Showing papers by "University of Dundee published in 1989"

The structure and regulation of protein phosphatases

Abstract: Four major serine/threonine-specific protein phosphatase catalytic subunits are present in the cytoplasm of animal cells. Three of these enzymes, PP-1, PP-2A, and PP-2B, are members of the same gene family, while PP-2C appears to be distinct. PP-1, PP-2A, and PP-2B are complexed to other subunits in vivo, whereas PP-2C has only been isolated as a monomeric protein. PP-1, PP-2A, and PP-2C have broad and overlapping specificities in vitro, and account for virtually all measurable activity in tissue extracts toward a variety of phosphoproteins that regulate metabolism, muscle contractility, and other processes. Their precise functions in vivo are unknown, although important clues to the physiological roles of PP-1 and PP-2A are provided by the effects of okadaic acid and by the subcellular localization of PP-1. The active forms of PP-1 are largely particulate, and their association with subcellular structures is mediated by "targetting subunits" that direct PP-1 to particular locations, enhance its activity toward certain substrates, and confer important regulatory properties upon it. This concept is best established for the glycogen-bound enzymes in skeletal muscle and liver (PP-1G) and the myofibrillar form (PP-1M) in skeletal muscle. The activities of PP-1 and PP-2B are controlled by the second messengers cyclic AMP and calcium. The activity of PP-2B is dependent on calcium and calmodulin, while PP-1 is controlled in a variety of ways that depend on the form of the enzyme and the tissue. PP-1 can be inhibited by cyclic AMP in a variety of cells through the A-kinase-catalyzed phosphorylation of inhibitor-1 and its isoforms. Phosphorylation of the glycogen-binding subunit of PP-1G by A-kinase promotes translocation of the catalytic subunit from glycogen particles to cytosol in skeletal muscle, inhibiting the dephosphorylation of glycogen-metabolizing enzymes. Allosteric inhibition of hepatic PP-1G by phosphorylase a occurs in response to signals that elevate cyclic AMP or calcium, and prevents the activation of glycogen synthase in liver. PP-1 can also be activated indirectly by calcium through the ability of PP-2B to dephosphorylate inhibitor-1. This control mechanism may operate in dopaminoceptive neurones of the brain and other cells. The inactive cytosolic form of PP-1 (PP-1I) can be activated in vitro through the glycogen synthase kinase-3-catalyzed phosphorylation of its inhibitory subunit (inhibitor-2), but the physiological significance is unclear.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism

Abstract: Okadaic acid is a polyether derivative of 38-carbon fatty acid, and is implicated as the causative agent of diarrhetic shellfish poisoning. It is a potent tumour promoter that is not an activator of protein kinase C, but is a powerful inhibitor of protein phosphatases-1 and -2A (PP1 and PP2A) in vitro. We report here that okadaic acid rapidly stimulates protein phosphorylation in intact cells, and behaves like a specific protein phosphatase inhibitor in a variety of metabolic processes. Our results indicate that PP1 and PP2A are the dominant protein phosphatases acting on a wide range of phosphoproteins in vivo. We also find that okadaic acid mimics the effect of insulin on glucose transport in adipocytes, which suggests that this process is stimulated by a serine/threonine phosphorylation event.

Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells.

Abstract: Addition of EGF to human epidermoid carcinoma A431 cells increases the rate of fluid-phase pinocytosis 6-10-fold as measured by horseradish peroxidase uptake (Haigler, H.T., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:82-90). We show here that in the absence of extracellular Na+ or in the presence of amiloride the stimulation of pinocytosis by EGF is substantially reduced. Amiloride had no effect on the endocytosis of EGF itself or of transferrin, demonstrating that the receptor-mediated endocytotic pathway operated normally under conditions that blocked stimulated pinocytosis. Amiloride blocked EGF-stimulated pinocytosis in both HCO3(-)-containing and HCO3(-)-free media. The EGF-stimulated pinocytotic activity can frequently be localized to areas of the cell where membrane spreading and ruffling are taking place. These results demonstrate that (a) EGF induces a distinct amiloride-sensitive endocytotic pathway on A431 cells; (b) occupied EGF receptors do not utilize this pathway for their own entry; (c) endocytosis of occupied EGF receptors is not in itself sufficient to stimulate pinocytosis.

Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay.

