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Biomedical subjects

L Crawford

Publications and source records attributed to L Crawford.

At least 91 records · Page 5Linked to original sources
PubMed1987

Expression of human papillomavirus type 6 and type 16 capsid proteins in bacteria and their antigenic characterization.

The L1 and L2 capsid proteins encoded by human papillomavirus types 6 and 16 (HPV-6 and HPV-16) have been synthesized in bacteria. Antisera were raised against the HPV-6 L1- and L2-beta-galactosidase fusion proteins and against an HPV-16 L1 C-terminal peptide which was 14 amino acids long. The HPV-16 L1 peptide antibodies have been shown to be highly reactive with the HPV-16 L1-beta-galactosidase protein but not against the equivalent HPV-6 L1-beta-galactosidase fusion protein. The effectiveness of these antibodies was compared with commercially available antibovine papillomavirus type 1 (BPV-1) antibodies and the results demonstrated that the anti-BPV-1 antibodies reacted well against HPV-6 L1-beta-galactosidase but not against HPV-16 L1-beta-galactosidase. In addition, the L2 portion of the HPV-6 L2-beta-galactosidase fusion protein appeared particularly immunogenic, since antibodies raised against this fusion protein were predominantly reactive with the L2 moiety. The HPV-16 L1 peptide antibodies described here will be preferred reagents for the specific detection of HPV-16 capsid antigens, which may be particularly important in early diagnosis of HPV-16 infection.

Antibodies, Viral↗
PubMed1987

Identification of human papillomavirus type 18 E6 polypeptide in cells derived from human cervical carcinomas.

We recently reported the expression of human papillomavirus type 18 (HPV-18) E6 protein in bacteria and the production of anti-E6 polyclonal antibodies. This work has now been extended with the production of a panel of monoclonal antibodies against the HPV-18 E6 protein. These antibodies demonstrate that there is little antigenic conservation in the E6 protein between HPV-16 and HPV-18, with only one antibody recognizing a cross-reactive epitope. We have used both the monoclonal and the polyclonal antibodies to look for E6 expression in a number of HPV DNA-containing cell lines. These reagents specifically detected a 16.5K mol. wt. polypeptide in cells derived from a human cervical carcinoma.

Antibodies, Monoclonal↗
PubMed1987

Alternative splicing of human p53 transcripts.

The human p53 gene is expressed in both normal and transformed human cells as a family of polypeptides. One type of heterogeneity, which results in variation in electrophoretic mobility, is found at amino acid residue 72 (Matlashewski et al, 1987). A second type of heterogeneity is described here which involves an alteration in the sequence of the open reading frame at the 5' end of the p53 mRNA. This heterogeneity is generated by alternative splicing at the acceptor site in intron 2 resulting in an altered 5' end coding region. This study represents the first description of an altered human p53 transcript arising by differential splicing.

Amino Acid Sequence↗
PubMed1986

Isolation of human-p53-specific monoclonal antibodies and their use in the studies of human p53 expression.

The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti-(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos-l cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with beta-galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognized by the antibodies. At least two previously unrecognized epitopes are involved and two of the antibodies are human-p53-specific. The epitopes are denaturation-resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme-linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos-l) cells cause expression of human p53 recognized by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.

Animals↗
PubMed1986

Analysis of human p53 proteins and mRNA levels in normal and transformed cells.

p53 mRNA and proteins were examined in a variety of human transformed cells and in normal human foreskin fibroblast cells. Both the steady-state and translatable levels of p53 mRNA were the same in normal and transformed human cells. In vitro synthesized p53, programmed by mRNA from normal and transformed human cells, revealed that there was heterogeneity in the primary structure of p53 from these cells. Pulse labeling of cells and immunoprecipitation analysis with a panel of human reactive anti-p53 antibodies demonstrated that the types of p53 synthesized in vitro corresponded to the types made in vivo from SV80 and COLO 320 cells. No p53 was detectable by similar pulse-labeling analysis of HeLa and normal foreskin fibroblast cells. Since it was necessary to use anti-p53 sera from cancer patients to carry out much of the immunoprecipitation analysis in this study we therefore further characterised these sera to determine if they reacted with one or more than one epitope. p53-beta-galactosidase fusion proteins were synthesized in Escherichia coli and used to analyse the anti-p53 antibodies produced by cancer patients. We demonstrate that the antisera contain antibodies directed against epitopes in both the N-terminal and C-terminal regions of the p53 molecule.

