Biomedical subjects

B F Cheetham

Publications and source records attributed to B F Cheetham.

32 records · Page 2Linked to original sources

PubMed1991

Structure-function studies of human interferons-alpha: enhanced activity on human and murine cells.

To identify functionally important regions of the human interferon (IFN)-alpha molecule, mutagenesis in vitro of human IFN-a genes was used to create analogs with deletions or specific amino acid replacements. These analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate protein synthesis system. Deletion of 7 highly conserved hydrophilic amino acids from the C-terminus of human IFN-alpha 4 reduced, but did not abolish, antiviral activity on human cells. However, analogs with deletions of 15 or 25 amino acids from the C-terminus, or 28 amino acids from the N-terminus, had no measurable antiviral activity. The antiviral activity of human IFN-alpha 4 was increased by substitution of cysteine for serine at position 86, and lysine for arginine at position 121. However, other amino acid substitutions at positions 121, 122 or 123 reduced antiviral activity. The size of the side chain of the amino acid residue at position 130 was shown to be important. Replacement of the absolutely conserved leucine residue at position 131 with glutamine had little effect on antiviral activity. However, the introduction of a proline residue at this position abolished antiviral activity, probably due to the formation of a beta turn in the polypeptide chain. The antiviral activity of human IFN-alpha 4 on murine cells was increased by substitutions at positions 86, 121 and 133. This study illustrates the utility of the in vitro mutagenesis and rabbit reticulocyte lysate systems for the investigation of structure-function relationships, and extends our knowledge of the biologically active regions and species specificity of the human IFN-alpha molecule.

Animals↗

PubMed1990

Pharmacokinetics, tissue distribution, and cell localization of [35S]methionine-labeled recombinant human and murine alpha interferons in mice.

The pharmacokinetics, tissue distribution, cell localization, and penetration into tumor xenografts of recombinant [35S]methionine-labeled human alpha interferon (HuIFN-alpha) and murine alpha interferon (MuIFN-alpha) were examined in mice. Both interferons (IFNs) were removed from the blood in a rapid biphasic manner; HuIFN-alpha was cleared faster than MuIFN-alpha. Tissues were analyzed for radioactivity and over 90% of the IFNs was accounted for. The IFNs were detected predominantly in liver, kidney, gastrointestinal tract, pancreas, spleen, and lung. The levels of MuIFN-alpha compared with HuIFN-alpha were greater in the liver, spleen, and lung and less in the kidney, pancreas, and gastrointestinal tract. Heart, brain, testes, thymus, lymph nodes, fat, skin, and skeletal muscle contained much lower but measurable levels of both IFNs. There was penetration of HuIFN-alpha into tumor xenografts. The pharmacokinetics of IFN-alpha were independent of the strain of mouse, BALB/c or CBA, immune deprivation, or the presence of a tumor xenograft. Autoradiography of tissue sections from mice given injections of HuIFN-alpha or MuIFN-alpha indicated focal radioactivity in proximal convoluted tubules in the kidney and diffuse radioactivity in the liver, gastrointestinal tract, and pancrease. MuIFN-alpha, but not HuIFN-alpha, showed intense localization in cells in hepatic sinusoids, marginal zones in the spleen, and pulmonary alveolar walls, suggesting uptake by cells of the monocyte/macrophage lineage in these sites. The study shows the utility of biosynthetic labeling for pharmacokinetic studies of cytokines, clear differences in tissue distribution of IFN-alpha according to its species of origin, and targeting of homologous IFN-alpha to cells of the monocytic lineage.

Animals↗

PubMed1990

Structure-function studies of interferon-alpha based on random mutagenesis and expression in vitro.

