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Biomedical subjects

A M Edwards

Publications and source records attributed to A M Edwards.

At least 19 recordsLinked to original sources
PubMed1992

Riboflavin-photosensitized anaerobic modification of rat lens proteins. A correlation with age-related changes.

When rat lens homogenate or its soluble protein fractions are irradiated in the presence of riboflavin, a photo-adduct is obtained between this vitamin and the lens proteins. Irradiation of these proteins in the presence of riboflavin also leads to a modification in the chromatographic elution pattern with an increase in the high-molecular-weight fraction. In an aging study with rats, it was shown that the proportion of the high-molecular-weight protein fraction significantly increased with age, whereas the proportion of the low-molecular-weight protein fraction concomitantly decreased. It is postulated that aging produces an increase in the accessibility of the tryptophan residues of the lens proteins, as established by iodide fluorescence quenching experiments.

Aging↗
PubMed1992

Effect of fatty acids on rat liver nuclear T3-receptor binding.

The effects of selected fatty acids (linoleic, oleic, and palmitic) on triiodothyronine (T3)-receptor binding were compared in isolated rat hepatocytes, rat liver nuclei, and receptor protein. Scatchard analysis indicated that the inhibition of T3-receptor binding by fatty acids was characterized by an increase in Kd and no change in maximum binding capacity (MBC). In isolated receptors, the rank order of potency for inhibition was linoleic acid greater than oleic acid greater than palmitic acid. The Ki for oleic acid in isolated receptors was the same as that for whole nuclei (15.4 +/- 1.3 v 16.3 +/- 1.9 mumol/L, respectively), indicating that the inhibition of nuclear T3 binding is probably at the level of the receptor protein itself. In isolated hepatocytes, linoleic acid was more potent than oleic acid in inhibiting T3 binding to nuclear receptors. Cell-associated T3 was not affected by the presence of fatty acids, implying that cellular uptake of T3 was not inhibited. High concentrations of fatty acids were necessary for inhibition of T3-receptor binding in isolated hepatocytes, with linoleic acid being one to two orders of magnitude less potent in isolated hepatocytes compared with isolated receptors (Ki, 179 +/- 12 v 4.4 +/- 0.5 mumol/L, respectively). It is concluded that the inhibitory effect of fatty acids on T3-receptor binding in isolated rat hepatocytes probably occurs at the level of the nuclear receptor, and does not involve an inhibition of the access of T3 to the receptor. However, in vivo it seems unlikely that fatty acids will have access to the nuclear receptors in sufficiently high concentrations to affect T3-receptor binding in liver cells.

Animals↗
PubMed1991

Two-dimensional and epitaxial crystallization of a mutant form of yeast RNA polymerase II.

A mutant form of yeast RNA polymerase II that lacks the fourth and seventh largest subunits, referred to as pol II delta 4/7, crystallized on positively charged lipid layers. Both single-layered (two-dimensional) crystals and several multi-layered crystal forms were obtained. The two-dimensional crystals, preserved in negative stain, diffracted strongly to about 1/20 A-1 and more weakly to 1/13 A-1 resolution. A projection map computed from averaged Fourier transforms revealed four pol II delta 4/7 complexes per unit cell and further revealed a cleft on the surface of the complex similar to that previously observed in the structure of Escherichia coli RNA polymerase. One of the multi-layered crystal forms, preserved in negative stain, diffracted strongly beyond 1/15 A-1 resolution. Coherent diffraction from the multi-layered crystal is indicative of protein-protein interactions between layers and ordering in the third dimension.

Crystallization↗
PubMed1991

Three-dimensional structure of yeast RNA polymerase II at 16 A resolution.

The structure of yeast RNA polymerase II has been determined by three-dimensional reconstruction from electron micrographs of two-dimensional crystals at approximately 16 A resolution. The most prominent feature of the structure is an arm of protein density surrounding a channel about 25 A in diameter, similar to that found previously for E. coli RNA polymerase. The 25 A-diameter channel bifurcates on one face of the protein, connecting with a 25 A-wide groove and with a channel about half as wide. The 25 A channel and groove, and the narrow channel, may bind double- and single-stranded nucleic acids, respectively. A finger of protein density projecting from the molecule adjacent to the arm-like feature may represent the C-terminal domain of the largest subunit. These results provide a structural basis for analyses of the transcription process and its regulation.

Microscopy, Electron↗
PubMed1991

Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro.

RNA polymerase II lacking the fourth and seventh largest subunits (pol II delta 4/7) was purified from Saccharomyces cerevisiae strain rpb-4, in which the gene for the fourth largest subunit is deleted. pol II delta 4/7 was indistinguishable from wild-type pol II (holoenzyme) in promoter-independent initiation/chain elongation activity (400-800 nmol of nucleotide incorporated/10 min/mg of protein at 22 degrees C), in rate of chain elongation (20-25 nucleotides/s), and in the recognition of pause sites in the DNA template. In contrast to pol II holoenzyme, pol II delta 4/7 was inactive in promoter-directed initiation of transcription in vitro. The addition of an equimolar complex of the fourth and seventh largest subunits, purified from pol II holoenzyme by ion-exchange chromatography in the presence of urea, restored promoter-directed initiation activity to pol II delta 4/7. The transcriptional activator protein Gal4-VP16 could also elicit promoter-directed initiation by pol II delta 4/7 from a promoter with a Gal4 binding site. Complementation was observed between extracts of strain rpb-4, lacking the fourth largest subunit, and strain Y260-1, with a defect in the largest subunit. These extracts were individually inactive, but a mixture would support promoter-directed initiation. The fourth and seventh largest subunits may, therefore, shuttle between polymerase molecules.

