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Biomedical subjects

M Waterfield

Publications and source records attributed to M Waterfield.

At least 19 recordsLinked to original sources
PubMed2007

Cracking the mild, difficult and fiendish codes within and downstream of the EGFR to link diagnostics and therapeutics.

Over the last 45 years, I have been working on growth factors, their receptors and signal transduction mechanisms. This period has seen a tremendous growth in knowledge and technology, and all of this, together with a focus interest in oncology, has steered me along a path designed to understand growth factor signalling so that we can see how drugs that target signalling pathways might be able to control cancer. The knowledge that we already have is likely to lead to cures for many common cancers within the next 25 years.

Animals↗
PubMed2001

Retroviral oncoprotein Tax induces processing of NF-kappaB2/p100 in T cells: evidence for the involvement of IKKalpha.

IkappaB kinase (IKK) is a key mediator of NF-kappaB activation induced by various immunological signals. In T cells and most other cell types, the primary target of IKK is a labile inhibitor of NF-kappaB, IkappaBalpha, which is responsible for the canonical NF-kappaB activation. Here, we show that in T cells infected with the human T-cell leukemia virus (HTLV), IKKalpha is targeted to a novel signaling pathway that mediates processing of the nfkappab2 precursor protein p100, resulting in active production of the NF-kappaB subunit, p52. This pathogenic action is mediated by the HTLV-encoded oncoprotein Tax, which appears to act by physically recruiting IKKalpha to p100, triggering phosphorylation-dependent ubiquitylation and processing of p100. These findings suggest a novel mechanism by which Tax modulates the NF-kappaB signaling pathway.

Amino Acid Sequence↗
PubMed2001

NF-kappaB signaling pathway governs TRAIL gene expression and human T-cell leukemia virus-I Tax-induced T-cell death.

The Tax oncoprotein encoded by human T-cell leukemia virus induces both T-cell activation and apoptosis. The mechanism by which Tax induces apoptosis has remained unclear. Using genetically manipulated T-cell lines, we demonstrate that Tax-induced T-cell death is dependent on NF-kappaB signaling. Tax fails to induce apoptosis in T cells lacking IkappaB kinase gamma (IKKgamma), an essential component of the NF-kappaB signaling pathway. This defect was rescued when the mutant cells were reconstituted with exogenous IKKgamma. We further demonstrate that the Tax-induced T-cell death is mediated by TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL), because this event can be effectively inhibited by a TRAIL-blocking antibody. Consistent with this functional aspect, Tax stimulates the expression of TRAIL mRNA. Finally, we provide genetic evidence demonstrating that the NF-kappaB signaling pathway is essential for TRAIL gene induction by both Tax and T-cell activation signals. These studies reveal a novel function of the NF-kappaB signaling pathway and suggest a key mechanism by which Tax induces T-cell death.

Apoptosis↗
PubMed2000

Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein.

Class I(A) phosphatidylinositol 3-kinase (PI 3-kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Ruk(l), which forms complexes with the PI 3-kinase holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of Ruk and the SH3 domain of the p85 alpha regulatory subunit of the class I(A) PI 3-kinase. In contrast to many other adaptor proteins that activate PI 3-kinase, interaction with Ruk(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Ruk(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of PI 3-kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3-kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The health of their nation: how would citizens develop England's health strategy?

BACKGROUND: England's first health strategy, The Health of the Nation, was formulated without systematic input from the citizens whose health it targeted. Several studies indicate that citizens, when asked to prioritize services, rank interventions for acute and life-threatening conditions highest. But how they view and what they want in the areas of prevention, public health, or care for the chronically and mentally ill is not known. AIM: To explore citizens' attitudes towards England's health strategy as set out in The Health of the Nation and to elicit their ideas for developing it further. METHOD: The study was conducted in four general practices and a secondary school in southwest England. The design was a qualitative analysis of testimony from 24 audiotaped focus groups. Twenty-three groups were drawn from lists of National Health Service registered patients, stratified by The Health of the Nation target category, and one group was drawn from 13 to 15 year-old girls at a mixed secondary school. In all, 173 citizens took part in the 24 focus group meetings. RESULTS: In group discussions, these citizens demonstrated an understanding of The Health of the Nation strategy, which enabled them to form views and develop relevant arguments. They produced 26 specific ideas for developing the strategy across its five key areas. There was congruence with the action plans of a national expert group convened by the Department of Health and charged with reviewing the strategy's progress. The focus groups went beyond the experts' proposals, with further practical ideas to achieve The Health of the Nation targets. CONCLUSION: Citizens in this study contributed a broad range of relevant, appropriate, and innovative ideas on how to develop health strategy. The use of focus groups to achieve this is practical and efficient.

Adolescent↗
PubMed1994

Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study.

Amino Acid Sequence↗
PubMed1994

Crystal structure of the pleckstrin homology domain from dynamin.

The pleckstrin homology (PH) domain is a conserved module present in many signal transducing and cytoskeletal proteins. Here we report the 2.8 A crystal structure of the PH domain from dynamin. This domain consists of seven beta-strands forming two roughly orthogonal antiparallel beta-sheets terminating with an amphipathic alpha-helix. The structure also reveals a non-covalent dimeric association of the PH domain and a hydrophobic pocket surrounded by a charged rim. The dynamin PH domain structure is discussed in relation to its potential role in mediating interactions between proteins.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of phosphatidylinositol 4-kinase from human erythrocyte membranes.

