Search PubMed⌕ Search

Biomedical subjects

L Crawford

Publications and source records attributed to L Crawford.

At least 37 records · Page 2Linked to original sources
PubMed1995

The interaction between human papillomavirus type 16 E1 and E2 proteins is blocked by an antibody to the N-terminal region of E2.

Replication of papillomavirus DNA requires two virally encoded proteins, E1 and E2. We expressed human papillomavirus (HPV) type 16 E1 and E2 in bacteria and showed that purified full-length E2 protein interacted directly with E1, in the absence of HPV16 DNA. It was established that the first 142 amino acids of E1 were not required for binding as E2 protein was able to interact with E1 devoid of this region. The interaction of E2 with E1 could be blocked by a monoclonal antibody that bound E2 in the region of amino acids 18-41 of E2 whereas a monoclonal antibody reactive with a nearby part of the molecule (amino acids 2-17) only partially blocked this interaction. These results suggest that a region in the N-terminus of E2 around amino acids 18-41 is a site of interaction with the E1 protein.

Adenosine Triphosphatases↗
PubMed1995

Is proximal demarcation of ulcerative colitis determined by the territory of the inferior mesenteric artery?

The sharp demarcation between diseased and normal mucosa often observed in ulcerative colitis remains unexplained by current hypotheses of disease pathogenesis. To investigate whether this demarcation occurs at the watershed of vascular territories, the colonic arterial anatomy of 10 patients with ulcerative colitis was studied by in-vitro angiography and macroscopic and histological examination of total colectomy specimens. Of the 10 perfusion-fixed colectomy specimens studied, 7 had pancolitis associated with a complete marginal artery (Arteria marginalis coli) that spanned the entire length of the large bowel. 3 specimens had sharply demarcated disease in which the marginal artery arose from the inferior mesenteric artery and ended abruptly at the point of mucosal demarcation. The colon proximal to this point was histologically normal. These findings suggest that the proximal extent of colitis is determined by the limit of the marginal artery. We suggest that some characteristic of the mucosal microvasculature in the territory of the inferior mesenteric artery, possibly embryological in origin, predisposes the dependent colon to develop ulcerative colitis.

Adult↗
PubMed1995

Synthesis and assembly of virus-like particles of human papillomaviruses type 6 and type 16 in fission yeast Schizosaccharomyces pombe.

We have synthesized capsid proteins of human papillomavirus types 6 (HPV 6) and 16 (HPV 16) in fission yeast Schizosaccharomyces pombe and produced virus-like particles (VLP). The capsid proteins were localized in the nucleus by indirect immunofluorescence and cell fractionation analyses. The VLP were produced in both yeast clones synthesizing L1 alone and L/L2 and purified by sulfato-cellulofine chromatography. Electron microscopic examination showed that these VLP were similar in structure to native HPV particles. Two HPV 16 L1 variants (16 B27L1 and 16 T3L1), isolated from benign cervical samples, produced many more (68- and 14-fold) VLP than the prototype L1 (16 PL1) derived from cervical carcinoma. Coexpression of the HPV 6 L2 protein with 6 L1 and 16 B27L1 proteins increased the production level of the VLP four- and twofold, respectively. The L2 was not detected in the VLP purified with sulfato-cellulofine column, although the L2 was purified in the same fraction containing HPV 6 and 16 B27-VLP by size-fractionation using Sepharose column. Interaction between 6 L2 and 6/16 L1 proteins was not detected by the coimmunoprecipitation assays with either L1 or L2 antibodies. These results suggest that the L2 is not incorporated into the VLP synthesized in yeast.

Base Sequence↗
PubMed1995

Human papillomavirus E6 and E7: proteins which deregulate the cell cycle.

Numerous clinical, epidemiological and molecular findings link some types of Human Papillomaviruses (HPV) with cancer of the genital tract. They share a common pathway of transformation with a number of DNA tumour viruses, such as Adenovirus and SV40. Although all these viruses are termed 'DNA tumour viruses' and have similar in vitro transforming activities, Human Papillomavirus is the only one so far clearly involved in human cancer. Extensive studies on HPV E6 and E7 proteins have demonstrated their involvement in malignant transformation. E6 and E7 bind the products of tumour suppressor genes, p53 and Rb1, respectively, modifying or inactivating their normal functions. The Rb1 and p53 genes are deleted or mutated in several cancers and both proteins regulate the transcription of genes involved in cell cycle progression control. The E6/p53 and E7/Rb1 interactions result in a deregulation of the cell cycle with loss of control of crucial cellular events, such as DNA replication, DNA repair and apoptosis.