Cell Nucleus↗
PubMed1991

A light-induced tryptophan-riboflavin binding: biological implications.

We review here the covalent photo-binding induced by visible light between the essential amino acid tryptophan and the vitamin riboflavin. We discuss the biological implications of this photoadduct in relation to the hepatotoxic and cytotoxic effect associated to parenteral nutrients and to culture media exposed to the action of light, respectively. We also analyze the formation of a photo-binding between riboflavin and the residues of tryptophan present in the proteins of the eye lens, a tissue which is permanently exposed to visible light.

Animals↗
PubMed1990

Enzymatic generation of triplet acetone by deglycosylated horseradish peroxidase.

A study of the effects of deglycosylation of horseradish peroxidase on protein conformation, as well as on its catalytic activity of oxidation of isobutyraldehyde or its enol form to triplet acetone and formic acid, was performed. The loss of carbohydrates leads to structural modifications of this enzyme. This is confirmed by a change in the circular dichroism spectrum, an increase in tryptophan's environment polarity, and a loss of the chiral specificity toward D- and L-tryptophan. Deglycosylation does not destroy either the peptide backbone or the amino acid residues and does not affect the heme group content of the protein. The rates of oxygen uptake and light emission observed when horseradish peroxidase oxidizes isobutyraldehyde or the trimethylsilyl enol ether form of the latter are reduced when the enzyme is 70% deglycosylated. Concomitantly, the acting deglycosylated enzyme becomes inactivated during the course of the reaction. It appears that the carbohydrate moiety plays an important role in the protection of the peroxidase from damaging effects induced by triplet acetone and in the stabilization of the three-dimensional structure of this enzyme.

1-Propanol↗
PubMed1990

Purification and lipid-layer crystallization of yeast RNA polymerase II.

Yeast RNA polymerase II was purified to homogeneity by a rapid procedure involving immunoaffinity chromatography. The purified enzyme contained 10 subunits, as reported for conventional preparations, but with no detectable proteolysis of the largest subunit. In assays of initiation of transcription at the yeast CYC1 promoter, the enzyme complemented the deficiency of an extract from a strain that produces a temperature-sensitive polymerase II. Mammalian RNA polymerase II was inactive in this initiation assay. The purified yeast enzyme formed two-dimensional crystals on positively charged lipid layers, as previously found for Escherichia coli RNA polymerase holoenzyme. Image analysis of electron micrographs of crystals in negative stain, which diffracted to about 30-A resolution, showed protein densities of dimensions consistent with those of single polymerase molecules.

Chromatography, Affinity↗
PubMed1990

Stimulation of DNA synthesis in primary rat hepatocyte cultures by liver tumor promoters: interactions with other growth factors.

Mechanism(s) of tumor promotion in liver by xenobiotics such as hexachlorocyclohexane (HCH), 1,1,1-trichloro-2,2-bis (4-chlorophenyl)ethane (DDT) and phenobarbital (PB) are not understood in detail although growth-stimulatory effects may be significant in their action. As a basis for studying mechanisms of growth control by liver tumor promoters, effects of xenobiotics on DNA synthesis have been examined in primary cultures of normal rat hepatocytes, maintained under fully-defined conditions. The xenobiotics alone were relatively ineffective but they exhibited synergism with epidermal growth factor (EGF), insulin and dexamethasone in stimulating DNA synthesis and were effective in moderate-to-low density cultures but not in confluent monolayers. Under conditions optimized for HCH or pregnenolone 16 alpha-carbonitrile (PCN) (i.e. subconfluent cultures exposed to insulin, EGF and dexamethasone), HCH, PCN, DDT or PB caused a transient stimulation of DNA synthesis, apparent after 2 days in culture. This probably reflected earlier entry of hepatocytes to S-phase. HCH was shown to increase total DNA, total numbers of nuclei and numbers of cells undergoing mitosis per culture. In optimized conditions, HCH or PCN were about additive with norepinephrine, dialyzed serum or pyruvate or with a small effect of tri-iodothyronine in stimulating DNA synthesis. Although conditions optimal for HCH or PCN were not necessarily optimal for detecting growth-stimulatory effects of other xenobiotics or steroids, these culture conditions were shown to support stimulation of DNA synthesis by a variety of known liver tumor promoters including barbiturates, estrogens, progestins, peroxisomal proliferators and bile acids. Several compounds known not to promote liver carcinogenesis failed to stimulate DNA synthesis in similar hepatocyte cultures.

Animals↗
PubMed1990

Two isoforms of myelin-associated glycoprotein accumulate in quaking mice: only the large polypeptide is phosphorylated.