Abstract: 1. We have synthesized two peptides, one based on the exact sequence around the unique site (Ser79) for the AMP-activated protein kinase on rat acetyl-CoA carboxylase (SSMS peptide) and another in which the serine residue corresponding to the site for cyclic-AMP-dependent protein kinase (Ser77) was replaced by alanine (SAMS peptide). 2. Both peptides were phosphorylated with similar kinetics by the AMP-activated protein kinase, but only the SSMS peptide was a substrate for cyclic-AMP-dependent protein kinase. The SAMS peptide was not phosphorylated by any of five other purified protein kinases tested. 3. The Km of AMP-activated protein kinase for the SAMS peptide is higher than that for acetyl-CoA carboxylase, but the Vmax for peptide phosphorylation is 2.5 times higher than that of its parent protein. This peptide therefore gives a convenient and sensitive assay for the AMP-activated protein kinase. 4. Acetyl-CoA-carboxylase kinase and peptide kinase activities copurify through six steps from a post-mitochondrial supernatant of rat liver, showing that the SAMS peptide is a specific substrate for the AMP-activated protein kinase in this tissue. We could not demonstrate AMP-dependence of the kinase activity in crude preparations, apparently due to endogenous AMP remaining bound to the enzyme. However, 8-bromoadenosine 5-monophosphate (Br8AMP) is a partial agonist at the allosteric (AMP) site, and inhibition by 2 mM Br8AMP can be used to test that one is measuring the AMP-stimulated form of the kinase. 5. Using this approach, we have examined the kinase activity in nine different rat tissues, plus a mouse macrophage cell line, and find that there is a correlation between tissues expressing significant levels of peptide kinase activity and those active in the synthesis or storage of lipids. 6. We also use the peptide assay to show that cyclic AMP-dependent protein kinase does not activate purified AMP-activated protein kinase, and does not affect the activation of partially purified AMP-activated protein kinase by endogenous kinase kinase.

Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities.

Abstract: 1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine. 2. The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of 63 kDa, is labelled using [14C]fluorosulphonylbenzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [gamma 32P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity. 3. In the absence of AMP the purified kinase has apparent Km values for ATP and acetyl-CoA carboxylase of 86 microM and 1.9 microM respectively. AMP increases the Vmax 3-5-fold without a significant change in the Km for either protein or ATP substrates. 4. The response to AMP depends on the ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 microM. Studies with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations. 5. These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.

Release of Glutamate, Aspartate, and γ‐Aminobutyric Acid from Isolated Nerve Terminals

Abstract: With the advent of cloning, sequencing, and patchclamping techniques, knowledge of the postsynaptic actions of amino acid neurotransmitters has undergone a dramatic advance. The primary sequences of the inhibitory receptors for γ-aminobutyric acid (GABA) (Schofield et al., 1987) and glycine (Grenningloh et al., 1987) are now established, and patch-clamp analysis has elucidated many of the factors that regulate the opening of their ion channels. The excitatory glutamate receptors are being extensively characterized at both the pharmacological (reviewed by Foster and Fagg, 1984) and the electrophysiological (reviewed by Cull-Candy and Usowicz, 1987) level. In this climate, it is perhaps surprising that the fundamental presynaptic release mechanism for the amino acid neurotransmitters remains controversial.

Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules

Abstract: THE four-way junction between DNA helices is the central intermediate in recombination1–10, and the manner of its interaction with resolvase enzymes can determine the genetic outcome of the process. A knowledge of its structure is a prerequisite to understanding the interaction with proteins, and there has been recent progress11–14. Here we use fluorescence energy transfer to determine the relative distances between the ends of a small DNA junction, and hence the path of the strands. Our results are consistent with the geometry of an 'X'. The interconnected helices are juxtaposed so that the continuous strands of each helix generate an antiparallel alignment, and the two interchanged strands do not cross at the centre. The acute angle of the X structure is defined by a right-handed rotation of the helical axes about the axis perpendicular to the X plane, as viewed from the centre of the X.

Repetitive action potentials in isolated nerve terminals in the presence of 4-aminopyridine: effects on cytosolic free Ca2+ and glutamate release.

Abstract: The mechanisms by which an elevated KCl level and the K+-channel inhibitor 4-aminopyridine induce release of transmitter glutamate from guinea-pig cerebral cortical synaptosomes are contrasted. KCl at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 +/- 13 nM above the polarized control. The Ca2+-dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 +/- 0.35 nmol/mg was released. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl-induced elevation in [Ca2+]c could be observed in both low-Na+ medium and in the presence of low concentrations of veratridine. 4-Aminopyridine at 1 mM increased [Ca2+]c by 143 +/- 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4-aminopyridine administration was slower than that following 30 mM KCl, and a transient spike was less apparent. Consistent with this, the 4-aminopyridine-induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4-Aminopyridine induced the Ca2+-dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4-aminopyridine action. In contrast to KCl, glutamate release and the increase in [Ca2+]c with 4-aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A possible antilipolytic mechanism.

