Biomedical subjects

M J Tymms

Publications and source records attributed to M J Tymms.

33 records · Page 2Linked to original sources

PubMed1994

Expression of copper/zinc superoxide dismutase and glutathione peroxidase in organs of developing mouse embryos, fetuses, and neonates.

The rise in antioxidant enzyme activity in the lungs of late-gestation fetuses is thought to be caused by the preparation of the pulmonary antioxidant system for birth. However, recent data have shown that such a rise also occurs in the livers of late-gestation fetuses. Consequently, this surge cannot solely be ascribed to the preparation of the pulmonary antioxidant system for birth. In this study we examine the expression of copper/zinc superoxide dismutase (Sod1) and glutathione peroxidase (Gpx1) in various organs of late-gestational mouse fetuses. Furthermore, we compare the expression of these genes in organs of fetuses, neonates, and adult mice. These studies were carried out to investigate whether the change in mRNA levels for these two genes is related to a developmental change in oxidant stress. Our data demonstrate that an increase in both Sod1 and Gpx1 mRNA occurs in lungs and livers of late-gestational mouse fetuses. The brain demonstrates an increase in Sod1 expression at or around the time of birth, the kidney shows an elevation in Gpx1 mRNA levels, and the heart fails to demonstrate a surge in both Sod1 and Gpx1 mRNA levels. Our data show that the liver is the organ with the highest levels of Sod1 and Gpx1 mRNA in embryos and neonates (immediately after birth). In the adult, the liver has the highest levels of Sod1 mRNA and the spleen the highest level of Gpx1 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Structure-function study of the region encompassing residues 26-40 of human interferon-alpha 4: identification of residues important for antiviral and antiproliferative activities.

The highly conserved amino acid residues Leu-30 and Arg-33 of human interferon-alpha 4 (IFN-alpha 4) have previously been identified as important for biological activity. In this study, the sequence around Arg-33 was targeted to determine the importance of other residues in this region. A library of analogues containing amino acid substitutions spanning residues 26-37 was generated using site-directed and random mutagenesis. Analogues were expressed in vitro and assayed for antiviral and antiproliferative activity on human cells. No significant separation between the antiviral and antiproliferative activities was observed for any of the analogues tested. Substitutions at positions 26, 27, 31, 32, 34, 35, and 37, did not substantially affect biological activity. However, substitution of Phe-36 with arginine resulted in a greater than 100-fold decrease in biological activity. Thus, together with previous data, the residues in this region identified as most important for biological activity include Leu-30, Arg-33, and Phe-36. Recently published models for the three-dimensional structure of human IFN-alpha and the X-ray crystallographic structure of murine IFN-beta, suggest that the region investigated in this study forms a loop at the surface of the protein. Thus, residues Leu-30, Arg-33, and Phe-36, could be involved in binding to the Type-I IFN receptor, or in interactions with signal-transducing molecules.

Base Sequence↗

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↗

PubMed1991

Universal antibodies to human interferon-alpha subtypes--the production of antipeptide antibodies to conserved regions of interferon-alpha.

Antibodies to three conserved regions of all human interferon (IFN)-alpha 1 subtypes were raised by immunizing rabbits with short synthetic peptides coupled to a carrier. These peptides correspond to amino acid residues 29-36, 31-36, 126-131, 139-151, and 142-151 of the consensus sequence of IFN-alpha 1. The antibodies were tested for reactivity with IFN-alpha 1, -alpha 2a, -alpha 2b, -alpha 4a, and -alpha 14 and IFN-beta. Most antipeptide antibodies react only weakly with IFN-alpha subtypes; only the antibodies raised against the peptide corresponding to residues 142-151 conjugated to keyhole limpet hemocyanin using glutaraldehyde react appreciably with all IFN-alpha subtypes tested. These antipeptide antibodies are potentially universal antibodies to the human IFN-alpha 1 subtypes, since they exhibit similar binding affinity to all the IFN-alpha subtypes tested and do not react with IFN-beta.

Amino Acid Sequence↗

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↗

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↗

PubMed1988

Conserved amino acid residues arginine 33 and tyrosine 123 are critical for the antiviral activity of murine interferon-alpha 1.

Analogues of murine interferon-alpha 1 were produced by site directed mutagenesis and expressed in vitro. Replacement of the conserved residue arginine 33 by glutamic acid resulted in an interferon with no detectable antiviral activity. Substitution of tyrosine residue 123 shows dramatic reductions in the antiviral activity. These analogues demonstrate the importance of the conserved residues arginine 33 and tyrosine 123 in maintaining the antiviral activity of murine interferon-alpha 1.

Amino Acid Sequence↗

PubMed1988

Amino acid substitutions which alter the antiviral activity of human interferon-alpha 1 on mouse cells.

Human interferon-alpha 1 (IFN-alpha 1) is one of only three human (Hu) IFN-alphas having significant antiviral activity on mouse cells. Specific amino acid substitutions in HuIFN-alpha 1 in the region from amino acids 121 to 136 indicate that this region is critical to the determination of mouse and human cell antiviral activities. Bovine cell activities are relatively unaffected by changes in this region. In particular we have identified the arginine residue at position 125 of human IFN-alpha 1 as a major mediator of the molecules antiviral activity on mouse cells. Various substitutions in the carboxy-terminal region of human IFN-alpha 1 are also evaluated and discussed in the context of recently published data.