Antibody Specificity↗
PubMed1986

Criteria for establishing that a virus is oncogenic.

To prove that a particular infectious agent causes a disease is much more difficult in human subjects than in other animals for both ethical and practical reasons. Where the disease is a malignant tumour with a long latent period the situation is even more difficult. For these reasons, it is often necessary to concentrate in the first instance on association of the virus with the disease, and this is discussed in the context of papillomaviruses. Association of a virus with a tumour may occur for a variety of reasons other than the virus being the cause of the tumour. This is illustrated by several examples of parvoviruses and DNA tumour viruses. Conversely, the absence of any sign of virus or viral nucleic acid in a tumour does not prove that the tumour was not induced by a virus. Apart from association of a virus with a tumour it is also necessary to show that the virus in question is oncogenic. Again this cannot normally be done directly, so that indirect evidence from animal experiments or from in vitro transformation is likely to be the best available alternative. In the final analysis the best proof of oncogenicity may be the effectiveness of intervention directed at the virus.

Adult↗
PubMed1986

Codex committee on veterinary drug residues.

There has been widespread international concern over residues of veterinary drugs in food. However, until recently, there was little international cooperation in trying to find solutions to the problem. Many nations have taken steps to control the increased use of veterinary drugs, but the rules governing their use vary widely from country to country. Sharing of scientific expertise and other resources between countries would markedly improve this situation. With the need for international cooperation mounting, concerned drug regulators from 20 countries met in 1982 and again in 1984 to discuss international use of veterinary drugs. The group repeatedly called for a Codex committee on veterinary drug residues. At its 1983 meeting in Rome the Codex Alimentarius Commission convened an Expert Consultation to consider the need for a new committee. That international group of experts strongly agreed that a standing committee under the sponsorship of the commission should be established. In July of 1985, the commission unanimously voted to establish a new Committee on Residues of Veterinary Drugs in Food. The United States was chosen as host country. The committee will work closely with several existing Codex committees, but it has a clear mandate of its own. Its responsibilities will include establishing a list of priority drugs for review, recommending maximum residue levels, developing codes of practice, and reviewing analytical methods used to control veterinary drug residues. This fall, a new Codex committee has met for the first time--the Codex Committee on Residues of Veterinary Drugs in Food.(ABSTRACT TRUNCATED AT 250 WORDS)

Agriculture↗
PubMed1986

The expression of human papillomavirus type 18 E6 protein in bacteria and the production of anti-E6 antibodies.

Human papillomavirus type 18 (HPV-18) has recently been closely linked with human malignant cervical lesions. The early region of the genome of the related bovine papillomavirus (BPV) has been shown to be important for the production of the transformed phenotype. BPV E6 has been shown to be a transforming protein. We report the primary structure of the HPV-18 E6 open reading frame and its predicted amino acid sequence. Both E6 protein and E6-beta-galactosidase fusion protein were synthesized in bacteria. Antisera were raised against the E6-beta-galactosidase fusion protein and against an E6 N-terminal peptide which was 14 amino acids long. We show that these antisera reacted on Western blots against E6 synthesized in bacteria. The HPV E6 antigen and antibodies described here will be useful in understanding HPV expression and its association with human malignancies and may also be diagnostically useful.

Amino Acid Sequence↗
PubMed1986

Characterization of the human p53 gene.