Abstract: Hormone-sensitive lipase is phosphorylated at a single site (site 2) in vitro by the AMP-activated protein kinase, without any direct effect on the activity of the enzyme. The amino acid sequence around this site has been determined. Ca2+/calmodulin-dependent protein kinase II also phosphorylates hormone-sensitive lipase predominantly at this site, whilst cyclic-GMP-dependent protein kinase phosphorylates exclusively the regulatory site (site 1) which is also phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of site 2 has been found to inhibit subsequent phosphorylation and activation of hormone-sensitive lipase by the cyclic-AMP-dependent and cyclic-GMP-dependent protein kinases, indicating that site-2 phosphorylation may have an antilipolytic role in vivo.

Occurrence of nodulation in the Leguminosae

Abstract: summary Reports of nodulation in the Leguminosae are examined in the light of current views on the taxonomy of the family. In the subfamily Caesalpinioideae, nodulation is largely restricted to the tribe Caesalpinieae and the genus Chamaecrista from the Cassieae. All nodules studied have rhizobia retained within infection threads during the nitrogen fixing period. In the Mimosoideae, nodulation is general, except for 4 groups within the tribe Mimoseae, and a very few species of Acacia. The only tribe from the Papilionoideae which appears not to nodulate is the Dipterygeae, although the monogeneric Euchresteae has not been examined. A number of genera in the Swartzieae do not nodulate. Taking tile family as a whole, nodulation appears to be very uniform – certain sections nodulate, others do not.

Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo.

Abstract: The ompC, ompD, and ompF genes encode the three major porins of Salmonella typhimurium. ompR encodes a positive regulator required for the expression of ompC and ompF. Transposon-generated mutations in ompC, ompD, ompF, and ompR were introduced into the S. typhimurium mouse virulent strain SL1344 by P22-mediated transduction. Following preliminary characterization in vitro, the strains were used to challenge BALB/c mice by using the oral or intravenous route. Strains harboring ompC or ompF mutations were as virulent as SL1344 after oral challenge. Strains harboring ompD mutations had a slight reduction in virulence. In contrast, ompR mutants failed to kill BALB/c mice after oral challenge and the intravenous 50% lethal dose was reduced by approximately 10(5). The ompR mutants persisted in murine tissues for several weeks following oral or intravenous challenge. Furthermore, mice orally immunized with these ompR mutant strains were well protected against challenge with virulent SL1344.

Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway.

Abstract: Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane. During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused. These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.

Increase in anterior tibialis muscle protein synthesis in healthy man during mixed amino acid infusion: studies of incorporation of [1-13C]leucine.

Abstract: 1. Anterior tibial muscle protein synthesis in seven healthy postabsorptive men was determined from increases in muscle protein bound leucine enrichment during a primed continuous infusion of L-[1-13C]leucine. Biopsies were taken 30 min after the beginning of leucine infusion (when plasma 13C enrichment was steady), 240 min later during continued fasting and again after 240 min of infusion of a mixed amino acid solution which increased plasma total amino acid concentrations by 37%. The mean enrichment of 13C in plasma alpha-ketoisocaproate was used as an index of the enrichment of the precursor pool for leucine metabolism. 2. Anterior tibial muscle mixed protein synthetic rate during fasting was 0.055 (SD 0.008)%/h and this increased by an average of 35% during infusion of mixed amino acid to 0.074 (SD 0.021)%/h (P less than 0.05). 3. Whole-body protein breakdown (expressed as the rate of endogenous leucine appearance in plasma) was 121 (SD 8) mumol h-1 kg-1 during fasting and decreased (P less than 0.01) by an average of 12% during amino acid infusion. Leucine oxidation was 18 (SD 3) mumol h-1 kg-1 during fasting and increased (P less than 0.001) by 89% during amino acid infusion. Whole-body protein synthesis (non-oxidative leucine disappearance) was 104 (SD 6) mumol h-1 kg-1 during fasting and rose by 13% (P less than 0.001) during mixed amino acid infusion. 4. 13C enrichment of muscle free leucine was only 61 (SD 19)% of that in plasma alpha-ketoisocaproate and this increased to 74 (SD 16)% (P less than 0.02) during mixed amino acid infusion. 5. The results suggest that increased availability of amino acids reverses whole-body protein balance from negative to positive and a major component of this is the increase in muscle protein synthesis.

