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Biomedical subjects

P Chambers

Publications and source records attributed to P Chambers.

At least 55 records · Page 3Linked to original sources
PubMed1988

Epidermal growth factor receptor (EGFr) status associated with failure of primary endocrine therapy in elderly postmenopausal patients with breast cancer.

We have used primary endocrine therapy for 61 elderly women with operable breast cancer (median age 77 years). Eleven patients (18%) had complete and 24 (39%) partial tumour regression, 12 (20%) had stable disease for a minimum of six months and 14 (23%) no response. Salvage surgery was undertaken in the 14 with no response and 8/9 with progressive disease following initial response, thus samples were available from relapse patients only. Assays for EGFr (two point radioreceptor assay) and oestrogen receptors (ER) (dextran coated charcoal method and an immunohistochemical method) were performed on 20/22 patients. Ten of these 20 tumours were EGFr+ (greater than 10 fmol mg-1 binding) and 9/13 patients progressing within six months had EGFr+ tumours. 15/22 were available for ER evaluation and there was no such association with ER status. EGFr status was also associated with early recurrence after surgery and death in the endocrine failure group (P less than 0.005 and P less than 0.05 respectively). Of a control population of 33 patients (median age 72 years) treated by primary surgery, only 6 were EGFr+. In this group early relapse was predicted by EGFr status, but not by ER status (median disease free survival for EGFr+ patients 15 months, and for EGFr- patients 40 months, P less than 0.01, logrank test). There was a significantly higher proportion of EGFr+ tumours in the endocrine failure group compared with the control population (P less than 0.001). EGFr status is a marker for rapid early progression on primary endocrine therapy and the development of non-excisional methods of EGFr analysis would allow better directed therapeutic decisions.

Aged↗
PubMed1988

Nucleotide sequence of the fusion and haemagglutinin-neuraminidase glycoprotein genes of Newcastle disease virus, strain Ulster: molecular basis for variations in pathogenicity between strains.

The nucleotide sequences of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoprotein genes of the extremely avirulent Newcastle disease virus (NDV) strain Ulster have been determined by sequencing cDNA clones derived from viral genomic RNA. Open reading frames, assumed to encode the F0 and HN0 glycoprotein precursors, were 553 and 616 amino acids long, respectively. Comparisons of the two glycoprotein sequences with those of more virulent NDV strains suggested an explanation for the molecular basis of the wide-ranging differences in virulence observed between strains of NDV. The open reading frame corresponding to the Ulster HN glycoprotein extended beyond the C terminus of more virulent strains. This C-terminal extension was assumed to be responsible for the origin of the HN precursor (HN0) found in strain Ulster and other extremely avirulent strains of NDV. There were fewer basic amino acids at the cleavage site of F0 in strain Ulster than are present in more virulent strains, which may be responsible for the absence of cleavage and activation of F0 from this strain in many host cells. In more virulent strains of NDV, as well as in other paramyxoviruses, a phenylalanine residue occurs at the N terminus of the F1 cleavage fragment. The occurrence of a leucine residue at this position in strain Ulster may be partly responsible for the lack of virulence of this strain.

Base Sequence↗
PubMed1988

Location of a neutralizing epitope for the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

The binding site of a monoclonal antibody to the haemagglutinin-neuraminidase (HN) polypeptide of Newcastle disease virus (NDV) has been located. Complementary DNA or synthetic oligonucleotides corresponding to portions of the HN gene were cloned into the Escherichia coli vector pUC19 and fragments of the HN protein were thereby fused to the alpha-peptide of beta-galactosidase. Western blot analysis of E. coli lysates containing expressed fragments of the HN cDNA or synthetic oligonucleotides identified an antibody-binding peptide (Asp-Glu-Gln-Asp-Tyr-Gln-Ile-Arg; amino acid residues 346 to 353). Nucleotide sequence analysis of an antibody-resistant mutant of NDV revealed a Glu (wild-type) to Lys (mutant) substitution within the above sequence. The methods described could be useful for the location of continuous epitopes of other polypeptides.

Animals↗
PubMed1987

Nucleotide sequence analysis of the L gene of Newcastle disease virus: homologies with Sendai and vesicular stomatitis viruses.

