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Biomedical subjects

D J Powell

Publications and source records attributed to D J Powell.

At least 19 recordsLinked to original sources
PubMed2006

Expression of p53 and p53/47 are controlled by alternative mechanisms of messenger RNA translation initiation.

P53 controls the growth and survival of cells by acting in response to a multitude of cellular stresses. It is, however, not yet fully understood how different p53 activation pathways result in either cell cycle arrest or apoptosis. We and others have described an N-terminally truncated p53 protein (p53/47) originating from a second translation initiation site in the p53 messenger RNA (mRNA), which can interact with p53 and impose altered stability and transactivation properties to p53 complexes. Here we show that cap-dependent and cap-independent mechanisms of initiation govern the translation of the p53 mRNA. Changes in synthesis of full-length p53 or p53/47 are regulated through distinct cell stress-induced pathways acting through separate regions of the p53 mRNA. We also show that some cytotoxic drugs require the presence of full-length p53 to induce apoptosis, whereas for others p53/47 is sufficient. This indicates that by harbouring alternative translation initiation sites, the p53 mRNA gives rise to different levels of the p53 isoforms which help to orchestrate the cell biological outcome of p53 activation in response to different types of cell stress. This sheds new light into the way p53 can integrate and differentiate a large multiplicity of changes in the cellular environment.

5' Untranslated Regions↗
PubMed2001

Altered gene expression in immunogenic poorly differentiated thyroid carcinomas from RET/PTC3p53-/- mice.

Cancers develop and progress via activation of oncogenes and loss of tumor suppressor genes, a progression that can be recapitulated through cross breeding mouse strains harboring genetic mutations. To define the role of RET/PTC3, p53 and Fhit in thyroid carcinogenesis, we intercrossed RET/PTC3 transgenics with p53-/- mice. This new strain, RET/PTC3p53-/-, succumb to rapidly growing and strikingly large multilobed thyroid tumors containing mixtures of both well and poorly differentiated, highly proliferative follicular epithelial cells. Interestingly, transplanted tumors from RET/PTC3p53-/- mice grew in SCID but not syngeneic immunocompetent mice indicating that these advanced tumors were immunogenic. RET/PTC3 protein expression was reduced to undetectable levels in tumors of older mice suggesting that the continued elevated expression of RET/PTC3 may not be necessary for tumor progression. Similarly, expression of Fhit protein was reduced in early tumors and undetected in older tumors irrespective of tumor histopathology. In contrast to RET/PTC3p53-/- mice, RET/PTC3Fhit-/- mice did not develop advanced thyroid carcinomas. These studies support a model of human thyroid cancer whereby thyroid epithelium expresses RET/PTC3 protein at early stages of tumor development, followed by the reduction of RET/PTC3 and loss of p53 function with progressive reduction of Fhit protein expression coincident with malignant progression.

Acid Anhydride Hydrolases↗
PubMed2001

Characterization of the glycosylation profiles of Alzheimer's beta -secretase protein Asp-2 expressed in a variety of cell lines.

Amyloid 39-42 beta -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39-42 beta-peptide is formed from amyloid precursor protein by the sequential action of beta- and gamma-secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have beta-secretase activity. Mature Asp-2 has four N-glycosylation sites. In this report we have characterized the carbohydrate structures in this glycoprotein expressed in three different cell lines, namely Chinese hamster ovary, CV-1 origin of SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary oligosaccharides of the "complex" type were released from glycoprotein expressed in the mammalian cells, whereas mannose-rich glycans were identified from glycoprotein synthesized in the baculovirus-infected cells. Site-directed mutagenesis of the asparagine residues at amino acid positions 153, 172, 223, and 354 demonstrate that the protease activity of Asp-2 is dependent on its glycosylation.

Alzheimer Disease↗
PubMed2000

The TRK-T1 fusion protein induces neoplastic transformation of thyroid epithelium.

