PubMed Health⌕ Search

Biomedical subjects

P Towner

Publications and source records attributed to P Towner.

At least 19 recordsLinked to original sources
PubMed2003

No change in apoptosis in skeletal muscle exposed acutely or chronically to alcohol.

The pathogenic mechanisms responsible for the deleterious changes in ethanol-exposed skeletal muscle are unknown, although apoptosis may be a causal process. We therefore investigated the responses of skeletal muscle to acute or chronic ethanol exposure in male Wistar rats. In acute studies, rats were dosed with ethanol (75 mmol (3.46 g)/kg BW) and killed after either 2.5 or 6 hours. In chronic studies, rats were fed ethanol as 35% of total dietary energy for 6 weeks. Apoptosis was determined by either DNA fragmentation or TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labelling) assays. The results showed that apoptosis was not increased in the ethanol-exposed muscle in both acute and chronic studies compared to appropriate controls.

Animals↗
PubMed2002

Alcohol increases c-myc mRNA and protein in skeletal and cardiac muscle.

The pathogenic mechanisms responsible for alcohol-induced muscle disease are unknown, although it is possible that increased proto-oncogene expression may be the causative process. Therefore, we investigated the responses of skeletal muscle c-myc protein and mRNA to a standard acute ethanol dosage regimen (75 mmol/kg/body weight [BW]) for 2.5 to 24 hours. Comparative studies were made on the heart. Acute ethanol administration in vivo led to an increase in c-myc proto-oncogene mRNA in rat skeletal and cardiac muscle. The changes in c-myc mRNA were mirrored by increases in the c-myc protein as demonstrated by immunohistochemistry. The changes in the c-myc protein were localized to the myonuclei, with no corresponding changes seen in the interstitial cell nuclei. This is the first report of altered proto-oncogene expression in muscle in response to ethanol. Increased c-myc mRNA and protein may reflect adaptive changes, a stress response, or another uncharacterized cellular adaptation.

Animals↗
PubMed2000

Complementation of a glucose transporter mutant of Schizosaccharomyces pombe by a novel Trypanosoma brucei gene.

The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect vector and the mammalian host. This is underscored by biochemical switches in its nutritional requirements. In the insect vector, the parasite relies on amino acid catabolism, but in the mammalian host, it derives its energy exclusively from blood glucose. Glucose transport is facilitated, and constitutes the rate-limiting step in ATP synthesis. Here, we report the cloning of a novel glucose transporter-related gene by heterologous screening of a lambdaEMBL4 genomic library of T. brucei EATRO 164 using a rat liver glucose transporter cDNA clone. Genomic analysis shows that the gene is present as a single copy within the parasite genome. The gene encodes a protein with an estimated molecular mass of 55.9 kDa, which shares only segmental homology with members of the glucose transporter superfamily. Several potential post-translational modification sites including phosphorylation, N-glycosylation, and cotranslational myristoylation sites also punctuate the sequence. It is distinguished from classical transporter proteins by the absence of putative hydrophobic membrane-spanning domains. However, this protein was capable of complementing Schizosaccharomyces pombe glucose transporter mutants. The rescued phenotype conferred the ability of the cells to grow on a broad range of sugars, both monosaccharides and disaccharides. The kinetics of glucose uptake reflected those in T. brucei. In addition to complementation in yeast, we also showed that the gene enhanced glucose uptake in cultured mammalian cells.

Amino Acid Sequence↗
PubMed1999

New growth hormone receptor exon 9 mutation causes genetic short stature.

A novel form of congenital growth hormone insensitivity syndrome (GHIS), which lacks the classic phenotype associated with this condition, is described. Dominant inheritance is shown to result from a heterozygous 876-1 G to C transversion of the 3' splice acceptor site preceding exon 9 in the growth hormone receptor (GHR) gene. The result of this mutation is a severely truncated cytoplasmic domain of the GHR, which is incapable of transmitting a signal. The mutant receptor is shown to form a heterodimer with the wild-type GHR, the activity of which is inhibited in a dominant-negative manner.

Base Sequence↗
PubMed1998

Cholecystokinin receptors in human pancreatic cancer cell lines.

