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P Chambers

Publications and source records attributed to P Chambers.

At least 19 recordsLinked to original sources
PubMed1997

Nucleotide sequence of the genes encoding the matrix and small hydrophobic proteins of pneumonia virus of mice.

The nucleotide sequences of the genes encoding the matrix (M) protein and the small hydrophobic (SH) protein of pneumonia virus of mice (PVM) are described. The matrix protein gene contains a large open reading frame encoding polypeptide of 257 residues which shows considerable (38.6-42.1%) amino acid identity with the matrix proteins of other pneumoviruses. The matrix gene also contains a second, smaller, open reading frame (ORF) as seen with the equivalent genes of other pneumoviruses. The PVM second open reading frame is capable of encoding a polypeptide of 46 residues and shows no significant similarity with the proteins encoded by the equivalent open reading frames of the other pneumoviruses. The gene adjacent to that encoding the matrix protein encodes a small, 92 residue, polypeptide which has a central hydrophobic domain and is structurally similar to the small hydrophobic protein of respiratory syncytial virus.

Amino Acid Sequence↗
PubMed1997

KTP laser-resistant properties of the reinforced laryngeal mask airway.

We have assessed, in vitro, the effect of KTP laser strike on the reinforced laryngeal mask airway (RLMA) under a variety of conditions. At power densities normally encountered in clinical practice, using a divergent KTP laser beam, the RLMA could not be penetrated and did not ignite with laser strike. The RLMA was penetrated at a high power density of 6.94 W mm-2 after 45-60 s. A flame appeared over the RLMA shaft at this power density after 12-35 s. The black marker line on the RLMA shaft was somewhat more vulnerable to the effects of laser strike. The flow of oxygen and nitrous oxide within the shaft did not appreciably alter the laser-resistant properties of the RLMA. The RLMA cuff was more vulnerable to laser strike than was the shaft and was penetrated at very low power densities. Filling the cuff with saline had a protective effect and penetration did not occur at power densities which caused penetration of air-filled cuffs (0.37 W mm-2).

Air↗
PubMed1997

Ca2+ mobilization in cultured human bladder smooth muscle cells in response to hypotonic stimuli.

The effects of hypotonic solutions on intracellular Ca2+ were determined on primary cultures of human detrusor smooth muscle cells. In the presence of external Ca2+, changing from a solution containing 290 mosmol l-1 to one containing 240 mosmol l-1 (80% normal tonicity) activated a rise in intracellular Ca2+. If the exposure to 240 mosmol l-1 was followed by exposure to 150 mosmol l-1 (50% normal tonicity) a further increase in Ca2+ was noted. Exposure to hypotonic solutions which contained nominally zero Ca2+ also resulted in a rise in intracellular Ca2+, suggesting that mechanical perturbation of the surface membrane can mobilize Ca2+ directly from intracellular stores. Stimulation of cells with a brief exposure to agonist (histamine, 5 microM) in the nominal absence of external Ca2+ produced a release of Ca2+ from intracellular stores. Under these conditions the response to a subsequent hypotonic stimulation was small or absent. These observations suggest that hypotonic stress induces a mobilization of Ca2+ from the same store as that activated by agonist. Detailed analysis of data from individual bladders revealed that there were significant differences between cells cultured from normal bladders and those from bladders with detrusor instability. On exposure to 240 mosmol l-1 cells from unstable bladders gave larger responses than cells from normal bladders. However, exposure of cells from unstable bladders to a subsequent stimulus of 150 mosmol l-1 resulted in a smaller rise in Ca2+ than was observed with normal cells. Overall, these data suggest that isolated detrusor smooth muscle cells are sensitive to membrane stretch by hyposmotic solutions and that the mechanisms involved include the direct mobilization of intracellular Ca2+. The data also suggest that there may be differences in the responsiveness of cells isolated from stable and unstable bladders.

Arachidonic Acid↗
PubMed1996

Transforming growth factor alpha and epidermal growth factor levels in bladder cancer and their relationship to epidermal growth factor receptor.

We have examined levels of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) in neoplastic and non-neoplastic bladder tissue using a standard radioimmunoassay technique. Tumour samples had much higher TGF-alpha levels compared with EGF and TGF-alpha levels in malignant tissue were significantly higher than in benign bladder samples. There was, in addition, a difference in mean EGF levels from 'normal' bladder samples from non-tumour bearing areas of bladder in patients with bladder cancer compared with 'normal' bladder tissue obtained at the time of organ retrieval surgery. Levels of EGF and TGF-alpha did not correlate with levels of EGF receptor (EGFR) as determined by a radioligand binding method but levels of TGF-alpha > 10 ng gm-1 of tumour tissue did correlate with EGFR positivity defined using immunohistochemistry. These data suggest that TGF-alpha is the likely ligand for EGFR in bladder tumours.