An efficient procedure for random chemical mutagenesis was used to create analogs of human interferon (IFN)-alpha 4. Unique restriction enzyme sites were introduced into the human IFN-alpha 4 gene to enable cassetting of the gene for localized random mutagenesis. Single-stranded IFN-alpha 4 DNA was treated with nitrous acid, followed by second-strand synthesis using reverse transcriptase. A 72 base pair cassette spanning the coding region for amino acid residues 120 to 136 (120-136 region) was isolated and cloned into a phagemid vector adjacent to a GC-rich sequence. A DNA segment comprising the IFN-alpha 4 cassette sequence and the GC clamp was excised and electrophoresed on a denaturing gradient gel, which allowed the separation from unmutated DNA of DNA fragments with single base pair changes. DNA fragments with mobility different from that of the unmutated fragment were pooled and cloned into an expression vector. Using this procedure, mutations were found in the DNA of 48% of the clones analyzed. However, mutations at two "hot spots" accounted for 89% of these clones. Four of the IFN-alpha 4 analogs with mutations in the 120-136 region were expressed in vitro. The antiproliferative activities on human Daudi cells of most of the analogs were less than 0.2% of the activity of unmodified IFN-alpha 4, suggesting that the integrity of the carboxy terminus is important for the antiproliferative activity of human IFN-alpha 4.

Amino Acid Sequence↗

PubMed1990

Receptors for human interferon alpha on bovine cells: specificity and tissue distribution.

Human interferon alpha 1 (HuIFN alpha 1) is known to protect bovine as well as human cells against viral infection. Hence, we investigated the specificity and tissue distribution of receptors for HuIFN alpha 1 on various cells. [35S]HuIFN alpha 1 bound specifically to homogenates of bovine tissues and particularly to bovine liver, but there was also specific binding to spleen, kidney, brain, adrenal gland, lung, thymus, skeletal muscle, heart, mammary gland and testis. There was no difference in the degree of binding of HuIFN alpha to foetal or adult liver. Competitive binding experiments showed that bovine interferon alpha C (BoIFN alpha C) competed with HuIFN alpha 1 for binding to a bovine liver plasma membrane preparation, indicating that these two IFNs bind to the same receptor. An 35S-labelled IFN alpha 1-receptor complex was isolated from bovine liver extracts by SDS/polyacrylamide gel electrophoresis, and shown to have a molecular weight of 153 kDa. Isolation of the bovine IFN alpha receptor would be a feasible approach to the characterization of the HuIFN alpha receptor.

Animals↗

PubMed1989

Functional domains of human interferon gamma probed with antipeptide antibodies.

Functional domains of biologically active polypeptide molecules can be sought by raising antibodies to synthetic peptides. Human interferon gamma (HuIFN gamma) was thus studied, using two peptides based on candidate regions representing amino acids 7-16 and 121-130 of the HuIFN gamma molecule. These were conjugated to bovine serum albumin prior to immunization of rabbits. High titres of antipeptide antibodies which recognized the synthetic peptides were elicited in all of the four rabbits injected. The antipeptide antibodies from one of the rabbits immunized with the C-terminal (121-130) peptide detected native HuIFN gamma at a concn as low as 300 IU/ml, but the antipeptide antibodies from the rabbit immunized with the N-terminal (7-16) peptide did not detect HuIFN gamma. The IFN gamma-reactive antipeptide antibodies (anti-121-130) did not neutralize the antiviral activity of HuIFN gamma in a cytoprotection assay. These data and other studies establish that the C-terminus of HuIFN gamma is not essential for full antiviral activity and indicate an application of antipeptide antibodies in the analysis of structure-function relationships of cytokine molecules.

Antibodies↗

PubMed1989

Functional significance of amino acid residues within conserved hydrophilic regions in human interferons-alpha.

Site-directed in vitro mutagenesis was used to create analogs of human interferons (IFNs)-alpha 1 and -alpha 4. Analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate cell-free protein synthesis system. Amino acid substitutions for the highly conserved residues at positions 33, 121, 122 and 123 greatly reduced the antiviral and antiproliferative activities on human cells of IFNs-alpha 1 and -alpha 4. In general, the amino acid substitutions had much less effect on the antiviral activities on bovine, compared with human, cells. Substitutions at positions 31, 41, 42, 124, 134, 135 and 136 had little or no effect on the biological activities of the IFN analogs. The abrogation of antiviral activity resulting from amino acid substitutions for the arginine residue at position 33 suggests that this arginine residue is required for binding to the IFN-alpha receptor on the cell surface.