Recent studies have suggested that cholecystokinin (CCK) receptors may play a role in the development and growth of pancreatic cancers. We detected the expression of mRNA encoding CCK-A and CCK-B receptors in eight human pancreatic tumour cell lines using reverse transcription-polymerase chain reaction (RT-PCR), but not by RNase protection assays. The K-ras gene, which can be activated by G-coupled protein receptors such as CCK receptors, was mutated in codon 12 in five of the cell lines. In addition, Mia PaCa-2 pancreatic cancer cells did not respond to CCK or gastrin in cell proliferation or focal adhesion kinase (FAK) phosphorylation assays. In contrast, mouse NIH3T3 fibroblasts transfected with human CCK-B receptor (NIH3T3CCK-BR) showed increased proliferation and phosphorylation to the peptides. Also, radioligand binding studies indicated that Mia PaCa-2 cells had approximately 12.5-fold less CCK-B receptors than NIH3T3CCK-BR. Our results suggest that in Mia PaCa-2 cells, CCK receptors may not play a crucial role in supporting cell growth.

3T3 Cells↗
PubMed1998

Characterization of a nicotinic acetylcholine receptor from the insect Manduca sexta.

Manduca sexta is a nicotine-insensitive insect, the larval form of which feeds on tobacco. It has been postulated that its nicotine insensitivity may reflect the presence of a modified nicotinic acetylcholine receptor whose alpha subunits lack the amino acid residues necessary for binding nicotine: we have performed ligand binding assays and molecular cloning to examine this hypothesis. [125I]alpha-bungarotoxin bound specifically to both larval and adult membranes, with Kd values of 7.6 and 6.5 nM and Bmax values of 119 and 815 fmol/mg protein, respectively. The pharmacological profile of [1251]alpha-bungarotoxin binding was similar in both tissues. In particular, nicotine (Ki values: 1.6 microM and 2 microM for larvae and adults, respectively) competed with an affinity similar to that found for nicotine-sensitive insects. No alpha-bungarotoxin-insensitive binding sites labelled by [3H]epibatidine could be detected. Using the alpha-like subunit from the locust Schistocerca gregaria to probe two cDNA libraries, and by inverse PCR on circularized genomic DNA from Manduca sexta, we have obtained overlapping cDNA clones that contain the complete coding sequence of a putative nicotinic subunit from Manduca sexta (MARA1). No other alpha-subunit cDNAs were isolated using this probe, although it hybridized to multiple bands on Southern blots. The sequence of MARA1 is consistent with an alpha-like subunit capable of binding alpha-bungarotoxin, and it retains all those amino acids implicated in nicotine binding to vertebrate nicotinic receptors. Taken together, these findings provide no support for the hypothesis that the nicotine insensitivity of Manduca sexta is the result of a nicotinic receptor with diminished nicotine binding.

Aging↗
PubMed1997

Molecular cloning of human GATA-6 DNA binding protein: high levels of expression in heart and gut.

Human GATA-6 has been cloned from foetal heart by a combination of PCR-based methods and cDNA library screening. The 3.8 kbp cDNA has a coding sequence of 1347 bp the 449 aa protein is virtually identical in the two zinc-finger binding domains to other human GATA sequences, but varies considerably in the amino and carboxy terminal regions. The sequence shows greatest similarity to GATA-6-like sequences from rat, mouse, chicken and Xenopus. Northern analysis and in situ hybridisation show that GATA-6 is expressed at high levels in human adult and foetal heart as well as in gut derivatives. It is postulated that GATA-6, in concert with GATA-4, plays a crucial role in the regulation of cardiac differentiation.

Amino Acid Sequence↗
PubMed1997

Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors.

Previous studies have shown that expression of the immune co-stimulator B7.1 reduces the tumorigenicity of some, but not all, malignant cell lines. However, B7.1-expressing tumor cells are not very effective in inducing the rejection of established tumors. This may in part be due to induction of anergy in the potentially reactive T cells. Previous studies have shown that IL-2 can reverse the anergic state both in vitro and in vivo. Therefore, we have examined the effect of retrovirus-mediated delivery and expression of murine B7.1 and interleukin-2 on tumor formation and rejection of established MHC class I+/II- NC adenocarcinomas. Neither the expression of B7.1 nor IL-2 alone had a significant effect on NC tumorigenicity. In contrast, combined expression of B7.1 and IL-2 substantially decreased the tumorigenicity of these cells in the immunecompetent syngeneic hosts. T-cell depletion studies show this to be dependent primarily on the activation of CD4+ cells. Furthermore, distant subcutaneous injection of irradiated NC/IL-2/B7.1 can induce, much more effectively than NC/B7.1 or NC/IL-2, the rejection of small NC tumors, and prevent the recurrence of large surgically resected tumors. Together, these results suggest that tumor cells genetically modified to express B7.1 and IL-2 can induce the immune-mediated rejection of established class II- tumors by a mechanism involving CD4+ cells.