Epidermal Growth Factor↗
PubMed1996

Ca2+ signalling in cultured smooth muscle cells from human bladder.

It has been suggested that the isolation and culture of human detrusor smooth muscle cells may provide useful insights into the physiology of the intact detrusor muscle. In the present paper, data are presented from cultured human bladder smooth muscle cells isolated from small, routinely available biopsies. Since the initiation of contractions involves a rise in intracellular Ca2+, this study has focused on the mechanisms involved in the rise of Ca2+ in cultured cells. Exposure of cells to bathing solutions with elevated K+ concentrations resulted in an increase in Ca2+ consistent with the presence of voltage-activated Ca2+ channels. Agonists, including carbachol, histamine and ATP, also activated repetitive transient increases in Ca2+ in the presence and absence of external Ca2+. Spontaneous Ca2+ transients were recorded in 31% of cells isolated from normal bladders. Such spontaneous and agonist-induced oscillations were not abolished in depolarized cells, suggesting that the mechanisms underlying the oscillations are not dependent on the cyclical operation of voltage-operated Ca2+ channels. However, the spontaneous activity was inhibited by the Ca2+ blocker verapamil, pointing to the presence of Ca2+ channels. The operation of an IP3-sensitive Ca2+ release mechanism was examined using saponin-permeabilized cells, which demonstrated that IP3 increased the rate of 45Ca2+ efflux. The conclusion from this study is that many of the mechanisms described in the intact tissue are operational in cultured cells.

Adenosine Triphosphate↗
PubMed1995

Nucleotide sequences of the genes encoding the putative attachment glycoprotein (G) of mouse and tissue culture-passaged strains of pneumonia virus of mice.

The sequences of the genes encoding the putative attachment (G) proteins of pathogenic (strain J3666) mouse lung-passaged and nonpathogenic (strain 15) tissue culture-passaged strains of pneumonia virus of mice (PVM) have been determined. In both cases the major polypeptide was synthesised from the second open reading frame (ORF), a feature also found in the G gene of respiratory syncytial (RS) virus, another pneumovirus. However, the ORFs of the G genes of the two PVM strains were initiated at different nucleotide positions in the mRNA and comparison of hydrophobicity profiles revealed the presence of the putative amino-terminal cytoplasmic domain in the strain J3666 G protein and its absence in the predicted G protein of PVM strain 15. In common with the G protein of RS virus, the gene product of both PVM strains contained a high serine, threonine, and proline content. Indirect immunofluorescence analysis of BSC-1 cells expressing the G gene products confirmed the surface location of the proteins. Thus, the absence of a cytoplasmic domain does not interfere with the translocation of the G protein of PVM strain 15. In vitro translation of mRNA from the two PVM genes directed the synthesis of a larger polypeptide with the G gene of PVM strain J3666 than was seen with strain 15 G gene. In addition, a second protein was seen with strain J3666 mRNA which was the same size as the strain 15 G protein.

Amino Acid Sequence↗
PubMed1994

Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping reading frames.

The gene encoding the phosphoprotein of the pneumovirus pneumonia virus of mice (PVM) has been cloned and sequenced. The gene is 903 nucleotides in length and contains a long open reading frame (ORF) capable of encoding a polypeptide of 295 amino acid residues. A smaller, second, overlapping ORF encoding a polypeptide 137 amino acids in length was also present. The large ORF directed the synthesis of a 39-kDa polypeptide and four additional polypeptides with masses of 37 kDa, 26 kDa, 23 kDa, and 16 kDa in vitro. The smaller polypeptides were generated by internal initiation on in-frame AUG initiation codons to generate carboxy co-terminal products. Western immunoblot analysis indicated that at least two of these proteins and several other related polypeptides are present in infected cells, and the possible origins of these are discussed. Western blot analysis using antiserum raised against a synthetic peptide and specific for the predicted second ORF product identified a polypeptide of 23 kDa in PVM-infected cells. The pattern of PVM P gene expression is unlike that of the closely related respiratory syncytial virus and is reminiscent of that of paramyxoviruses such as Sendai virus. This is the first example of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo.

Amino Acid Sequence↗
PubMed1992

Different interactions of interferon-alpha subtypes at the surface of epithelial and lymphoid cells.