The nucleotide sequence of the L gene of the Beaudette C strain of Newcastle disease virus (NDV) has been determined. The L gene is 6704 nucleotides long and encodes a protein of 2204 amino acids with a calculated molecular weight of 248822. Mung bean nuclease mapping of the 5' terminus of the L gene mRNA indicates that the transcription of the L gene is initiated 11 nucleotides upstream of the translational start site. Comparison with the amino acid sequences of the L genes of Sendai virus and vesicular stomatitis virus (VSV) suggests that there are several regions of homology between the sequences. These data provide further evidence for an evolutionary relationship between the Paramyxoviridae and the Rhabdoviridae. A non-coding sequence of 46 nucleotides downstream of the presumed polyadenylation site of the L gene may be part of a negative strand leader RNA.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of the gene encoding the matrix protein of Newcastle disease virus.

The nucleotide sequence of the gene encoding the matrix (M) protein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from overlapping cDNA clones. Control sequences typical of paramyxovirus mRNA start and polyadenylation signals have been identified. Assuming that the M gene starts and finishes at these sequences, the M gene is 1241 nucleotides long and encodes one long open reading frame of 364 amino acids, corresponding to a polypeptide of molecular weight 39605, in good agreement with estimates from SDS gels. The M protein has an amino acid sequence that is both hydrophobic and highly basic. The NDV M protein has sequence homologies to the M proteins of Sendai, measles, canine distemper and respiratory syncytial viruses.

Amino Acid Sequence↗
PubMed1986

A direct study of the relative synthesis of petite and grande mitochondrial DNA in zygotes from crosses involving suppressive petite mutants of Saccharomyces cerevisiae.

Work in recent years has produced indirect evidence to support the view that the phenomenon of suppressiveness in yeast is the result of the ability of the petite mtDNA to out-replicate the wild-type genome. We have developed a method, based on fluorography of gels containing restriction fragments of radioactively labelled zygotic mtDNA, by which it has been possible to follow directly the incorporation of label into the two mtDNA species and hence their relative synthesis. Four petite isolates of 70%, 43%, 23% and 12% suppressiveness were tested by this method in crosses with a grande strain. Only the mtDNA from the 70% suppressive petite showed a replicative advantage over the grande mtDNA. The mtDNA from the 43% and 23% suppressive actually appeared to undergo, if anything, less replication in the zygote than the grande mtDNA. It is concluded that while some petites may exhibit suppressiveness as a result of enhanced replicative efficiency of their mtDNA, this cannot be the explanation for all suppressive petite strains.

DNA Replication↗
PubMed1986

Nucleotide sequence of the gene encoding the fusion glycoprotein of Newcastle disease virus.

The nucleotide sequence of the gene encoding the fusion (F) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from cDNA clones obtained from virion RNA. The gene is 1792 nucleotides long, including mRNA start and polyadenylation signals typical of paramyxoviruses. The single open reading frame encodes a polypeptide of 553 amino acids, with a predicted molecular weight of 59042. The F polypeptide has three regions of high hydrophobicity: an N-terminal signal peptide, the N terminus of F1 (known from protein sequencing) and a C-terminal membrane-spanning region by which the F glycoprotein is anchored to the membrane. The cleavage site of F0 is located in a highly basic region of the F polypeptide. Five potential asparagine-linked glycosylation sites are present in the amino acid sequence, of which one is in F2 and the others in F1. Comparison of the NDV F amino acid sequence to those from other paramyxoviruses reveals homology to Sendai virus, simian virus 5 and human respiratory syncytial virus. There is also limited homology between the N terminus of F1 of NDV and the N termini of HA2 of influenza viruses. Post-translational modifications of the NDV F polypeptide are discussed in the light of information provided by the amino acid sequence.

Amino Acid Sequence↗
PubMed1986

Molecular cloning of complementary DNA to Newcastle disease virus, and nucleotide sequence analysis of the junction between the genes encoding the haemagglutinin-neuraminidase and the large protein.

Complementary DNA clones to 90% of the Newcastle disease virus (NDV) genome have been produced and mapped. These clones cover the entire HN, F and M genes, most if not all of the L gene and parts of the NP and P genes. The map of overlapping clones gives the gene order 3'-NP-P-M-F-HN-L-5' for NDV, identical to the gene order of Sendai virus, on the assumption that the NP gene of NDV is at the 3' end of the genome as previously suggested by inactivation of NDV transcription by u.v. light. The nucleotide sequence of 453 bases covering the junction between the HN and L genes has been determined. There is nucleotide sequence homology to the consensus polyadenylation and mRNA start sites of Sendai virus and vesicular stomatitis virus. The deduced amino acid sequence of the C terminus of the HN protein of NDV shows homology to the C-terminal amino acid sequences of the HN proteins of simian virus 5 and Sendai virus. An explanation for the presence of HN0, the precursor to HN in some strains of NDV, is suggested by the presence of a long non-coding region at the 3' terminus of the mRNA encoding the HN protein of NDV that could, by mutation, allow synthesis of a larger polypeptide.

