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Biomedical subjects

P Whyte

Publications and source records attributed to P Whyte.

At least 37 records · Page 2Linked to original sources
PubMed1993

Conformational constraints of conserved neutralizing epitopes from a major antigenic area of human respiratory syncytial virus fusion glycoprotein.

To study the conformational requirements of epitopes from a conserved antigenic area (area II) of respiratory syncytial (RS) virus fusion (F) glycoprotein, peptides of increasing length containing amino acids essential for these epitopes were synthesized. The synthetic peptides were tested for binding to a panel of neutralizing monoclonal antibodies (MAbs) for this area as well as to rabbit hyperimmune and human convalescent antisera. Antibody binding was dependent on peptide length; thus, a 61-residue peptide spanning amino acids 215 to 275 of the F1 subunit (peptide F215-275) reacted with more antibodies than a shorter (41-residue) peptide F235-275, and this one with more than the (21-residue) peptide F255-275. Most human convalescent sera contained antibodies that reacted with peptides F215-275 and F235-275 but failed to react with F255-275. The results of antibody binding could be related to the structure adopted by the peptides in solution, as determined by circular dichroism spectroscopy and susceptibility of peptides to trypsin digestion. Pretreatment of peptide F215-275 with SDS abolished reactivity with certain MAbs, supporting the notion that higher order structures were needed for antibody binding. High titre anti-peptide antisera were induced in rabbits inoculated with the peptides; however, these sera failed to react with the native F molecule. In mice, only the largest F215-275 peptide induced an anti-peptide response, but their sera reacted poorly with the native F protein and the animals were not protected against an RS virus challenge. These results illustrate the potential use of synthetic peptides in studies of the F protein physical and antigenic structures as well as the problems in designing synthetic RS virus vaccines.

Amino Acid Sequence↗
PubMed1993

The adenovirus E1A-associated 130-kD protein is encoded by a member of the retinoblastoma gene family and physically interacts with cyclins A and E.

The adenovirus E1A proteins form complexes with a group of cellular proteins, including a protein of 130 kD. E1A-associated p130 was purified through coimmunoprecipitation with E1A, and sequence data from four tryptic peptides was obtained. Oligonucleotides derived from the peptide sequences were used to clone a 4.85-kb cDNA. The cDNA contained an 1139-amino-acid open reading frame with homology to the retinoblastoma protein and E1A-associated p107 but was more closely related to p107. In vitro-translated p130 bound to E1A, and anti-p130 antibodies detected p130 in immunoblots of E1A immunoprecipitates. p130 was also detected in immunoprecipitates of cyclins A and E and was an efficient substrate in vitro for kinase activities associated with these cyclins. The p130 gene mapped to chromosome 16q12.2-13, a region that undergoes allelic loss in several types of tumors, including hepatocellular, prostate, and breast carcinomas.

Adenovirus E1A Proteins↗
PubMed1993

Cell cycle-specific association of E2F with the p130 E1A-binding protein.

Association of the E2F transcription factor with the pRb and p107 proteins appears to regulate the activity of E2F and, in turn, affect cell cycle progression. We found, however, that pRb and p107 are only minor E2F-associated proteins in G0/G1 mouse fibroblasts, and we sought to identify the major E2F partner protein in these cells. Because the adenovirus E1A oncoprotein seemed able to bind to the G0 E2F partner, we enriched for proteins that associated both with an E2F-binding site DNA column and with E1A. The major species in G0 and early G1 fibroblasts detected with this approach had properties identical to the pRb- and p107-related p130 protein. In serum-stimulated cells, p107 replaced p130 as the major E2F-associated protein near the G1/S border, concomitant with an increase in p107 protein levels. p130-E2F complexes resembled p107-E2F complexes in their ability to bind to cyclin-cdk kinases, and they appeared to be associated with the cyclin E-cdk2 kinase in late G1 cells. These observations indicate that E2F transcription factors are regulated by a succession of partner proteins with which they associate during defined stages of the cell cycle.

3T3 Cells↗
PubMed1993

Five year study of prenatal testing for Huntington's disease: demand, attitudes, and psychological assessment.

Adult predictive and prenatal testing programmes for Huntington's disease (HD) in Canada have been available since 1986. However, the demand for prenatal testing and the reasons why some people choose not to have the prenatal test for this late onset disorder have not been well documented. In addition, the knowledge and attitudes of adult predictive testing candidates and their partners about prenatal testing are not well known nor are the psychological effects of prenatal testing well understood. As of September 1991, 425 subjects had entered the Canadian Collaborative Study of Predictive Testing and, of these, 47 subjects or their partners had become pregnant. Of this group, 14 (30%) couples requested prenatal testing, 24 (51%) couples did not want prenatal testing, and nine (19%) at risk subjects had already received a decreased risk through adult predictive testing and, therefore, were not eligible for the prenatal test. Of the 14 couples who initially requested prenatal testing, seven withdrew. Thus, demand for the prenatal test by eligible candidates was 7/38 or 18%, which is much lower than the 32 to 65% expected based on early survey data. The most frequently cited reason for declining prenatal testing was the hope that a cure would be found in time for their children. While the majority of adult predictive testing candidates (71%) in our study had accurate information about definitive prenatal testing, many (63%) did not have a correct understanding of exclusion prenatal testing. Although no serious adverse events such as suicide planning or admission to psychiatric hospital have occurred, a particular need for careful counselling was identified for those at risk candidates and their partners who have one prenatal test and feel compelled to use the test again in future pregnancies. Even though prenatal testing for HD is not requested as often originally expected, it still remains a desired option for some at risk persons and their partners.