Adenocarcinoma↗
PubMed1997

Primary structure of locust opsins: a speculative model which may account for ultraviolet wavelength light detection.

The sequences of two locust opsins have been determined by dideoxy nucleotide sequencing of PCR products from cDNA derived from eyecup tissue. The opsins (Lo1 and Lo2) are encoded by 381 and 380 amino acid residues, respectively, with hydropathy profiles and placement of key amino acid residues suggestive of a typical seven-transmembrane rhodopsin structure. The sequence alignment of Lo1 reveals significant homology to mantid opsin. These opsins contain retinal as their visual chromophore and have similarity to the Rh1 type sequences from Drosophila and Calliphora which use 3-hydroxy retinal. Lo2 is most closely related to the Rh3/4 type of visual pigments from Drosophila. The retinal-based opsins show reduced numbers of charged amino acids in the loop region connecting transmembrane segments V and VI compared to the 3-hydroxy retinal opsins. Sequence alignment of all the known insect visual pigments has shown that only those with maximal sensitivity in the blue/UV spectral range, Lo2 and the Rh3/4 opsins of Drosophila, have three charged amino acids in transmembrane segments II, IV and VII. The charged residue in transmembrane VII is two helical turns away from the positively charged Schiff base and could act directly as a counterion to it. From the secondary structure analysis of opsin, the two charged residues in transmembrane II and IV would be in close proximity to form a dipole. These polar motifs in Lo2 and Rh3/Rh4 could act in wavelength modulation of short wavelength sensitive pigments and substantiate the proposed external two-point charge model which accounts for the spectral sensitivity of visual pigments [Honig, B., Dinur, U., Nakanishi, K., Balogh-Nair, V., Gawinowicz, M.A. and Motto, M. (1979). Journal of the American Chemical Society, 101, 7084-7086].

Amino Acid Sequence↗
PubMed1997

In vitro immune modulation by antibodies coupled to tumour cells.

Modification of autologous tumour cells to express the immune costimulator B7.1 is a potential strategy for immunotherapy of cancer. Previously, this has involved introduction of genetic material into cells, in vitro culture, and confirmation of the protein product on the cell surface. This is possible only if sufficient tumour is obtainable and efficiently modified in a short time. Whilst progress has been made on ex vivo tumour cell culture and transfection/infection procedures there are still tumour types for which the present means of gene transfer are not efficient enough. We describe a highly efficient in vitro procedure for the modification of over 99% of the cells in a population, allowing the expression of cell surface proteins with potential immune modulatory activities. This procedure, which can be completed in as little as 24 h with no upper limit on cell number, utilizes succinimide esters to label cell surface proteins with biotin covalently. Biotinylated cell membrane proteins then anchor an avidin bridge for immobilizing protein G'-biotin. This can serve to bind immunoglobulin (Ig) molecules via their Fc region such that the variable region of the antibody is freely and functionally available. In the present study the binding of a stimulatory mouse anti-human CD28 monoclonal antibody to the surface of tumour cells is used to show that the modified cells are capable of co-stimulating T cells in vitro. The simplicity of the method, and the use of common reagents, represents a further step towards a realistic, truly 'off-the-shelf', nongene immunotherapy protocol.

Animals↗
PubMed1997

Human skin fibroblasts as a model of growth hormone (GH) action in GH receptor-positive Laron's syndrome.