Genetic analysis of human papillary thyroid carcinomas (PTC) has revealed unique chromosomal translocations that form oncogenic fusion proteins and promote thyroid tumorigenesis in up to 60% of tumors examined. Although, the majority of thyroid specific translocations involve the growth factor receptor c-RET, variant rearrangements of the receptor for nerve growth factor, NTRK1 have also been described. One such translocation, TRK-T1, forms a fusion protein composed of the carboxyl terminal tyrosine kinase domain of NTRK1 and the amino terminal portion of TPR (Translocated Promoter Region). To determine if TRK-T1 expression can cause thyroid cancer in vivo, we developed transgenic mice that express the human TRK-T1 fusion protein in the thyroid. Immunohistochemical analysis of TRK-T1 transgenic mouse thyroids revealed TRK-T1 staining within the thyroid follicular epithelium. In contrast to nontransgenic littermates, 54% of transgenic mice developed thyroid abnormalities that included follicular hyperplasia and papillary carcinoma. Furthermore, all transgenic mice examined greater than 7 months of age developed thyroid hyperplasia and/or carcinoma. These data support the conclusion that TRK-T1 is oncogenic in vivo and contributes to the neoplastic transformation of the thyroid.

Animals↗
PubMed2000

ASP1 (BACE2) cleaves the amyloid precursor protein at the beta-secretase site.

Sequential proteolytic processing of the Amyloid Precursor Protein (APP) by beta- and gamma-secretases generates the 4-kDa amyloid (A beta) peptide, a key component of the amyloid plaques seen in Alzheimer's disease (AD). We and others have recently reported the identification and characterisation of an aspartic proteinase, Asp2 (BACE), as beta-secretase. Here we describe the characterization of a second highly related aspartic proteinase, Asp1 as a second beta-secretase candidate. Asp1 is expressed in brain as detected at the mRNA level and at the protein level. Transient expression of Asp1 in APP-expressing cells results in an increase in the level of beta-secretase-derived soluble APP and the corresponding carboxy-terminal fragment. Paradoxically there is a decrease in the level of soluble A beta secreted from the cells. Asp1 colocalizes with APP in the Golgi/endoplasmic reticulum compartments of cultured cells. Asp1, when expressed as an Fc fusion protein (Asp1-Fc), has the N-terminal sequence ALEP..., indicating that it has lost the prodomain. Asp1-Fc exhibits beta-secretase activity by cleaving both wild-type and Swedish variant (KM/NL) APP peptides at the beta-secretase site.

Amyloid Precursor Protein Secretases↗
PubMed1998

Napsins: new human aspartic proteinases. Distinction between two closely related genes.

cDNA sequences were elucidated for two closely related human genes which encode the precursors of two hitherto unknown aspartic proteinases. The (pro)napsin A gene is expressed predominantly in lung and kidney and its translation product is predicted to be a fully functional, glycosylated aspartic proteinase (precursor) containing an RGD motif and an additional 18 residues at its C-terminus. The (pro)napsin B gene is transcribed exclusively in cells related to the immune system but lacks an in-frame stop codon and contains a number of polymorphisms, one of which replaces a catalytically crucial Gly residue with an Arg. Consideration is given to whether (pro)napsin B may be a transcribed pseudogene or whether its putative protein product undergoes rapid intracellular degradation.

Amino Acid Sequence↗
PubMed1998

The RET/PTC3 oncogene: metastatic solid-type papillary carcinomas in murine thyroids.

Our research goal is to better understand the mechanisms controlling the initiation and progression of thyroid diseases. One such disease, papillary thyroid carcinoma (PTC), is the leading endocrine malignancy in the United States. Recently, a family of related fusion proteins, RET/PTC1-5, has been implicated in the early stages of PTC. Although all five members of this family have the c-RET proto-oncogene kinase domain in their COOH terminus, little is known about how these genes alter follicular cell biology. Consequently, to answer questions related to the mechanism of the RET/PTC fusion protein action, we have devised a molecular genetic strategy to study PTC using a mouse model of thyroid disease. A new member of this fusion oncogene family, RET/PTC3, which has been implicated in more cases of solid tumor carcinoma (79%) than PTC1 or PTC2 and predominates (80%) in radiation-induced thyroid cancer of children, was investigated in our study. We have generated transgenic mice expressing human RET/PTC3 exclusively in the thyroid. These mice develop thyroid hyperplasia, solid tumor variants of papillary carcinoma and metastatic cancer. This new transgenic line will be useful in deciphering the molecular and biological mechanisms that cause PTC and histological variations in humans.