The interaction of different interferon (IFN)-alpha subtypes with different cell types was investigated using a unique monoclonal antibody (MAb), I-4-A. This MAb reacts in immunoassays equally with IFN-alpha 2b and IFN-alpha 4a, but does not inhibit the binding of IFN to cell receptors. 125I-labeled I-4-A reacted with IFN-alpha 4a and IFN-alpha 2b bound to receptors on Daudi cells. However, in a "double assay" developed using Daudi cells to measure antiviral and antiproliferative activity, I-4-A neutralized both activities of IFN-alpha 4a, but neither of IFN-alpha 2b. Similarly, in studies on the activation of natural killer (NK) cells, I-4-A neutralized the effect of IFN-alpha 4a but not that of IFN-alpha 2b. In contrast, when cell lines other than lymphoid were studied, e.g., HEp 2 and WISH cells, I-4-A neutralized the antiviral activity of both IFN-alpha subtypes. The neutralization of one IFN-alpha subtype but not another on lymphoid cells suggests a difference either in the receptor-bound form of the subtypes, or in subsequent interactions prerequisite for activation of these cells. Furthermore, the neutralization of a particular IFN subtype, alpha 2b, on epithelial-derived but not lymphoid cells suggests differences in the IFN-receptor complex or the mechanisms of cell activation between these cell types. An implication from these studies is that some IFN-alpha subtypes can exert different functions on lymphoid and epithelial cells.

Amino Acid Sequence↗
PubMed1992

Sequence analysis of the gene encoding the fusion glycoprotein of pneumonia virus of mice suggests possible conserved secondary structure elements in paramyxovirus fusion glycoproteins.

The gene encoding the fusion (F) glycoprotein of pneumonia virus of mice consists of 1657 bases and contains an open reading frame encoding 537 amino acids which is more similar to the F proteins of pneumoviruses than to those of other paramyxoviruses. Computer-assisted sequence analyses can be combined with data on the antigenicity of various F proteins to suggest a possible arrangement of secondary structure elements common to all pneumovirus and paramyxovirus F proteins.

Amino Acid Sequence↗
PubMed1991

The nucleotide sequences of intergenic regions between nine genes of pneumonia virus of mice establish the physical order of these genes in the viral genome.

We have cloned eight intergenic regions from the Pneumovirus pneumonia virus of mice that link the nine small and medium sized genes previously described (Chambers et al., 1990). The nucleotide sequences of the clones confirm the locations of these genes and their mRNA transcripts in the viral genome. The intergenic regions vary in size from 2-56 nucleotides and show only faint homology to each other or to their analogues in respiratory syncytial virus. Sequence alignments suggest that the location of the transcriptional start site for the mRNA encoding the major nucleocapsid protein of pneumonia virus of mice and respiratory syncytial virus may have altered during virus evolution by gain or loss of a transcriptional start signal.

Base Sequence↗
PubMed1991

Genes 1 and 2 of pneumonia virus of mice encode proteins which have little homology with the 1C and 1B proteins of human respiratory syncytial virus.

Genes 1 and 2 of pneumonia virus of mice (PVM) consist of 410 and 571 nucleotides and encode proteins of 113 and 156 amino acids respectively. The proteins show no extensive (gene 1 analogous to 1C) or low (gene 2 analogous to 1B) homology to their presumed counterparts in human respiratory syncytial virus (HRSV). The strongest homology is between regions of approximately 35 amino acids located near the carboxy termini of the gene 2 product and the 1B protein with 29% identity, although a lower level of homology can be detected throughout much of these proteins (18% identity overall). These observations contrast with the conservation of 1C and 1B proteins between subgroups of HRSV and with the conservation of nucleocapsid proteins between HRSV and PVM.

Amino Acid Sequence↗
PubMed1991

Sequence of the major nucleocapsid protein gene of pneumonia virus of mice: sequence comparisons suggest structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdoviruses and filoviruses.

The complete nucleotide sequence of gene 3 of pneumonia virus of mice has been determined, and the 5' end of the mRNA mapped using a modification of the polymerase chain reaction technique. The gene contains a single open reading frame, beginning with a 5'-proximal AUG initiation codon, encoding a polypeptide with a predicted Mr of 43141. Expression of the gene 3 protein in Escherichia coli and in vitro showed that it reacted with virus-specific antiserum and comigrated with the major nucleocapsid (N) polypeptide. The predicted amino acid sequence has extensive identity with that of the N protein of human respiratory syncytial virus. Comparisons with the amino acid sequences of N proteins of other paramyxoviruses, vesicular stomatitis virus and Ebola virus suggest that these proteins may have retained much of the same structure. These regions of conserved structure would most likely have the common functions of RNA binding and protein/protein interactions in the virus nucleocapsid.

Amino Acid Sequence↗
PubMed1990

Heptad repeat sequences are located adjacent to hydrophobic regions in several types of virus fusion glycoproteins.

Extensive regions of heptad repeat units consistent with an alpha-helical coiled coil conformation are located adjacent to hydrophobic, potentially fusion-related regions in the amino acid sequences of paramyxovirus fusion and retrovirus envelope glycoproteins. Similar arrangements of hydrophobic peptides and heptad repeat units exist in coronavirus peplomer proteins and influenza virus haemagglutinins. This suggests that there may be similarities in the structures of these proteins and in the functions of the hydrophobic fusion-related regions during virus entry.