Base Sequence↗
PubMed1986

Nucleotide sequence analysis of the haemagglutinin-neuraminidase gene of Newcastle disease virus.

The nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV) has been determined. The HN gene is 2031 nucleotides long, approximately 13.5% of the viral genome. The nucleotide sequence contains a single long open reading frame which would encode a protein of 577 amino acids, with a mol. wt. of 63,149. This is in good agreement with estimates of the molecular weight of the unglycosylated HN protein. Analysis of the amino acid sequence reveals six potential glycosylation sites and shows the major hydrophobic region to be close to the N terminus. This provides evidence for the N-terminal attachment of HN to the viral membrane. The hydrophilic nature of the extreme N-terminal amino acids suggests the absence of a cleaved signal sequence. Analysis of the long non-coding region at the 3' end of the mRNA encoded by the HN gene of NDV suggests a possible explanation for the origin of HN0 in extremely avirulent strains of NDV. There are regions of high homology between the deduced amino acid sequence of the NDV HN glycoprotein and the HN glycoproteins of two other paramyxoviruses, Sendai virus and simian virus 5 (SV5). An alignment of the HN amino acid sequences of these viruses shows 32% of amino acid residues are conserved between NDV and SV5, and 23% between NDV and Sendai virus. In contrast, only very limited homology is found between NDV HN and the influenza virus glycoproteins.

Amino Acid Sequence↗
PubMed1985

Identification of the Escherichia coli recN gene product as a major SOS protein.

The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified.

Bacterial Proteins↗
PubMed1982

Non-structural proteins in Newcastle disease virus-infected cells.

Examination of pulse-labelled Newcastle disease virus (NDV)-infected chick embryo fibroblasts (CEF) by two-dimensional polyacrylamide gel electrophoresis revealed the presence of two-virus-coded non-structural polypeptides of mol. wt. 36K and 33K. Longer pulses and pulse-chase incubations revealed the production of an additional, glycosylated, non-structural polypeptide of mol. wt. 40K (gp40). Kinetic arguments suggest that 36K and 33 K are primary translation products but that gp40 is not. 36K was stable in chase incubations, but 33K was not. Partial digest peptide analysis showed that gp40 and an additional glycosylated polypeptide gp62, which is sometimes present (Chambers & Samson, 1980), are related to the HN polypeptides. Partial digest peptide analysis of the 36K polypeptide generated only a few peptides, which were not sufficient to conclude whether 36K was related to the major virus polypeptides, and since polypeptides 33K was metabolically unstable, insufficient radioactivity was incorporated for peptide studies. Extensive strain-dependent variation in the isoelectric points and mol. wt. of all the NDV polypeptides which are soluble in the isoelectric focusing gels, including 36K and 33K, is reported. This variation, and the insensitivity of the synthesis of 36K and 33K to actinomycin D, show that both non-structural polypeptides are virus-coded.

Animals↗
PubMed1981

Temperature-sensitive mutant of Newcastle disease virus which has an altered nucleocapsid-associated protein.

Analysis of six temperature-sensitive (ts) mutants of Newcastle disease virus (NDV) representing each of six complementation groups by both SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis revealed that in only one mutant was there an alteration in the isoelectric point of a protein. This altered protein was the nucleocapsid-associated protein, NAP. In addition, the mobility of the haemagglutinin-neuraminidase protein, HN, was decreased on non-reduced SDS-PAGE in this mutant. All independent ts+ clones derived from this mutant had normal NAP but HN protein migrated at the decreased rate. Haemagglutinating activity of wide-type (ts+) and ts virions was equally thermostable. Wild-type and ts+ clones derived from this ts mutant were RNA(+) at both permissive and non-permissive temperatures, whereas the ts mutant was RNA(-) at the non-permissive temperature. This ts mutant appears to be a double mutant in both HN and NAP genes, the latter only being a temperature-sensitive lesion which affects virus-directed RNA synthesis.