Amino Acid Sequence↗
PubMed1995

A preliminary assessment of the toxic and mutagenic potential of steroidal alkaloids in transgenic mice.

Impregnated CD2 transgenic mice, which contain multiple copies of a lambda gt10lacZ construct integrated into the genome of each cell, were given a predetermined estimated maximum tolerated dose of several steroidal alkaloids: Solanum glycoalkaloids from potato, alpha-chaconine and alpha-solanine; aglycones, solanidine and solasodine, and a Veratrum alkaloid, jervine. Observations were made of dams and foetuses for indications of toxicity and/or terata; some dam livers and foetuses were assayed for mutagenicity using the lacZ gene. Other dams were gavaged with a single dose of 75 mg all-trans-retinol/kg to serve as a reference teratogen. Unexpectedly, this level of retinol was not clearly teratogenic. The results of both positive and non-positive selection systems showed that the mutation frequencies in the livers of the dams dosed with alpha-chaconine, alpha-solanine and solanidine were three to four times higher than historically normal in the livers of this transgenic mouse strain.

Alkaloids↗
PubMed1995

Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity.

We demonstrate a novel and efficient bioprocess for production of the cephalosporin intermediates, 7-aminocephalosporanic acid (7-ACA) or 7-amino deacetoxycephalosporanic acid (7-ADCA). The Streptomyces clavuligerus expandase gene or the Cephalosporium acremonium expandase-hydroxylase gene, with and without the acetyltransferase gene, were expressed in a penicillin production strain of Penicillium chrysogenum. Growth of these transformants in media containing adipic acid as the side chain precursor resulted in efficient production of cephalosporins having an adipyl side chain, proving that adipyl-6-APA is a substrate for either enzyme in vivo. Strains expressing expandase produced adipyl-7-ADCA, whereas strains expressing expandase-hydroxylase produced both adipyl-7-ADCA and adipyl-7-ADAC (aminodeacetylcephalosporanic acid). Strains expressing expandase-hydroxylase and acetyltransferase produced adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA. The adipyl side chain of these cephalosporins was easily removed with a Pseudomonas-derived amidase to yield the cephalosporin intermediates.

Acetyltransferases↗
PubMed1995

Cell cycle-dependent disruption of E2F-p107 complexes by human papillomavirus type 16 E7.

The human papillomavirus type 16 (HPV-16) E7 and adenovirus (Ad) E1A oncoproteins share a common pathway of transformation. They disrupt the cell cycle G1 phase-specific protein complex containing the E2F transcription factor and the regulatory protein Rb1, the retinoblastoma tumour suppressor gene product. In the G1 and S phases of the cell cycle, E7 and E1A bind two other cellular complexes containing the Rb1-related protein p107 and E2F. Ad E1A disrupts both complexes and releases active E2F. In contrast, HPV-16 E7, although it efficiently binds both E2F-p107 complexes, causes dissociation of the G1 phase complex only. Using chimeric proteins of HPV-16 E7 and Ad E1A we were able to demonstrate that the ability of E1A to disrupt both G1 and S phase E2F-p107 complexes is not due to the higher concentration of Ad E1A in the cell, but is an intrinsic property of the Ad E1A transforming region. These data suggest that E1A and E7 may function in cellular transformation in similar, but not identical ways.

3T3 Cells↗
PubMed1995

Both immunization with protein and recombinant vaccinia virus can stimulate CTL specific for the E7 protein of human papilloma virus 16 in H-2d mice.

