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Biomedical subjects

D C Burke

Publications and source records attributed to D C Burke.

At least 37 records · Page 2Linked to original sources
PubMed1983

Characterization and properties of a modified human interferon-alpha containing an additional 18 amino acids at the N-terminus.

A modified human interferon-alpha 2 was produced in Escherichia coli cells infected with phage M13 mp7 containing an interferon-alpha gene. After purification by immunochromatography with the monoclonal antibody NK2, the N-terminal amino acid sequence was determined. The N-terminal methionine was absent but an additional sequence of 18 amino acids at the N-terminus was retained. The modified interferon-alpha 2 was indistinguishable from authentic interferon-alpha 2 in its ability to activate natural killer cells, to slow the growth of Daudi cells, and to confer virus resistance on heterologous cells.

Amino Acid Sequence↗
PubMed1982

The mechanism of interferon production.

Interferons are formed when most cells are treated with viruses or double-stranded RNA (to form IFN-alpha or beta, or both) or when lymphoid cells are treated with mitogens or the appropriate antigen (to form IFN-gamma). Interferon-alpha and beta are formed as a result of transcription of cellular genes--probably in response to double-stranded RNA in the cytoplasm. The process can be controlled at three levels. (1) In mouse teratocarcinoma stem cells or early mouse embryos the interferon system is inaccessible and only becomes inducible as differentiation proceeds. (2) The target(s) responding to double-stranded RNA probably involve sequences upstream from the 5' end of the interferon genes, sequences now becoming accessible by gene cloning. (3) Levels of interferon mRNA can be regulated either by an increased rate of transcription or by an increased half-life of the mRNA.

Animals↗
PubMed1982

Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae.

The 5' control region of the yeast phosphoglycerate kinase gene (PGK) was fused to the coding sequence of a human interferon-alpha. This PGK-interferon fusion was then introduced into yeast on a high copy number 2mu-based plasmid vector. Strains containing this plasmid produced a PGK-interferon-alpha fusion protein as 1-2% of cell protein and the expression of interferon activity was regulated by the availability of a fermentable carbon source. The system is capable of making as much as 15 mg of human interferon-alpha per litre of batch culture.

Amino Acid Sequence↗
PubMed1982

High-level expression of an interferon alpha 2 gene cloned in phage M13mp7 and subsequent purification with a monoclonal antibody.

A cloned interferon alpha 2 (IFN-alpha 2) gene was partially digestd with Pvu II to give a fragment that was inserted into the HincII site of the lacZ gene of bacteriophage M13mp7. Two recombinant phages containing the IFN-alpha 2 sequences in the correct orientation for expression from the lac promoter were characterized in detail. DNA sequence analysis showed that the inserted IFN-alpha 2 gene was in phase with the initiation codon of the lacZ gene. The polypeptide product has an additional 19 amino amino acids at the amino terminus of the mature IFN-alpha 2. The first 11 amino acids originate from the amino terminus of beta-galactosidase, and the remaining 8 amino acids are part of the signal sequence of pre-IFN-alpha 2. Infection of Escherichia coli with these phage followed by induction of the lac promoter with isopropyl thiogalactoside gives high yields (up to 10(9) units/liter with an average of 1.5 X 10(8) units/liter) of the modified IFN-alpha 2. This was purified to homogeneity in a single step by immunochromatography using the monoclonal antibody NK2. The nonreduced product had an apparent molecular weight of 20,500 and was shown by immunoradiometric assay to have the same specific activity as IFN made in Namalwa cells. It exhibited the characteristic cross-species antiviral activity of IFN-alpha 2.

Amino Acid Sequence↗
PubMed1982

Control of interferon mRNA levels and interferon yields in butyrate and 5'-bromodeoxyuridine-treated Namalwa cells.

Treatment of Namalwa cells with butyrate or 5'-bromodeoxyuridine (BrdUrd) before induction with Sendai virus caused an increase in the production of both interferon (IFN) and interferon mRNA (IFN mRNA). However, the increase in IFN mRNA did not completely account for the increase in IFN yield. The treatments did not affect the time course of IFN mRNA transcription and translation, or the association of IFN mRNA with polysomes. Likewise, the treatments did not alter the post-translational fate of the IFN produced. We conclude that butyrate and BrdUrd affect IFN production at the level of transcription or processing of IFN mRNA and suggest that increased efficiency of translation provides an additional level of control.

Bromodeoxyuridine↗
PubMed1982

Analysis and purification of human lymphoblastoid (Namalwa) interferon using a monoclonal antibody.

Highly purified interferon-alpha (IFN-alpha) prepared from a human lymphoblastoid line (Namalwa) was analysed by gel filtration and polyacrylamide gel electrophoresis (PAGE). Gel filtration separated the IFN-alpha into two peaks (A and B). All the components of peak A were retained by a monoclonal antibody (NK2) column, but some of those from peak B were not retained. The IFN that was not bound was active on mouse cells and could be resolved into two major bands by PAGE. The bound fraction (about 75% of the interferon protein) was purified by means of the monoclonal antibody column, although complete purification of crude interferon was not achieved in one passage.