Cosmid and lambda clones containing the human p53 gene were isolated and characterized in detail. The gene is 20 kilobases (kb) long and has 11 exons, the first and second exons being separated by an intron of 10 kb. Restriction fragments upstream of sequences known to be within the first identified exon were tested for promoter activity by cloning them in front of the chloramphenicol acetyltransferase gene and transfecting the resulting constructs into HeLa cells. A 0.35-kb DNA fragment was identified that had promoter activity. Results of primer extension experiments indicated that the mRNA cap site falls within this fragment, as expected. Analysis of the sequence upstream of the presumptive cap site indicated that the human p53 promoter may be of an unusual type.

Amino Acid Sequence↗
PubMed1986

Migration in vitro by blood and exudate neutrophils assessed serially during an inflammatory response.

Migration in vitro by blood and inflammatory neutrophils has been compared serially during an inflammatory response. Using an experimental pig model, neutrophils are isolated from the peripheral blood and from the pleural space at hourly intervals after an intrapleural challenge with zymosan activated pig serum (ZAS). Following acepromazine sedation and halothane anesthesia, blood neutrophil migration was transiently reduced. By 1 hour random and directed migration of blood neutrophils returned to normal. Directed and random migration of exudate neutrophils was markedly decreased to both a stimulus-specific (ZAS) and an unrelated (LTB4) chemoattractant. After 3 hours, migration by exudate neutrophils was similar to migration by blood neutrophils examined in parallel. These findings emphasize the importance of performing serial evaluations of cell function during an inflammatory response.

Animals↗
PubMed1985

Permanent expression of p53 in FR 3T3 rat cells but cell cycle-dependent association with large-T antigen in simian virus 40 transformants.

The p53 oncogene is thought to play a role in the proliferation of normal and transformed cells and its expression was postulated to be cell cycle dependent. Using flow cytofluorimetry sorting of populations of exponentially growing cells, coupled to a radioimmune assay, we have investigated the accumulation of p53 along the cell cycle in normal FR 3T3 rat cells as well as in two types of SV40-transformed derivatives, one of which only expresses the large-T protein during the G2 phase of the cell cycle. p53 was accumulated at a constant level throughout the cell cycle in FR 3T3 cells. Its level and stability increased to different extents in the two types of transformants. However, the formation of complexes between p53 and large-T was modulated by the G2-restricted accumulation of large-T, thus leading to a differential increase in the levels of p53 both in exponentially growing cells and along the cell cycle. This increase in the levels of p53 appeared to be regulated at a post-transcriptional level.

Animals↗
PubMed1985

Malakoplakia and immunosuppressive therapy. Reversal of clinical and leukocyte abnormalities after withdrawal of prednisone and azathioprine.

Malakoplakia is a chronic granulomatous inflammatory disorder. It is suspected clinically by the presence of chronic infection and diagnosed by histologic examination of affected tissues. Studies of 4 patients with malakoplakia--2 renal transplant recipients, 1 patient with systemic lupus erythematosus, and 1 patient with polymyositis--are reported. All patients were receiving prednisone and azathioprine at the time of diagnosis and had an infection caused by Escherichia coli. Leukocytes from all patients failed to kill Staphylococcus aureus and E coli normally in vitro. Cholinergic agonists had no apparent effect on bacterial killing in vitro or in vivo in the 2 patients examined. Clinically, malakoplakia improved significantly when immunosuppressive therapy was tapered or discontinued, and leukocyte function returned to normal in all 4 patients. The cases reported here and those documented previously suggest that the pathogenesis of malakoplakia and its treatment may not be the same for all patients. Malakoplakia may be more common than previously thought, particularly with the increased use of immunosuppressive therapy.

Abscess↗
PubMed1984

Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene.