Capsid↗
PubMed1980

Location of post-translational cleavage events within F and HN glycoproteins of Newcastle disease virus.

The biologically active form of the fusion glycoprotein F from Newcastle disease virus (NDV) comprises two polypeptides, F1 and F2 (derived from a precursor polypeptide F0 by a post translational cleavage event), which are covalently linked together (F1,2) by disulphide bonds. This feature was exploited in a two-dimensional SDS-polyacrylamide gel electrophoretic analysis to orientate the position of the cleavage event within F0. Separation of proteins from NDV-infected CEF in the first dimension in the absence of reducing agent resolved F1,2 protein from all NDV-induced proteins other than F0. Reduction of the first dimension gel with 2-mercaptoethanol, followed by electrophoresis in the second dimension, resolved F1 (55K), F2 (12.5K) and F0 (64K) proteins. The only polypeptides other than F1 and F2 which fell below the diagonal, indicating the positions of the polypeptides from infected cells, were two minor glycoproteins designated HN1 (51.5K) and HN2 (27.5K) which took up positions vertically beneath the major haemagglutinin-neuraminidase glycoprotein HN (75K). Dual isotope labelling experiments with NDV-infected chick embryo fibroblasts, which had previously received a salt shock to effect synchronization of polypeptide initiation upon release of salt shock, revealed the following orientations within the parent molecules: NH2-F2-F1-COOH and NH2-HN1-HN2-COOH. The existence of intermolecular disulphide bonds, orientation and relative lengths of the two NDV HN fragments is analogous to the HA1 and HA2 proteins of influenza virus haemagglutinin.

Animals↗
PubMed1980

A new structural protein for Newcastle disease virus.

Proteins induced in Newcastle disease virus (NDV)-infected chick embryo fibroblasts (CEF) and proteins incorporated into virions grown in ovo were analysed by modified versions of a two-dimensional polyacrylamide gel electrophoresis system. The following previously described NDV-induced proteins were detected and resolved from host proteins: L (mol. wt. approx. 200K), HN (75K), F0 (66K), F1 (56K), NP (55K) and M (39K). Two additional polypeptides, NAP (nucleocapsid-associated protein, mol. wt. 56K) and a 36K mol. wt. protein were induced in NDV-infected cells. NAP but not 36K was found in purified virions. Radioactive labelling studies with 3H-glucosamine and 32P-orthophosphate demonstrated that neither NAP nor 36K is glycosylated, but that both are phosphorylated. Variation in the isoelectric point and apparent mol. wt. of NAP among different strains was seen and exactly reproduced in both CEF and baby hamster kidney (BHK) cells. The synthesis of NDV-induced proteins including NAP and 36K was unaffected by actinomycin D, whereas the synthesis of host cell proteins was drastically reduced. These data are evidence that NAP and 36K are virus coded. Peptide analysis indicated that NAP, NP and F1 are unrelated polypeptides. The demonstration that NAP is virus coded, together with its phosphorylation and association with the nucleocapsid, suggest that NAP may be the NDV analogue of the P protein of Sendai virus.

Animals↗
PubMed1976

Concentration of oestrogens and androgens in human ovarian venous plasma and follicular fluid throughout the menstrual cycle.

The concentrations of androstenedione, testosterone, oestrone and oestradiol-17beta were measured in peripheral and ovarian venous blood and follicular fluid of women at various stages of the menstrual cycle. The concentration of oestradiol was similar in small follicles (diameter less than 8 mm) at all stages of the menstrual cycle and in large follicles (diameter greater than or equal to 8 mm) except during the mid- and late follicular phase when the concentration reached a peak (approximately 1500 ng/ml). The concentration of androstenedione was lowest in large preovulatory follicles at midcycle at a time when the secretion into the ovarian vein was markedly increased. The concentration of testosterone in large follicles (greater than or equal to 8 mm) was unchanged during the follicular phase whereas in small follicles there was a peak at mid-cycle. The rise in the concentration of testosterone and androstenedione at mid-cycle in peripheral plasma may be due to increased secretion by the preovulatory follicle into the ovarian vein. It is suggested that the relatively low concentration of androstenedione in follicular fluid of the preovulatory follicle arises from increased aromatization by granulosa cells in the course of oestrogen synthesis.

Adult↗