Amino Acid Sequence↗
PubMed1990

Insertion of the fusion gene from Newcastle disease virus into a non-essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity induced by the recombinant.

In this paper we report on the identification of non-essential genes in the terminal repeats of the avipox-virus fowlpox virus and the use of these as insertion sites in a vector system. Foreign genes inserted into these sites are shown to be present in two copies in the resultant recombinant virus. To test the potential use of this vector as a live vaccine the fusion gene of Newcastle disease virus has been inserted into a vaccine strain of fowlpox virus and inoculated into chickens. The experiments demonstrate the ability of the recombinant to protect chickens against challenge by a virulent strain of Newcastle disease virus and to elicit the formation of an anti-fusion protein antibody.

Animals↗
PubMed1990

Molecular cloning of pneumonia virus of mice.

cDNA clones representing nine genes of pneumonia virus of mice (PVM) have been generated. The sizes of the corresponding mRNAs and a provisional transcriptional map of the virus genome have been determined. The apparent gene order is very similar to that of respiratory syncytial virus. The sequences adjacent to the 3' termini of the PVM genes were determined and are very similar to those of respiratory syncytial virus. Several PVM gene polypeptide products have been assigned.

Animals↗
PubMed1989

Expression of epidermal growth factor receptors associated with lack of response to endocrine therapy in recurrent breast cancer.

Epidermal growth factor receptors (EGFR) and oestrogen receptors (ER) were analysed in 221 patients with primary operable breast cancer by means of radioligand assays. After median follow-up of 24 months (range 3-60 months), there had been recurrences in 99 patients, of whom 72 (median age 56 years, range 32-77 years) received tamoxifen alone as first-line treatment for recurrence. 20 patients (28%) showed a response to this therapy and 52 (72%) did not. Of 32 ER-positive tumours, 12 (37.5%) showed an objective response to tamoxifen compared with only 2 of 40 (5%) ER-negative tumours (p less than 0.005). Of 35 EGFR-positive tumours, 3 (8.5%) achieved an objective response compared with 11 of 37 (30%) EGFR-negative tumours (p less than 0.05). Only 1 of 28 EGFR-positive, ER-negative tumours achieved an objective response. Including patients whose disease remained stable for more than 6 months with the responders, however, EGFR status was a better predictor of response to tamoxifen; 15 of 37 EGFR-negative patients and 5 of 35 EGFR-positive patients responded (p less than 0.01), compared with 13 of 32 ER-positive and 7 of 40 ER-negative patients (not significant). EGFR expression is a highly significant marker of poor prognosis in patients with breast cancer; it appears to be as good a predictor as ER for objective response and better for overall response to endocrine therapy on relapse.

Adult↗
PubMed1989

Quinazoline antifolate thymidylate synthase inhibitors: nitrogen, oxygen, sulfur, and chlorine substituents in the C2 position.

The synthesis of 16 new N10-propargylquinazoline antifolates with methylamino, ethylamino, (2-aminoethyl)amino, [2-(dimethylamino)ethyl]amino, (2-hydroxyethyl)amino, (carboxymethyl)amino, dimethylamino, imidazol-1-yl, methoxy, ethoxy, phenoxy, 2-methoxyethoxy, 2-hydroxyethoxy, mercapto, methylthio, and chloro substituents at C2 is described. In general, the synthetic route involved the coupling of diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate (5a) with 6-(bromomethyl)-2-chloro-3,4-dihydro-4-oxoquinazoline in N,N-dimethylformamide with calcium carbonate as the base, displacement of the C2-chloro substituent with nitrogen and sulfur nucleophiles, and deprotection using mild alkali. The C2-ether analogues were most conveniently prepared by coupling 5a with 6-(bromomethyl)-2,4-diakoxy(or diphenoxy)quinazolines. In this series the final deprotection step with aqueous alkali gave simultaneous selective hydrolysis of the C4-alkoxy or C4-phenoxy substituent. The compounds were tested as inhibitors of partially purified L1210 thymidylate synthase (TS). As a measure of cytotoxicity, they were examined for their inhibition of the growth of L1210 cells in culture. The C2-methoxy analogue 11a was equivalent to the previously described tight binding TS inhibitor N10-propargyl-5,8-dideazafolic acid (CB3717, ICI 155387, 1a) against the TS enzyme and exhibited enhanced potency in culture. The C2-methoxy substituent also gave a 110-fold enhancement in aqueous solubility relative to the C2-amine. These results suggest that 11a will be an interesting compound for further study as a potential antitumor agent in vivo. A further series of 2-methoxyquinazoline antifolates with modified alkyl substituents at N10 is also described. None of these analogues equalled the activity of 11a. Thus the propargyl group appears to be the optimum N10 substituent in both 2-amino- and 2-methoxyquinazoline antifolates.

Animals↗