Amino Acid Sequence↗

PubMed1989

Structure-function studies of interferons-alpha: amino acid substitutions at the conserved residue tyrosine 123 in human interferon-alpha 1.

Analogs of human interferon-alpha 1 (IFN-alpha 1) were created in vitro by site-directed mutagenesis to investigate the structural requirements at amino acid position 123 for binding to the IFN receptor, antiviral activity, and antiproliferative activity. The tyrosine residue 123, which is conserved in all known mammalian IFNs-alpha and -beta, was replaced by each of 6 amino acids or was deleted from the protein. Several of the substitutions at position 123 partly or completely abrogated antiviral and antiproliferative activities of human IFN-alpha 1 when human or murine cells were used but not when bovine cells were used. However, with analogs in which amino acids structurally related to tyrosine, phenylalanine, or tryptophan were substituted at position 123, there was retention of antiviral and antiproliferative activities using homologous cells. Thus, although there is not an absolute requirement for tyrosine at position 123, conformational changes associated with alterations of this residue are prejudicial to the biological functions of the IFN-alpha molecule.

Amino Acids↗

PubMed1989

Characterization of interferons produced by peripheral blood mononuclear cells from healthy subjects in response to Corynebacterium parvum or poly I: poly C.

The biological significance of acid labile interferon alpha is presently unknown. We examined the putative production of acid labile interferon in vitro from human peripheral blood mononuclear cells induced with Corynebacterium parvum or poly I: poly C. Both agents induced up to 1200 IU/ml interferon, and the interferon was 80 to 90% acid labile. The interferons were typed by antibody neutralization of their antiviral activity. Contrary to previous reports, C. parvum induced predominantly interferon gamma, which is normally acid labile, whereas poly I: poly C induced an acid labile interferon alpha activity with characteristics similar to those of acid labile interferon alpha reported in serum in certain human diseases.

Adult↗

PubMed1988

Interferons in rheumatoid arthritis: alterations in production and response related to disease activity.

Various markers associated with the production of and response to interferons (IFNs) were studied in patients with either inactive rheumatoid arthritis (RA) or active RA, and in healthy subjects. The IFN markers assessed were serum and synovial fluid (SF) levels, the activity in peripheral blood leukocytes (PBL) of (2'-5') oligoadenylate synthetase (OAS), and the production in vitro by PBL of IFN-alpha/beta in response to Sendai virus or Poly(I):Poly(C) as inducers, and of IFN-gamma using PHA or Con A as inducers. IFN activity, tested by antiviral assays using two different cell lines, was not demonstrable in the serum of any patient with RA. The activity of (2'-5') OAS in PBL, which may indirectly indicate exposure of leukocytes to IFN, was increased in RA compared with healthy subjects, more so in patients with inactive RA. The production of IFN-alpha/beta by PBL in response to Sendai virus was low in active RA but high in inactive RA, relative to production in healthy subjects. The production of IFN-gamma by PBL in RA was lower than in healthy subjects, more so in active RA. Thus inactive RA (remission status) is marked by evidence of PBL having been influenced by interferon and being a state of augmented inducibility to an IFN-alpha/beta stimulus, whereas active RA is associated with low inducibility of PBL to an IFN stimulus, but no evidence of IFN production in vivo. Our findings underscore the relevance of interferon to remission/activity in rheumatoid arthritis.

2',5'-Oligoadenylate Synthetase↗

PubMed1983

Adenovirus-induced alterations of the cell growth cycle: a requirement for expression of E1A but not of E1B.