Antibodies, Monoclonal↗
PubMed1981

Production and screening of cell hybrids producing a monoclonal antibody to human interferon-alpha.

Mice and rats were immunized with human lymphoblastoid interferon, a mixture of human leukocyte (interferon-alpha) and human fibroblast (interferon-beta) interferons, and their serum anti-interferon levels measured. Anti-interferon activity was detected in all animals, although the mouse sera had higher titers than the rat sera. The spleens of two mice were removed and used to prepare hybrid myelomas. Anti-interferon activity in tissue culture supernatants was measured using a direct neutralization assay and a clone (NK2) producing antibody to human interferon-alpha (leukocyte) was isolated. The anti-interferon activity of this monoclonal antibody was confirmed using a new indirect immunoprecipitation (IIP) assay, which shows some advantages over the direct neutralization assay. The antibody did not neutralize human interferon-beta (fibroblast) or mouse interferon.

Animals↗
PubMed1981

Partial reversion of the transformed phenotype of murine sarcoma virus-transformed cells in the presence of interferon: a possible mechanism for the anti-tumour effect of interferon.

The effect of interferon (IFN) on a series of cellular properties which characterize the transformed state was studied, using normal mouse fibroblasts (both established lines and embryo cells) and cloned derivatives transformed by murine sarcoma virus. It was found that the transformed cells behaved in a more normal fashion in the presence of IFN. This was indicated by reduction in saturation density, decreased DNA synthesis in crowded cultures, decreased formation of foci on monolayers of normal cells, and decreased growth in soft agar. These effects were not due to generalized inhibition of cell growth, since IFN had little effect on the growth and cloning efficiency of the cells used. In the presence of IFN, cellular morphology also appeared more normal. These results suggest that growth control of transformed cells may be at lease partly restored by IFN, an effect which could in part account for the anti-tumour effect of IFN

Animals↗
PubMed1981

General principles of management of the paraplegic.

The management of patients with traumatic paraplegia and tetraplegia has improved markedly in recent years. Survival now approaches the normal life expectancy, due to modern intensive care and understanding of potential complications of the spinal paralysis. Rehabilitation techniques lead to excellent physical and psychological adjustment, and community-based follow up with cooperation from the specialized spinal units has ensured continued good care after discharge.

Aftercare↗
PubMed1980

Interferon inhibits transformation by murine sarcoma viruses before integration of provirus.

Neoplastic transformation by C-type retroviruses requires synthesis of a DNA copy (the provirus) of the RNA genome and its integration into the host cell DNA. We have previously shown that interferon (IFN) can stably prevent transformation of murine fibroblasts by the Kirsten strain of murine sarcoma virus (KiMSV), a murine leukaemia virus (MLV). A series of cell clones (IFN clones), isolated in the presence of IFN (10(4) U ml-1) from cultures of NIH-3T3 cells which had been treated with IFN, and then infected with KiMSV (KiMLV) in conditions where every cell was infected, were shown to be phenotypically untransformed. These untransformed cells did not produce virus or contain rescuable KiMSV. However, cells isolated using an identical procedure, but in the absence of IFN, were uniformly transformed and all produced KiMSV (KiMLV) or contained rescuable KiMSV. It was concluded that IFN either prevents synthesis or integration of the provirus, or else that in the presence of IFN the provirus is integrated such that it is not expressed. We now show that five representative clones contain no detectable KiMSV proviral DNA, and also that the initial stages of infection by KiMSV (KiMLV) are inhibited by IFN treatment. IFN seems to act before integration, preventing either the synthesis or the integration of proviral DNA.

Animals↗
PubMed1980

A monoclonal antibody for large-scale purification of human leukocyte interferon.

A clone of hybrid myelomas (NK2), secreting a mouse monoclonal antibody to human leukocyte interferon, has been isolated. The antibody neutralizes the antiviral activity of the interferon and, when covalently attached to a solid support and used as an immunoadsorbent, allows interferon purification of up to 5,000-fold in a single step.

Antibodies↗
PubMed1980

Effects of sodium butyrate on a human lymphoblastoid cell line (Namalwa) and its interferon production.

Treatment of lymphoblastoid cells with sodium butyrate before infection with Sendai virus increases the subsequent yield of interferon and of its messenger RNA. This treatment caused almost complete inhibition of cellular DNA synthesis and consequently cell division, and some inhibition of cellular RNA and protein synthesis. It also caused increased acetylation of histones and alterations in the plasma membrane. There was no decrease in cell viability or change in cell morphology but the cells were smaller than untreated cells and were all in the G1 phase. Sodium butyrate treatment had no effect on the course of Sendai virus infections. The observed increase in interferon yields after butyrate treatment did not correlate with its effects on DNA synthesis, size, cell division and histone acetylation, but did with its effects on RNA and protein synthesis.

Acetylation↗
PubMed1980

Production of chick interferon by reactivating chick erythrocytes.

When chick erythrocytes were fused with mouse L929 or A9 cells and the heterokaryons induced for interferon production on consecutive days, considerable amounts of mouse interferon were produced every day. Small amounts of chick interferon were produced 2 to 3 days after fusion, coincident with the appearance of a chick enzyme, appearance of nucleoli and increase in chick cell nuclear diameter.

Animals↗