The transforming protein E7 of human papilloma virus type 16 can stimulate cytotoxic T lymphocytes (CTL) which can protect experimental animals against growth of E7 expressing tumour cells. In this study we compared CTL responses in mice immunized with either E7 protein in MF59 adjuvant or with recombinant vaccinia virus expressing E7 (Vac-E7). We have chosen H-2d mice because no E7-specific CTL responses have been described in this MHC haplotype. Immunization of these mice with Vac-E7 generated CTL which lysed target cells infected with Vac-E7 or transfected with the E7 gene. CTL from mice immunized with E7 protein in MF59 adjuvant showed specificity for the same target cells. Antibody blocking experiments revealed that both immunization with Vac-E7 and E7 protein stimulated CD8+ effector CTL. The find specificity of CTL induced by the two immunization protocols was similar. A major CTL epitope was mapped to the carboxyl terminal amino acids 48-98 of the E7 protein. Peptide isolation from E7 expressing cells followed by HPLC separation indicated that CTL induced by immunization with protein and Vac-E7 recognized the same HPLC purified peptide fractions. Together, the study suggests that vaccines based on protein can activate CTL with similar fine specificity to CTL induced by vaccines based on recombinant vaccinia virus.

Adjuvants, Immunologic↗
PubMed1994

Strategies for studying mouse and human immune responses to human papillomavirus type 16.

Cytotoxic T lymphocytes (CTL) are an important protective mechanism in viral infection and can be effective against tumours. We have investigated the tumour-associated E6 and E7 genes of human papillomavirus type 16 as CTL targets. In H-2b mice we have defined epitopes in E6 and E7 which can readily generate CTL in vivo and we have shown that HLA-A2.1 transgenic mice can generate an HLA-A2.1-restricted response. We have been unable to reveal a primed CTL response in humans. These paradoxical findings imply that human papillomavirus may fail to stimulate a systemic CTL response and/or employ strategies for evading or down-regulating such a response.

Amino Acid Sequence↗
PubMed1994

Limitations of predictive motifs revealed by cytotoxic T lymphocyte epitope mapping of the human papilloma virus E7 protein.

Human papilloma virus (HPV) type 16 is found in the majority of cervical cancer patients and the transforming protein E7 is consistently expressed in cancer cells, making it a potential target for immune attack. In this study we have investigated whether E7 gains access to the MHC class I processing pathway and provides cytotoxic T lymphocyte (CTL) stimulating peptide epitopes. CTL were induced in H-2b mice by immunization with recombinant vaccinia virus expressing E7 (Vac-E7). To map CTL recognition, natural peptides were purified from cells expressing either intact or truncated E7 protein. Following peptide separation by HPLC one major CTL epitope was detected and truncated constructs localized this epitope to the C-terminal region. Mapping with synthetic peptides indicated that residues 49-57 (RAHYNIVTF) were recognised by anti-E7 CTL. Synthetic 49-57 peptide was used to induce CTL, which recognized the same HPLC purified natural peptide fractions as anti-E7 CTL. Binding motifs for H-2b class I molecules did not predict residues 49-57 to be a CTL epitope, but instead the sequence 21-28 (DLYCYEQL) which contains a Kb anchor motif. Synthetic 21-28 peptide was found to bind to Kb class I molecules and readily induced CTL, indicating that the T cell repertoire of H-2b mice can recognize this epitope. However, these CTL did not recognize peptides isolated from E7 expressing cells, showing that natural processing did not produce detectable levels of the 21-28 epitope. Together, the data demonstrate that an unexpected E7 peptide can function as a major CTL epitope.

Amino Acid Sequence↗
PubMed1994

Immune response to human papillomavirus type 16 E6 gene in a live vaccinia vector.

Immunization of mice with a recombinant vaccinia virus expressing the human papillomavirus type 16 (HPV-16) E6 gene elicits specific antibody, proliferative and cytotoxic T lymphocyte responses. T and B cell epitopes were mapped by using synthetic peptides. This study provides the background to future investigation aimed at developing prophylactic and therapeutic vaccines against HPV-16 infection and cervical cancer.

Amino Acid Sequence↗
PubMed1994

Sequence variation in the capsid protein genes of human papillomavirus type 16.

