradation or remodelling. Rather, time-course analyses of PtdCho and lyso-PtdCho pools showed that both arachidonate-containing DAG incorporation and turnover of PtdCho is increased in hCEPT1 overexpressors. Increased choline-d9 incorporation into arachidonyl lyso-PtdCho shows that both phospholipase A1- and A2-mediated turnover is involved. Spatially distinct molecular specificity of DAG incorporation into cellular PtdCho at the level of hCEPT1 exists in situ.
Selective elimination of mitochondria from living cells induced by inhibitors of bioenergetic functions
Authors: Lyamzaev K.G. , Pletjushkina O.Y. , Saprunova V.B. , Bakeeva L.E. , Chernyak B.V. , Skulachev V.P.
Published: October 2004

The inhibitors of oxidative phosphorylation induced fragmentation of mitochondria without any signs of apoptosis in CV-1 and HeLa cells. Prolonged treatment with the uncouplers (alone or in combination with the inhibitors of respiration) caused perinuclear clusterization of mitochondria, followed by their selective elimination. The fraction of mitochondria-depleted cells remained viable.
Sab (SH3BP5), a novel mitochondria-localized JNK-interacting protein
Authors: Wiltshire C. , Gillespie D.A.F. , May G.H.W.
Published: October 2004

The JNK (c-Jun N-terminal kinase) pathway is activated by diverse stresses and can have an effect on a number of different cellular processes. Protein–protein interactions are critical for efficient signalling from JNK to multiple targets; through a screen for interacting proteins, we identified a novel JNK-interacting protein, Sab (SH3BP5). Sab has previously been found to interact with the Src homology 3 domain of Bruton's tyrosine kinase; however, the interaction with JNK occurs through a mitogen-activated protein KIM (kinase interaction motif) in a region distinct from the Bruton's tyrosine kinase-binding domain. As with c-Jun, the presence of this KIM is essential for Sab to act as a JNK substrate. Interestingly, Sab is associated with the mitochondria and co-localizes with a portion of active JNK after stress treatment. The present study and previously reported work may suggest a possible role for Sab in targeting JNK to this subcellular compartment and/or mediating crosstalk between different signal-transduction pathways.