A cDNA clone for human p53 cellular tumor antigen has been isolated and characterized. This clone contains the complete 3'-untranslated region and most of the open reading frame for the protein. Nucleotide sequence analysis revealed that p53 mRNA contains an Alu repeat in the 3'-untranslated region. Hybridization selection experiments showed this clone was capable of selectively binding p53 mRNA. In vitro translation of SV80 mRNA resulted in the synthesis of two immunoreactive p53 polypeptide species. Northern blot analysis showed that human p53 mRNA was 2.8 kb in length and was present in cell lines containing high and low levels of p53 protein. There appears to be only a single p53 gene in human cells and Southern blot analysis demonstrated no major genomic rearrangements or amplification of the p53 gene in the transformed cell lines examined.

Antigens, Neoplasm↗
PubMed1984

The kinetics of [3H]-dsDNA/anti-DNA immune complex formation, binding by red blood cells, and release into serum: effect of DNA molecular weight and conditions of antibody excess.

[3H]dsDNA/anti-DNA immune complexes (IC) formed, fixed complement, and bound rapidly to red blood cells (RBC) in whole blood (less than 5 min), but were released from the cells more slowly. The rate of release was dependent on both the antibody:DNA ratio and the m.w. of the DNA in the complex. For example, complexes formed with high m.w. DNA (6 X 10(6) daltons) were released more slowly (t1/2 = 60 min) than complexes formed with lower m.w. DNA (2 to 6 X 10(5) daltons, t1/2 = 15 to 20 min). The [3H]dsDNA/anti-DNA complexes, which were released from the cells as intact antigen/antibody/complement complexes, did not rebind to RBC, but did bind to Raji cells and could be precipitated by monoclonal antibody to C3d. When these released IC (RIC) containing high m.w. DNA were incubated with additional anti-DNA antibody and fresh complement, they rebound to RBC. However, RIC containing lower m.w. DNA (5 X 10(5) daltons) did not rebind to RBC under the same conditions. These data suggest that IC containing high m.w. DNA bind to and remain bound to RBC more effectively than IC containing lower m.w. DNA, and thus may be more easily cleared from the circulation by the RBC IC clearance mechanism. Thus, the size of the DNA in the IC may be a significant factor in the pathogenicity of DNA/anti-DNA complexes in SLE.

Antibodies, Antinuclear↗
PubMed1983

Monoclonal antibodies displaying a novel species specificity for the primate transformation-related protein, p53.

SV40 large T antigen associates with a cellular phosphoprotein, p53, in virus-transformed cells. We have raised three new monoclonal antibodies, PAb1101, PAb1102 and PAb1103, to this cellular protein, derived from SV40-transformed human fibroblasts. These define at least two non-overlapping determinants on human p53 that are in different areas of the molecule from those recognised by previously available antibodies. Unlike those antibodies, PAb1102 and PAb1103 do not react with rodent p53. PAb1101 reacts far more weakly with rodent p53 than with primate p53. All three antibodies show a preference for binding to the large T-associated form of p53, an effect that is particularly marked with PAb1102. The novel specificity of these antibodies allows further probing of the nature and function of the large T/p53 complex in human cells.

Animals↗
PubMed1983

Purification and partial amino acid sequence analysis of the cellular tumour antigen, p53, from mouse SV40-transformed cells.

The cellular tumour antigen p53 is implicated in the transformation process. To compare p53 from transformed cells and their normal counterparts in detail, and so to identify any structural differences that might alter p53 function, requires information on the primary structure of the protein. By making use of immunochemical techniques we have been able to purify nanomole amounts of p53. This was sufficient, using high sensitivity automated gas-phase sequencing techniques to determine the amino acid sequence of two tryptic peptides from p53. Their sequences agree completely with the predicted polypeptide sequence derived from a cloned cDNA for p53 mRNA and provide the first data on the amino acid sequence of p53. A combination of the high sensitivity amino acid sequencing procedures used here and cDNA sequence analysis should provide the complete amino acid sequence of p53.

Amino Acid Sequence↗