Two species of PtdIns 4-kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X-100, followed by Cibacron-blue-Sephadex, phosphocellulose and Mono Q anion-exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000-fold purification of the enzyme from erythrocyte membranes. Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D-4 position of the inositol ring. The Km values of both PtdIns 4-kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. The enzymes are both activated by Mg2+, and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Isolation of a polymorphic genomic clone from chromosome 7. Physical and genetic linkage studies to markers around the cystic fibrosis locus.

A peptide prepared from purified factor 13B (F13B) was sequenced, and a single, long oligonucleotide corresponding to its cognate DNA sequence was constructed and used to screen a chromosome 7 specific genomic library. The positive clone isolated, designated pKV13, was only related to F13B at the oligonucleotide region, but has proved to be a valuable chromosome 7 marker. pKV13 maps to 7pter-q22 in hybrid cell lines, and is present in a chromosome-mediated gene transfer (CMGT) cell line that also contains met and other 7q probes. pKV13 defines a common MspI restriction fragment length polymorphism (RFLP), and is genetically linked to two markers on the long arm of chromosome 7, B79a and COLIA2, both themselves linked to the cystic fibrosis locus. Multipoint linkage analysis demonstrates that KV13 maps centromeric to both B79a and COLIA2. pKV13 has been used to demonstrate the existence of rearrangements within CMGT hybrids, and will also prove valuable in multipoint linkage studies of other 7q markers. Finally, pKV13 provides a new polymorphic locus for the characterisation of 7q deletions in myeloid disorders such as myelodysplastic syndrome.

Amino Acid Sequence↗
PubMed1986

Sequence and expression of human estrogen receptor complementary DNA.

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.

Amino Acid Sequence↗
PubMed1986

Cloning of the human oestrogen receptor cDNA.

Poly A+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and those fractions enriched in oestrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt11 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kbase) and lambda OR8 (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains DNA sequences which cross-hybridize with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Using lambda OR8 as a hybridization probe revealed a single poly A+ RNA band of approx. 6.2 kbase in the ER containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-cell line HeLa. The same probe hybridizes to a chicken gene which is expressed in oviduct tissue as a 7.5 kbase poly A+ RNA.

Animals↗
PubMed1985

Cloning of the human estrogen receptor cDNA.

Poly(A)+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and fractions enriched in estrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt10 and lambda gt11 vectors. Clones corresponding to ER sequence were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five were isolated after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kilobase pairs) and lambda OR8 (2.1 kilobase pairs), isolated by using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains the DNA sequence expected from the two ER peptides and crosshybridizes with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Use of lambda OR8 as a hybridization probe revealed a single poly(A)+ RNA band of approximately equal to 6.2 kilobase pairs in the ER-containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-negative cell line HeLa. The same probe hybridizes to a chicken gene that is expressed in oviduct tissue as a 7.5-kilobase-pair poly(A)+ RNA.

Animals↗
PubMed1983

Purification and partial amino acid sequence analysis of the cellular tumour antigen, p53, from mouse SV40-transformed cells.

The cellular tumour antigen p53 is implicated in the transformation process. To compare p53 from transformed cells and their normal counterparts in detail, and so to identify any structural differences that might alter p53 function, requires information on the primary structure of the protein. By making use of immunochemical techniques we have been able to purify nanomole amounts of p53. This was sufficient, using high sensitivity automated gas-phase sequencing techniques to determine the amino acid sequence of two tryptic peptides from p53. Their sequences agree completely with the predicted polypeptide sequence derived from a cloned cDNA for p53 mRNA and provide the first data on the amino acid sequence of p53. A combination of the high sensitivity amino acid sequencing procedures used here and cDNA sequence analysis should provide the complete amino acid sequence of p53.

Amino Acid Sequence↗
PubMed1981

Disulphide bonds of haemagglutinin of Asian influenza virus.

The complete amino acid sequences of the haemagglutinins (HAs) of one strain of each of the Hav1 and H2 subtypes and of three strains of the H3 subtype of influenza virus have been established. A large external fragment (BHA) of HA can be released from virus particles using the protease bromelain that cleaves the protein close to its carboxyl terminus, which is inserted in the virus membrane (see Fig 1). Here we show that a single disulphide bond joins the two component polypeptide chains BHA1 and BHA2 and establish the location of five intrachain disulphides. These disulphide bonds have been located in the three-dimensional structure of HA which is known to 3 A resolution.

Amino Acid Sequence↗
PubMed1980

Cloning and DNA sequence of double-stranded copies of haemagglutinin genes from H2 and H3 strains elucidates antigenic shift and drift in human influenza virus.

Double-stranded DNA copies of the RNA gene coding for the haemagglutinin glycoproteins from human H2 and H3 pandemic strains of influenza virus have been cloned. DNA sequence analysis provides the first reported complete nucleotide sequence of an H2 haemagglutinin gene and a partial sequence (45%) of the H3 gene. The H2 haemagglutinin gene consists of 1,773 nucleotides containing an uninterrupted coding sequence of 1,686 nucleotides specifying a protein of 562 amino acids. Comparison of the amino acid sequences of these haemagglutinins with those of other H3 and avian strains reveals the extent of sequence changes in antigenic shifts and drifts.

Amino Acid Sequence↗