Adult↗
PubMed1992

The psychological consequences of predictive testing for Huntington's disease. Canadian Collaborative Study of Predictive Testing.

BACKGROUND: Advances in molecular genetics have led to the development of tests that can predict the risk of inheriting the genes for several adult-onset diseases. However, the psychological consequences of such testing are not well understood. METHODS: The 135 participants in the Canadian program of genetic testing to predict the risk of Huntington's disease were followed prospectively in three groups according to their test results: the increased-risk group (37 participants), the decreased-risk group (58 participants), and the group with no change in risk (the no-change group) (40 participants). All the participants received counseling before and after testing. Standard measures of psychological distress (the General Severity Index of the Symptom Check List 90-R), depression (the Beck Depression Inventory), and well-being (the General Well-Being Scale) were administered before genetic testing and again at intervals of 7 to 10 days, 6 months, and 12 months after the participants received their test results. RESULTS: At each follow-up assessment, the decreased-risk group had lower scores for distress than before testing (P < 0.001). The increased-risk group showed no significant change from base line on any follow-up measure, but over the year of study there were small linear declines (P < 0.023) for distress and depression. The no-change group had scores lower than at base line on the index of general well-being at each follow-up (P < or = 0.045). At the 12-month follow-up, both the increased-risk group and the decreased-risk group had lower scores for depression and higher scores for well-being than the no-change group (P < or = 0.049). CONCLUSIONS: Predictive testing for Huntington's disease has potential benefits for the psychological health of persons who receive results that indicate either an increase or a decrease in the risk of inheriting the gene for the disease.

Adult↗
PubMed1992

Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial virus.

Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.

Antibodies, Monoclonal↗
PubMed1992

Dephosphorylation of the retinoblastoma protein during differentiation of HL60 cells.

Immunoprecipitated retinoblastoma protein from HL60 cells migrated as a series of bands during electrophoresis. The heterogeneity appeared to be generated by phosphorylation of the retinoblastoma protein. Treatment of the cells with the phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in both a loss of the heterogeneity of the pRB species and a significant decrease in the level of pRB phosphorylation. These changes accompanied differentiation of the HL60 cells into macrophages. Treatment of the cells with dibutyryl cAMP also resulted in dephosphorylation of pRB as well as cell cycle arrest, although no recognizable differentiation occurred. These results are consistent with a model in which TPA and dibutyryl cAMP dependent pathways can activate pRB by altering its phosphorylation.

Bucladesine↗
PubMed1991

Handling the pressure on an offshore rig.

The chief instigator of offshore stress is time which in oil parlance is money, writes Patrick Whyte, an offshore medical officer. He explains that medical personnel by showing that they practice a healthy lifestyle on the offshore oil rigs can set a practical example of coping with the stressful conditions.

Adaptation, Psychological↗
PubMed1991

Human cyclin A and the retinoblastoma protein interact with similar but distinguishable sequences in the adenovirus E1A gene product.

The adenovirus early region 1A (E1A) proteins associate with several cellular proteins in adenovirus infected or transformed cells. Recently, two of the cellular proteins that bind to E1A encoded proteins have been identified. p105 has been shown to be the product of the retinoblastoma tumor suppressing gene. p60 has been shown to be a human cyclin A. Previously studies have shown that E1A protein sequences encoded by conserved domains 1 and 2 are required for interactions with the retinoblastoma protein (pRB). We have demonstrated here that amino acids 30 to 60 and 121 to 127 within the E1A proteins are required for interaction with p60/cyclin A. These are the same sites within conserved domains 1 and 2 that are required for E1A protein association with pRB. However, the association of p60/cyclin A does not appear to require pRB. We also demonstrate that another cellular protein, 130K, interacts with E1A at essentially the same sites. It is interesting that mutations in these regions destroy the ability of E1A to function as an oncogene, thereby raising the possibility that interaction with several different cellular proteins may be needed for transformation by E1A.

Adenoviridae↗
PubMed1990

Cellular proteins that are targets for transformation by DNA tumour viruses.

Small DNA tumour viruses produce proteins that redirect cellular gene expression and growth control. The E1A polypeptides of adenovirus perform the functions of transcriptional activation and cellular transformation. These two functions are carried out by different domains within the E1A protein. The E1A protein associates with several cellular proteins, including the product of the retinoblastoma gene, pRb-1. Mutational analysis correlates transformation with the sites required for binding pRb and two other cellular proteins, p107 and a 300 kDa polypeptide. This correlation suggests that these proteins are targets for E1A-mediated transformation. Transforming proteins from other small DNA tumour viruses interact with pRb, raising the possibility that a common event in viral transformation is the inactivation of proteins that inhibit cellular proliferation. The role of the E1A-associated 60 kDa protein, p60, in transformation is being investigated. In the absence of E1A, p60 binds to the human homologue of the Schizosaccharomyces pombe cdc2 gene product, p34, to form a complex that has kinase activity that oscillates during the cell cycle. Ongoing studies of the effect of adenovirus infection, and specifically E1A expression, on this cellular kinase may provide clues to how E1A overcomes cell cycle controls and transforms cells.