We have shown that the developmentally regulated appearance of the two myelin-associated glycoprotein (MAG) polypeptides in normal mouse brain myelin does not reflect the developmental pattern of differential splicing of primary gene transcripts as determined earlier by RNase protection assays. Contrary to expectation, the large (L-MAG) and small (S-MAG) polypeptides are present in about equal amounts at a relatively early stage of myelination, day 24 or earlier. In quaking (qk) mutant mouse brain myelin, both MAG polypeptides are evident at all ages examined; the relatively greater abundance of S-MAG compared to L-MAG at early ages (days 18 and 22) confirms our earlier observation on in vitro translations of mRNA. At later ages (day 27 and beyond) both isoform are present in approximately equal amounts. The L-MAG but not the S-MAG polypeptide can be phosphorylated by kinases that are endogenous to isolated qk myelin, analogous to the phosphorylation we have observed in vivo in normal mice and in their isolated myelin.

Animals↗
PubMed1989

Interaction of myelin basic protein and proteolipid protein.

The interaction of myelin basic protein (MBP) and proteolipid protein (PLP) was studied using a microtitre well binding assay and the ligand-blot overlay technique. The binding of iodinated PLP to MBP that was immobilized on microtitre wells was saturable and reversible. Its selectivity was investigated by the ligand-blot overlay technique. Iodinated PLP was found to bind MBP but not any other CNS myelin proteins. This interaction was not dependent on the phosphoryl moiety of MBP. Binding of PLP to histone H4 also occurred, but the amount of PLP bound per unit MBP was greater than for this histone.

Animals↗
PubMed1989

Chemical deglycosylation on a micro-scale of membrane glycoproteins with retention of phosphoryl-protein linkages.

We provide here an easy and convenient procedure for the chemical deglycosylation of membrane glycoproteins that can be done rapidly and inexpensively on large numbers of membrane samples on a microscale without prior isolation of the glycoprotein in question. We have used the procedure successfully on three different glycoproteins and additionally have shown that covalent linkages of the polypeptide to phosphoryl residues are preserved.

Animals↗
PubMed1989

Phosphorylation of myelin-associated glycoprotein in vivo and in vitro occurs only in the cytoplasmic domain of the large isoform.

Myelin-associated glycoprotein (MAG) was radioactively labelled with 32P both in intact brain and in myelin membrane preparations. Chemical deglycosylation of the phosphorylated products revealed that only one of the MAG isoforms (L-MAG) is labelled in vitro. Furthermore, the phosphorylation events in vivo and in vitro are confined to the cytoplasmic portion of the L-MAG isoform. Tryptic mapping of L-MAG labelled both in vivo and in vitro revealed that the majority of the sites phosphorylated in intact brain are also phosphorylated in myelin membrane preparations; however, the extent of phosphorylation at individual sites is variable. The results demonstrate that partially purified myelin membrane preparations can be used to study the enzymes responsible for MAG phosphorylation and dephosphorylation events in vivo.

Animals↗
PubMed1989

Comparison of a lactose-free formulation of sodium cromoglycate and sodium cromoglycate plus lactose in the treatment of asthma.

A double-blind, parallel group, 7-centre trial was carried out to compare the clinical efficacy and patient acceptability of two formulations of sodium cromoglycate for inhalation in patients suffering from asthma. Each single-dose capsule for use in a breath-actuated inhaler contained either a blend of sodium cromoglycate (20 mg) plus lactose (20 mg) or a lactose-free pelletized formulation of sodium cromoglycate (20 mg). Data were summarized from 529 asthmatic patients who had been using the blend formulation for at least 3 months previously. Two hundred and sixty-five patients then received pelletized sodium cromoglycate and 264 patients remained on sodium cromoglycate plus lactose for at least 3 months. Regular assessments were made by patients and clinicians during the trial period of treatment effectiveness. No clinically significant differences were observed between the two formulations after 3 months on test treatment. After a treatment period of 6 months, the pelletized formulation was shown to have some advantages over the blend formulation which were not observed at 3 months, with a significantly higher proportion of 'very effective' assessments being recorded by both patients and clinicians. The capsules of pelletized sodium cromoglycate required significantly less inhalations to empty compared to the capsules of the blend. No differences were observed between the two formulations with regard to the incidence of transient cough and throat irritation after inhalation.

Adolescent↗
PubMed1989

Transrectal ultrasound in monitoring response to treatment of prostate disease.

Transrectal ultrasound provides a safe method for repeated assessments of the prostate gland in the follow-up of prostatic cancer. Changes in the echo pattern of the gland may be observed following treatment, but these may be difficult to interpret. The capsule may be restored; the tumor may appear smaller, and in some cases, ultrasound identification of the tumor may become impossible. The total gland volume decreases with chemotherapy and hormonal manipulation (subcapsular orchidectomy or drug-induced). The volume generally decreases after radiotherapy but may show a transient increase if there is some associated radiation proctitis. The rate of decrease of total prostatic volume has not been established as a reliable indicator of the subsequent prognosis. Future studies should assess whether sequential measurements of the tumor volume rather than the total prostatic volume may be a more useful guide to prognosis.

Humans↗