Congenital GH insensitivity (Laron's syndrome, LS) is often associated with a dysfunctional GH receptor (GHR) causing complete insensitivity to GH and absent serum GH-binding protein (GHBP). However, a proportion of children with LS have normal GHBP levels. We have identified four girls from two families with this condition (height SD score, -3.4 to -6.8) and undertaken studies on 1) their GHR genes and 2) their GH responses in cultured skin fibroblasts to define the etiology of their GH insensitivities. No GHR gene mutations were identified in one family. In the other family, the affected siblings, an unaffected brother, and the father were heterozygous for a point mutation within exon 6 (D152H). In addition, use of intron 9 haplotypes to determine linkage to the GHR gene implied inheritance of different maternal GHR alleles in the two affected girls of the latter family. It is unlikely, therefore, that the D152H mutation alone could account for the LS phenotype. End points of GH action [DNA synthesis, insulin-like growth factor-binding protein-3 (IGFBP-3) messenger RNA (mRNA) and peptide production] in skin fibroblast cultures established from three of the LS subjects and four normal children were examined. Whereas normal fibroblasts incorporated [3H]thymidine dose dependently in response to 10-1000 ng/ml GH (increment at 1000 ng/ml, 77 +/- 19%), LS fibroblasts failed to respond significantly above basal levels (P < 0.01). In normal fibroblasts, IGFBP-3 mRNA and peptide increased maximally at 48 h in response to 200 ng/ml GH, as determined by ribonuclease protection assay, Western ligand blotting, and RIA. In comparison, LS fibroblasts produced significantly less IGFBP-3 peptide than normal fibroblasts in response to GH, whereas IGFBP-3 mRNA failed to increase above basal levels. These studies have shown that 1) cultured human skin fibroblasts can be used to define the end points of GH action; 2) fibroblast cultures from the LS children show absent or reduced responses to GH; and 3) GH insensitivity in these children does not appear to be caused exclusively by GHR mutations, but is probably due to dysfunctional GHR signalling. Such patients may prove particularly important to elucidation of the key events in GH signaling.

Cells, Cultured↗
PubMed1996

The effect of combined expression of interleukin 2 and interleukin 4 on the tumorigenicity and treatment of B16F10 melanoma.

The recent use of interleukin 2 (IL-2) and interleukin 4 (IL-4) single cytokine modified tumour cells in rodent models has demonstrated a potential use of these cytokines to produce autologous cancer cell vaccines. Here we compare the potential therapeutic benefit of transduction with IL-2 or IL-4 alone, and combined IL-2 + IL-4 in B16F10 cells, a murine malignant melanoma of poor immunogenicity. Transduction of B16F10 cells (MHC class I and II negative) to express either IL-2 or IL-4 alone delays the formation of tumours, IL-4 being more effective than IL-2. However, combined expression of IL-2 + IL-4 reduces tumorigenicity more than either cytokine alone. The eventual formation of tumours may result from loss of gene expression, and preliminary results suggest methylation of the retroviral long terminal repeat (LTR), rather than loss of the transduced DNA sequences. Histological examination of tumours expressing either IL-2 or IL-4 alone shows a non-specific inflammatory reaction with an increased tissue infiltrate of immune effectors (monocytes/macrophages, lymphocytes, granulocytes) localised around the tumour. In comparison, when cells expressing combined IL-2 + IL-4 were injected there were more granulocytes present, and perhaps more importantly, these were mainly localised within the tumour. The benefit of combined IL-2 + IL-4 expression results from a local rather than systemic effect as the growth of tumours from cells expressing IL-2 or IL-4 alone injected at distant sites was comparable with a single inoculation of cells expressing either cytokine alone. However, when cells expressing single cytokines IL-2 or IL-4 were mixed and injected at the same site, in comparison with the clonal population of cells expressing combined IL-2 + IL-4, tumour growth was characteristic of IL-4 alone rather than IL-2 + IL-4. Treatment of established tumours with a single injection of lethally irradiated tumour cells expressing IL-2 + IL-4 was sufficient to either reject tumours, or at least delay further tumour development. Furthermore, treatment stimulated an initial non-specific immune reaction that lead to a systemic immunity. Lethally irradiated wild-type cells were also successful in treating some established tumours, although this did not induce any systemic immunity. However, although successful in treatment studies, neither wild-type nor combined IL-2 + IL-4 expressing cells were able to vaccinate animals against a subsequent challenge with live wild-type tumour. These results indicate a potential therapeutic benefit with the use of combination IL-2 + IL-4 transduction of autologous cancer cells.