Mutants dl312, dl314, hr1, and hr3 with mutations in region E1A of adenovirus type 5 were defective for the induction of cell cycle abnormalities detectable by flow cytometry, cell DNA replication, thymidine kinase production, and chromosome aberrations and did not synthesize the viral DNA-binding protein (E2A) in rat cells. dl311, a leaky E1A mutant, induced cell cycle effects at high multiplicity in only one of three experiments, and synthesized the DNA-binding protein. hr7 (E1B) gave a wild-type response in all tests. dl313 was also positive in all tests, although it induced fewer polyploid cells than did wild-type virus, probably because of the leftward extension of the dl313 E1B deletion into E1A. sub315 and sub316, with mutations which also span the E1A-E1B border, synthesized DNA-binding protein, but caused no cell cycle alterations detectable by flow cytometry in rat or mouse cells. Although the participation of other viral early regions cannot be completely excluded, our results suggest that alteration of cell cycle progression is a direct effect of E1A unrelated to its control of other viral early regions, and may be the function of E1A in transformation.

Adenoviruses, Human↗

PubMed1983

An inhibitor of polyamine synthesis arrests cells at an earlier stage of G1 than does calcium deprivation.

Methylglyoxal bis(guanylhydrazone) completely inhibits the induction of thymidine kinase after serum stimulation of quiescent fibroblasts only if added within 3 h after serum, whereas calcium deprivation blocks this induction up to 12 h after serum stimulation. Experiments in which one of these blocks was imposed as the other was released confirmed that cells blocked by methylglyoxal bis(guanylhydrazone) are arrested at an earlier stage in G1 than cells blocked by calcium deprivation.

Animals↗

PubMed1982

A biochemical investigation of the adenovirus-induced G1 to S phase progression: thymidine kinase, ornithine decarboxylase, and inhibitors of polyamine biosynthesis.

Biochemical events were investigated in the G1 to S phase progression induced in quiescent rodent cells by human adenovirus type 5 (Ad5) and by serum. Thymidine kinase activity increased after infection of cells with Ad5 or addition of 10% serum. These stimulations were additive. An early viral gene was responsible for induction by Ad5, but the early mutants ts36, ts37, and ts125 induced thymidine kinase at the permissive and nonpermissive temperatures. Several differences were found between cells stimulated by serum compared with Ad5. Induction of thymidine kinase was delayed in Ad5-infected cells, insensitive to 0.01 microgram/ml actinomycin D and relatively resistant to reduced Ca2+ compared with induction by serum. Ornithine decarboxylase was induced by serum, but not by Ad5, alpha-Methylornithine had little effect on the induction of thymidine kinase by Ad5, but reduced the induction of thymidine kinase by serum, suggesting that Ad5-induced entry into S phase is uncoupled from polyamine biosynthesis. Methylglyoxal bis(guanylhydrazone), however, prevented the induction of thymidine kinase by both serum and Ad5. Adenovirus infection appears to induce cellular DNA synthesis and thymidine kinase in G1-arrested cells by a mechanism different from serum, and bypasses events in the normal G1 to S phase progression.

Adenoviruses, Human↗

PubMed1982

Spermine and aminoguanidine protect cells from chromosome aberrations induced by adenovirus during the G2 phase of the cell cycle.

Adenovirus uncouples DNA replication from polyamine biosynthesis and causes chromosome aberrations in rodent cells. Addition of polyamines protected infected cells from this chromosome damage. Spermine was the only individual polyamine which protected. The diamine oxidase inhibitor aminoguanidine also protected. Neither compound detectably reduced synthesis of viral early proteins. The protective effects of spermine and aminoguanidine were not additive. Maximal protection was obtained when the compounds were added 4.5 h before mitosis, but significant protection was observed up to 1.25 h before mitosis. This suggests that the compounds act in G2. In vitro, spermine bound strongly to DNA and protected it from mild endonuclease attack, but aminoguanidine did neither. We propose that viral infection causes a deficiency in spermine during a critical period G2, possibly accompanied by an increase in endonuclease activity. The resulting chromosome damage can be prevented by adding exogenous spermine, or by inhibiting the oxidative degradation of endogenous spermine.

Adenoviridae↗

PubMed1982

Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation.

Adenoviruses, Human↗