Amino Acids↗

PubMed1986

Antiviral and antiproliferative activities of interferon-alpha 1: the role of cysteine residues.

Site-specific in vitro mutagenesis was used to direct serine for cysteine substitutions within the sequence of human interferon-alpha 1 (IFN-alpha 1). Antiviral specific activities and antiproliferative activities of IFN-alpha 1 analogs, expressed in M13 as fusion proteins, were assessed following purification by monoclonal antibody affinity chromatography. Based on analysis of IFN-alpha 2, IFN-alpha 1 contains two disulfide bridges between cysteine residues 29 and 139 and cysteine residues 1 and 99. IFN-alpha 1 also contains a fifth cysteine residue at position 86. The series of serine for cysteine substitutions performed indicated that IFN-alpha 1 molecules unable to form the residue 29 to residue 139 disulfide bridge have substantially reduced antiviral and antiproliferative activities, IFN-alpha 1 molecules unable to form the residue 1 to residue 99 disulfide bridge have only marginally altered antiviral and antiproliferative activities, the low antiviral activity of IFN-alpha 1 compared with other human IFN-alpha subtypes is not due to the formation of nonnative disulfide bridges involving the fifth cysteine residue at position 86, which the other subtypes lack, and (iv) the reduced biological activities of certain analogs may be due to the formation of nonnative disulfide bridges.

Amino Acid Sequence↗

PubMed1985

Tandemly repeated nonribosomal DNA sequences in the chloroplast genome of an Acetabularia mediterranea strain.

A purified chloroplast fraction was prepared from caps of the giant unicellular green alga Acetabularia mediterranea (strain 17). High molecular weight DNA obtained from these chloroplasts contains at least five copies of a 10-kilobase-pair (kbp) sequence tandemly arranged. This unique sequence is present in DNA from chloroplasts of all stages of the life cycle examined. A chloroplast rDNA clone from mustard hybridized with some restriction fragments from Acetabularia chloroplast DNA but not with the repeated sequence. An 8-kbp EcoRI-Pst I fragment of the repeated sequence was cloned into pBR322 and used as a hybridization probe. No homology was found between the cloned 8-kbp sequence and chloroplast DNA from related species Acetabularia crenulata or chloroplast DNA from spinach.

Acetabularia↗

PubMed1985

Single amino acid substitutions at conserved residues of human interferon-alpha can effect antiviral specific activity.

Site-specific in vitro mutagenesis was used to direct various amino acid substitutions at conserved positions within the sequence of human interferon-alpha 1 (IFN-alpha 1). The antiviral specific activity of IFN-alpha 1, expressed in M13 as a fusion protein [IFN-alpha 1 (phi WT)], could be altered by single amino acid substitutions. The substitution of glycine for tyrosine at position 123 results in a loss of more than 99% of the antiviral specific activity on human cells, but causes no significant change in the antiviral specific activity on primary bovine cells. The tyrosine at position 123 is thus implicated in determining human cell specificity. Based on analysis of IFN-alpha 2, IFN-alpha 1 contains two dulsulphide bridges between cysteine residues 29 and 139 and cysteine residues 1 and 99. IFN-alpha 1 also contains a fifth cysteine residue at position 86. IFN-alpha 1 (phi WT) carrying three serine for cysteine substitutions at positions 1, 86 and 99 retains 23% of the antiviral specific activity of IFN-alpha 1 (phi WT) on human cells. However, the antiviral activity on bovine cells is not significantly affected by this modification. The presence of the disulphide bridge between residues 1 and 99 thus appears to be required for full antiviral activity on human but not bovine cells. A single serine for cysteine substitution at position 29 reduces the antiviral specific activity on both human and bovine cells by some 95%. This data shows that the disulphide bridge between residues 29 and 139 is critical for the antiviral activity of IFN-alpha's.

Amino Acid Sequence↗

PubMed1983

DNA Content of Beta vulgaris Chloroplasts during Leaf Cell Expansion.

During the growth of beet leaves from 2 to 3 to 25 to 30 centimeters, the leaf cells increase in size, the average number of chloroplasts per cell increases from 11 to 65 and the amount of chloroplast DNA per cell increases from 1100 to 1900 plastome copies. The average number of copies of the plastome per chloroplast decreases from 104 in 2 to 3-centimeter leaves to 29 in 25 to 30-centimeter leaves during a period when the chloroplasts undergo two to three rounds of division and increase diameter from 1.5 to 4.9 micrometers. This result is at variance with previously published studies of beet chloroplasts but agrees with the conclusions reached in more recent studies of pea and spinach and wheat leaf cell expansion.

Journal Article↗

PubMedDate not supplied

Efficient in vitro expression of interferon alpha analogs using SP6 polymerase and rabbit reticulocyte lysate.

We have investigated the use of in vitro expression as a quick and convenient means of screening large numbers of interferon (IFN) analogs generated using in vitro mutagenesis. The IFN-alpha 1 mRNA generated from DNA template using SP6 RNA polymerase is efficiently translated in rabbit reticulocyte lysate (RRL). The antiviral specific activity of this RRL-synthesized IFN-alpha l is equivalent to the yeast-synthesized protein. In contrast with the yeast-expression system, where some IFN-alpha analogs are poorly expressed, all analogs tested were well expressed in RRL.

Animals↗