We have cloned and sequenced the L1 and L2 genes from human papillomavirus type 16 (HPV16) DNA-containing cervical cytology samples collected from the U.K. and Trinidad. Samples containing high copy numbers of HPV16 DNA were selected as being likely to contain fully functional virus DNA molecules in an episomal state, rather than in an integrated and possibly altered state. In comparison with the previously published sequence of HPV16 isolated from an invasive cancer a variety of differences were detected in both L1 and L2. The pattern of changes appears to be different in samples from the two geographic regions. One of the differences (resulting in D at position 202 of the L1 protein) reported recently to be functionally important for virus particle assembly was found to occur in all the samples examined. Variations in L1 found within known immunoreactive regions or hydrophobic domains should be taken into account in design of prophylactic vaccines for HPV16 based on virus-like particles. All variations within L2 protein were found in hydrophilic domains in the carboxy-terminal half of L2. These positions were highly variable among other types of papillomavirus and are located outside the known L2 immunoreactive region.

Amino Acids↗
PubMed1994

Functional studies of E7 proteins from different HPV types.

We have performed comparative studies on the E7 proteins from malignant and non-malignant Human Papillomavirus types HPV 1, 6, 11, 16, 18, 33). GST/E7 fusion proteins from all these HPV types associate with Rb1, p107 and the cyclin A/CDK2 complex. As has been shown for Rb1, the association with p107 and Cyclin A was weaker for the 'low risk' HPV6 and 11 E7 proteins as compared to 'high risk' HPV16, 18 and 33 E7 proteins. In contrast the E7 protein of the benign type HPV1 bound Rb1, p107 and cyclin A with the same affinity as the 'high risk' E7 proteins. The affinities of the E7/Rb1 interaction have been confirmed in vivo by the 'two hybrid' method in the yeast Saccharomyces cerevisiae. Although HPV1 E7 showed the same affinity in vitro and in vivo for Rb1 as the high risk HPV E7s, it did not have the ability to activate the E2F-1 transcription factor inhibited by Rb1, nor did it have any transforming activity when coexpressed with activated ras in primary rodent cells.

Animals↗
PubMed1994

Neutralizing antibody responses to autologous and heterologous isolates of human immunodeficiency virus.

Although laboratory-adapted strains of human immunodeficiency virus (HIV) are generally highly sensitive to neutralization by HIV-positive patient sera, we have found a more complex pattern of cross-neutralization and neutralization resistance among low-passage clinical isolates. These HIV isolates, like many other lentiviruses, resisted neutralization by the patient's own (autologous) antibodies. We assessed the degree of antigenic relatedness between different patient isolates of HIV through cross-neutralization with heterologous sera and virus isolates. Complicated patterns emerged, with variation in breadth of neutralization among individual plasmas and variation in frequency of neutralization among isolates. In longitudinal studies of individuals, we found that some but not all such patients develop a neutralizing response that "catches up" with their earlier isolates after a lag period. Taken together, these data suggest that an individual's immune response broadens with time because of cumulative exposure to multiple antigenic variants that arise throughout HIV disease.

Cells, Cultured↗
PubMed1994

Interaction of the E6 protein of human papillomavirus with cellular proteins.

The E6 proteins of the oncogenic Human Papillomavirus (HPV) types 16 and 18 are known to bind two cellular proteins, the tumor suppressor protein p53 and a 100 kDa protein named E6-AP. In this paper we describe the expression and purification of biologically active E6 fusion proteins and their specific association with additional cellular proteins. HPV16E6 specifically associated with at least seven cellular proteins which have been designated pp212, pp182, p100, p81, p75, p53 and p33 respectively, on the basis of molecular mass and phosphorylation. We have also shown that the complex of cellular proteins associated with HPV16, 18, 6 and 11 E6 proteins contains a protein kinase. This protein kinase phosphorylated exogenous histone H1 and the E6 associated protein pp182.

Papillomaviridae↗
PubMed1994

The trials, tribulations and achievements of implementing quality management.

Quality initiatives in the 1960s included Mortality and Morbidity Studies. In the 1970s Retrospective Audit was developed. In the 1980s Quality Assurance was the favoured option and was strongly promoted by The Australian Council on Healthcare Standards. In the 1990s the trend established across business and industrial organizations has been towards Quality Management. Health care services today are taking up this challenge and embarking on a new Quality journey. The importance of Quality Leadership in preventing frustration, rework and demotivation for individual participants of the organization should not be underestimated in implementing this Quality process.

Education, Continuing↗