Animals↗
PubMed1998

Toward a universal inhibitor of retroviral proteases: comparative analysis of the interactions of LP-130 complexed with proteases from HIV-1, FIV, and EIAV.

One of the major problems encountered in antiviral therapy against AIDS is the emergence of viral variants that exhibit drug resistance. The sequences of proteases (PRs) from related retroviruses sometimes include, at structurally equivalent positions, amino acids identical to those found in drug-resistant forms of HIV-1 PR. The statine-based inhibitor LP-130 was found to be a universal, nanomolar-range inhibitor against all tested retroviral PRs. We solved the crystal structures of LP-130 in complex with retroviral PRs from HIV-1, feline immunodeficiency virus, and equine infectious anemia virus and compared the structures to determine the differences in the interactions between the inhibitor and the active-site residues of the enzymes. This comparison shows an extraordinary similarity in the binding modes of the inhibitor molecules. The only exceptions are the different conformations of naphthylalanine side chains at the P3/P3' positions, which might be responsible for the variation in the Ki values. These findings indicate that successful inhibition of different retroviral PRs by LP-130 is achieved because this compound can be accommodated without serious conformational differences, despite the variations in the type of residues forming the active-site region. Although strong, specific interactions between the ligand and the enzyme might improve the potency of the inhibitor, the absence of such interactions seems to favor the universality of the compound. Hence, the ability of potential anti-AIDS drugs to inhibit multiple retroviral PRs might indicate their likelihood of not eliciting drug resistance. These studies may also contribute to the development of a small-animal model for preclinical testing of antiviral compounds.

Antiviral Agents↗
PubMed1996

Expression, characterisation and mutagenesis of the aspartic proteinase from equine infectious anaemia virus.

The gene encoding the proteinase from equine infectious anaemia virus (EIAV) was cloned and expressed in Escherichia coli. The recombinant EIAV proteinase was purified to homogeneity and shown to have the ability to process polyprotein and synthetic peptide substrates of human immunodeficiency virus (HIV) origin with an efficiency that can approach that exhibited by HIV proteinase. EIAV proteinase, however, was not susceptible to inhibition by a wide variety of inhibitors of HIV-1 proteinase, including those which have been licenced as anti-AIDS drugs. In this respect, EIAV proteinase behaves like an extreme case of a drug-resistant mutant of HIV-1 proteinase that has arisen under selective drug pressure. Only one potent inhibitor (HBY-793) of HIV-1 proteinase showed comparable efficiency against the EIAV enzyme; the compounds A-77003 and A-76889, which differ only in their stereochemistry and which are otherwise structurally identical to HBY-793 from residues P2 to P2' [nomenclature of Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162], were not effective inhibitors of EIAV proteinase. Mutant forms of EIAV proteinase (Thr30-->Asp and Ile54-->Gly) were generated and their ability to interact with substrates and inhibitors was characterised. HBY-793 inhibited [Gly54]proteinase as effectively as the wild-type proteinase but was tenfold less potent against [Asp30]proteinase. Data interpretations are presented, based on the structure solved for the complex between HBY-793 and EIAV [Gly54]proteinase [Gustchina A., Kervinen, J., Powell, D. J., Zdanov, A., Kay, J. & Wlodawer, A. (1996) Protein Sci. 5, 1453-1465].

Amino Acid Sequence↗
PubMed1996

Structure of equine infectious anemia virus proteinase complexed with an inhibitor.

Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus-encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X-ray structure of a complex of the 154G mutant of EIAV PR with the inhibitor HBY-793 was solved at 1.8 A resolution and refined to a crystallographic R-factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and the inhibitor are symmetric, the interactions between the central part of the inhibitor and the active site aspartates are asymmetric, and the inhibitor and the two flaps are partially disordered. The overall fold of EIAV PR is very similar to that of other retroviral proteinases. However, a novel feature of the EIAV PR structure is the appearance of the second alpha-helix in the monomer in a position predicted by the structural template for the family of aspartic proteinases. The parts of the EIAV PR with the highest resemblance to human immunodeficiency virus type 1 PR include the substrate-binding sites; thus, the differences in the specificity of both enzymes have to be explained by enzyme-ligand interactions at the periphery of the active site as well.