Adenoviridae↗
PubMed1989

A 60 kd cdc2-associated polypeptide complexes with the E1A proteins in adenovirus-infected cells.

p60 is a cellular protein that binds to the adenovirus E1A protein complex in virally infected or transformed human cells. In both infected and uninfected cells, p60 was found in a complex with the cdc2 protein kinase. Immune complexes containing p60 and cdc2 display a cell cycle-dependent histone H1 kinase activity that is most active in interphase. The previously described cdc2-p62/cyclin complex also acts as a histone H1 kinase but is maximally active in mitotic metaphase. The shift in the timing of activation of different cdc2-containing complexes suggests that each might play a distinct role in regulation of the cell cycle.

Adenovirus Early Proteins↗
PubMed1989

The cellular 107K protein that binds to adenovirus E1A also associates with the large T antigens of SV40 and JC virus.

The association between the retinoblastoma protein (p105-RB) and either the large T antigen of SV40 or the E1A proteins of adenovirus is thought to be an important step in transformation by these viral oncogenes. E1A and large T antigen share a small region of amino acid homology that is necessary for high affinity binding with p105-RB. Mutations of this homology region were shown to reduce drastically the frequency of transformation mediated by the E1A or large T oncogenes. Previously, this small region in E1A was shown to be sufficient for interaction with a second cellular protein of 107,000 daltons (107K). Here we show that in human cells, the large T antigens of SV40 or JC virus also form complexes with 107K. Demonstration of complexes between 107K and the large T antigens of SV40 and JC virus suggests that these associations may represent another component of a common mechanism for transformation between adenoviruses and polyoma viruses.

Adenovirus Early Proteins↗
PubMed1989

Point mutational inactivation of the retinoblastoma antioncogene.

The retinoblastoma (Rb) antioncogene encodes a nuclear phosphoprotein, p105-Rb, that forms protein complexes with the adenovirus E1A and SV40 large T oncoproteins. A novel, aberrant Rb protein detected in J82 bladder carcinoma cells was not able to form a complex with E1A and was less stable than p105-Rb. By means of a rapid method for the detection of mutations in Rb mRNA, this defective Rb protein was observed to result from a single point mutation within a splice acceptor sequence in J82 genomic DNA. This mutation eliminates a single exon and 35 amino acids from its encoded protein product.

Adenovirus Early Proteins↗
PubMed1989

Cellular targets for transformation by the adenovirus E1A proteins.

Three cellular proteins, including species of 300,000 daltons and 107,000 daltons as well as p105-RB, the product of the retinoblastoma susceptibility gene, stably interact with the adenovirus E1A proteins. To help determine the functional basis of these interactions, the regions of E1A that participate in these interactions were mapped using a series of deletion mutants. The 300,000 dalton and the 107,000 dalton proteins interacted with sequences within amino acids 1 to 76 and 121 to 127, respectively. Interaction with the third cellular protein, p105-RB, required the presence of sequences from two noncontiguous regions of the E1A polypeptide chain, amino acids 30 to 60 and 121 to 127. The regions of E1A that are required for these interactions coincided precisely with the regions of E1A that are required for its transforming function. These results suggest that the interactions with these cellular proteins are fundamental to the transforming activity of E1A.

Adenovirus Early Proteins↗
PubMed1989

Cellular proteins that are targetted by DNA tumor viruses for transformation.

Tumor suppressor genes are genetic loci whose loss is associated with tumor development. Because the inactivation of these genes is a key feature in the genesis of certain tumors, it has been postulated that the protein products of tumor suppressor genes function in the negative regulation of cell proliferation. Tumor suppressor genes have been identified by genetic analysis either as loci associated with an inherited predisposition to certain tumors or by mapping studies that demonstrate allelic loss (reduction to homozygosity or loss of heterozygosity) during tumor development. The retinoblastoma gene, RB-1, was originally identified and cloned through its association with childhood retinoblastoma and is one of the best studied examples of the tumor suppressor genes. It has been shown that RB protein is also a key target for transformation by the oncogenes of several small DNA tumor viruses. The E1A proteins of adenovirus, the large T antigens of polyomaviruses, and E7 proteins of papillomaviruses all bind to pRB. Genetic studies of all three viruses have shown that any mutation that destroys binding to pRB also destroys the ability of these proteins to transform cells, suggesting that interaction with the RB gene product is a key event in viral transformation. In addition to interacting with pRB, the adenovirus E1A proteins and the polyomavirus large T antigens also bind to other cellular proteins. One of these, a protein with a molecular weight 0f 107,000 daltons, 107K, binds to E1A and large T at the same amino acid region as pRB, suggesting that the 107K and pRB proteins may have structural similarities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