Animals↗
PubMed1994

Expression of phytochrome apoprotein from Avena sativa in Escherichia coli and formation of photoactive chromoproteins by assembly with phycocyanobilin.

Phytochrome DNAs from oat (Avena sativa L.) encoding the full-length 124-kDa polypeptide, a 118-kDa fragment lacking the first 65 amino acids, and two N-terminal fragments of 65 kDa and 45 kDa were subcloned and expressed in Escherichia coli. Reducing the temperature to 25 degrees C during cell growth and the coexpression of chaperones improved the folding into a functional conformation for most of the polypeptides, and in one case the yield of polypeptides was also enhanced. A maximum yield of reconstitutable apoprotein was obtained by expressing the 65-kDa fragment consisting of 595 amino acids. The apoproteins could be assembled in the dark with phycocyanobilin into photoreversible chromoproteins. The yield of photoreversible pigment could be further increased by far-red/red irradiation cycles, indicating that the presence of the chromophore promotes the correct folding of the binding site. The chromoproteins with an intact N-terminal domain exhibit Pr and Pfr absorption bands, which are blue-shifted relative to the corresponding bands of native phytochrome due to the particular phycocyanobilin structure. The 118-kDa fragment, only lacking the 6-kDa N-terminus, exhibits a strong Pr band, but only a weak Pfr absorbance. This indicates an essential role of the front 6-kDa region of the protein in the formation of the far-red absorbing chromophore-protein complex. Otherwise, the C-terminal region seems to be less important for photoreversibility as indicated by the function of the shorter fragments.

Base Sequence↗
PubMed1994

The primary structure of mantid opsin.

The sequence encoding opsin from the mantid Sphodromantis sp. has been determined by dideoxynucleotide sequencing of PCR products from a cDNA derived from eye cup tissue. The 376-amino-acid (aa) residues show approx. 56% identity and 85% similarity to known insect opsins (Drosophila melanogaster and Calliphora erythrocephala). The predicted protein structure, based on the hydropathy profile and placement of key aa residues, reveals a seven-transmembrane structure typical of a rhodopsin. Unlike the previously characterised insect visual pigments which have 3-hydroxy retinal in their binding sites, mantid rhodopsin contains 11-cis retinal. Comparison of transmembrane sequences from the opsin family was performed in order to identify any specific aa substitutions which are able to account for the selection of retinal or its 3-hydroxy derivative by insect opsins.

Amino Acid Sequence↗
PubMed1994

Cloning of a putative inhibitory amino acid receptor subunit from the parasitic nematode Haemonchus contortus.

A cDNA, HGl, encoding an inhibitory amino-acid receptor subunit has been cloned from a mixed egg population of the parasitic nematode Haemonchus contortus. The predicted amino-acid sequence of the subunit shows 24% to 32% homology with other vertebrate and invertebrate GABAA and glycine receptor subunits and has all the expected motifs for a member of the ligand-gated ion channel superfamily. When expressed in Xenopus oocytes HGl gives a small response to 1 mM glycine, but not to 1 mM GABA, glutamate, taurine or L-alanine.

Alanine↗
PubMed1993

Does Escherichia coli possess a second citrate synthase gene?

Escherichia coli possesses a hexameric citrate synthase that exhibits allosteric kinetics and regulatory sensitivity, and for which the gene (gltA) has previously been cloned and sequenced. A citrate-synthase-deficient strain of E. coli (K114) has been mutated to generate a revertant (K114r4) that produces a dimeric citrate synthase with altered kinetic and regulatory properties. On cloning and sequencing the gltA gene from both K114 and K114r4, a single mutation was found that caused the replacement of Asp362 with Asn. Asp362 has been previously shown to be a catalytically essential residue in E. coli citrate synthase, and we demonstrate that the hexameric enzyme produced on expression of the gltA gene from K114 and K114r4 is inactive. The dimeric citrate synthase from K114r4 has been purified and shown to be immunologically distinct from the wild-type hexameric enzyme. Determination of its N-terminal amino acid sequence demonstrates that the mutant citrate synthase is encoded by a gene distinct from the E. coli gltA gene. The N-terminal sequence is compared with those of other eukaryotic, eubacterial and archaebacterial citrate synthases.

Amino Acid Sequence↗