Amino Acid Sequence↗
PubMed1992

The fate of DNA injected into mammalian oocytes and zygotes at different stages of the cell cycle.

The stability of exogenous DNA microinjected into the cytoplasm at different stages of the meiotic cycle and after pronuclear formation was examined in ungulate species. Metabolism of the injected 1201 base pair (bp) DNA was examined by Southern blotting. Similar levels of metabolism of the injected DNA were detected in pig, sheep and bovine oocytes before germinal vesicle breakdown, in which about 30-40% of detected DNA was ligated into higher-molecular-weight forms. Porcine metaphase oocytes and pronuclear zygotes showed a reduced ability to ligate the exogenous DNA. In contrast, sheep and bovine metaphase oocytes and zygotes showed increased levels of ligation and, at the pronuclear stage, generated significant amounts of extremely large (greater than 15 kbp) ligation products. These results are discussed in the context of maternal precursors and metabolic activities in the egg.

Animals↗
PubMed1991

Molecular cloning of the mouse CCK gene: expression in different brain regions and during cortical development.

In this paper we describe experiments that address specific issues concerning the regulation of the mouse cholecystokinin gene in brain and intestine. The mouse cholecystokinin gene was cloned and sequenced. Extensive homology among the mouse, man and rat genes was noted particularly in the three exons and the regions upstream of the RNA start site. RNAse protection assays for each of the three exons were used to demonstrate that CCK is expressed in only a subset of tissues and that the same cap site and splice choices are used in brain, intestine as well as in cerebellum, cortex, midbrain, hypothalamus and hippocampus. CCK RNA was also noted to be detectable in kidney. Thus the same gene using the same promoter is expressed in subsets of cells that differ in their biochemical, morphologic and functional characteristics. The level of expression of CCK was also monitored during mouse cortical development and the appearance of CCK RNA was compared to glutamate decarboxylase (GAD), enkephalin and somatostatin. It was noted that each of these cortical markers was first expressed at different times during cortical development. The appearance of CCK RNA during intestinal development was also measured and found to precede appearance in cortex by several days.

Amino Acid Sequence↗
PubMed1991

Survival of cancer of the larynx related to treatment.

Analysis of 3445 cases of cancer of the larynx has been made with reference to primary (first) and secondary treatments. It is easy to confirm that while radiotherapy is inferior to laryngectomy in the cure of larger glottic tumours, it is preferable in many cases because salvage surgery is possible and successful. Salvage surgery cannot be shown to be successful in larger supraglottic tumours, which should be considered for primary surgery. Radiotherapy appears better than surgery for small tumours.

Carcinoma, Squamous Cell↗
PubMed1989

Gastric cancer: a 25-year review.

Between 1957 and 1981, 31,716 cases of gastric cancer were registered in the West Midlands, UK. The age-standardized incidence has shown a decrease from 17.42 per 100,000 population during the first quinquennium to 15.30 per 100,000 in the last. There was an apparent increase in the proportion of proximal lesions with a decrease in the proportion of distal, antral cancers. The stage of disease at diagnosis remained constant with 79 per cent of patients having stage IV disease. Less than 1 per cent presented with stage I disease. As a result, the curative resection rate was 21 per cent. The operative mortality rates for curative partial gastrectomy and total gastrectomy were 13 and 29 per cent respectively. Surgeons undertaking more than nine total gastrectomies annually had an overall mean operative mortality rate of 22 per cent. Overall age-adjusted survival at 5 years was 5 per cent. Survival at 5 years for stage I, II and III disease was 72, 32 and 10 per cent respectively. There was a significant increase in survival time for those treated by curative resection between 1972 and 1981 compared with the previous decade. The implications for the management of gastric cancer are discussed.

Age Factors↗