Preliminary characterization of neurotoxic cyanobacteria blooms and strains from Finland

Abstract: Two hundred fifteen cyanobacteria bloom samples collected from different parts of Finland were studied, 35 of which proved to be neurotoxic. Toxicity was determined by mouse bioassay. Anabaena species were present in all neurotoxic samples except one, in which Oscillatoria dominated. The presence of anatoxin-a in the blooms and in the isolated strains was studied from freeze-dried materials by gas chromatography/mass spectrometry. The simultaneous occurrence of neurotoxicity and hepatotoxicity in some samples was studied by high performance liquid chromatography and high performance thin layer chromatography. Thirteen out of 30 bloom samples contained anatoxin-a. In the remaining samples, neurotoxicity was caused by unknown toxin(s). Strains producing anatoxin-a were isolated from the genera Anabaena, Aphanizomenon, Oscillatoria, and Cylindrospermum. Anatoxin-a content of the blooms varied from 12 to 4360 μg/g freeze-dried material. Some strains were able to produce about three times as much anatoxin-a as was detected in natural blooms. Simultaneous occurrence of neurotoxicity and hepatotoxicity was found in some samples as well as atypical toxic responses in mouse bioassay.

Amorphous silicon analogue memory devices

Abstract: Amorphous silicon M-p+ ni-M and M-p+ -M memory devices have been prepared. The characteristics are critically dependent on the metal used for the top contact. Devices with Cr top contacts exhibit the fast digital behaviour reported previously, whereas those using V exhibit fast analogue switching. The paper reports results for these and other metals.

Epitope-directed processing of specific antigen by B lymphocytes.

Abstract: Proteolytic processing of specific antigen was studied using Epstein Barr virus transformed B-lymphoblastoid cells expressing membrane IgG against tetanus toxin. As previously reported (Watts, C., and H.W. Davidson. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1937-1945), receptor-mediated endocytosis of monovalent antigen bound at 0 degrees C began immediately upon shifting the cells to 37 degrees C. In contrast, degradation of antigen, assessed either by the release of acid-soluble radiolabel into the incubation medium, or by SDS-PAGE analysis of total cell-associated antigen, proceeded after a lag of 10-20 min. Degradation was abolished by exposure of the cells to metabolic inhibitors, or by incubation at 20 degrees C, and inhibited in a dose-dependent fashion by chloroquine and by the lysosomal protease inhibitors leupeptin, E-64, and pepstatin A. Analysis of the cell-associated radiolabel by SDS-PAGE and autoradiography after incubations at 37 degrees C revealed the time-dependent generation of distinct antigen fragments. Virtually quantitative immunoprecipitation of these fragments was obtained using a monoclonal anti-human IgG antibody, indicating that the antigen/mIg complex is the initial substrate for processing. We show that the pattern of fragmentation observed varies from one B cell line to another (a) depending on the epitope through which the antigen is bound and endocytosed and (b) depending on whether additional epitopes in the antigen are complexed with anti-tetanus Fabs. The implications of these results for the presentation of major histocompatibility complex restricted antigen fragments, and for intracellular trafficking of ligand/receptor complexes are discussed.

The contrasting structures of mismatched DNA sequences containing looped-out bases (bulges) and multiple mismatches (bubbles)

Abstract: We have studied the structure and reactivities of two kinds of mismatched DNA sequences--unopposed bases, or bulges, and multiple mismatched pairs of bases. These were generated in a constant sequence environment, in relatively long DNA fragments, using a technique based on heteroduplex formation between sequences cloned into single-stranded M13 phage. The mismatched sequences were studied from two points of view, viz 1. The mobility of the fragments on gel electrophoresis in polyacrylamide was studied in order to examine possible bending of the DNA due to the presence of the mismatch defect. Such bending would constitute a global effect on the conformation of the molecule. 2. Sequences in and around the mismatches were studied using enzyme and chemical probes of DNA structure. This would reveal more local structural effects of the mismatched sequences. We observed that the structures of the bulges and the multiple mismatches appear to be fundamentally different. The bulged sequences exhibited a large gel retardation, consistent with a significant bending of the DNA at the bulge, and whose magnitude depends on the number of mismatched bases. The larger bulges were sensitive to cleavage by single-strand specific nucleases, and modified by diethyl pyrocarbonate (adenines) or osmium tetroxide (thymines) in a non-uniform way, suggesting that the bulges have a precise structure that leads to exposure of some, but not all, of the bases. In contrast the multiple mismatches ('bubbles') cause very much less bending of the DNA fragment in which they occur, and uniform patterns of chemical reactivity along the length of the mismatched sequences, suggesting a less well defined, and possibly flexible, structure. The precise structure of the bulges suggests that such features may be especially significant for recognition by proteins.

Present and future applications of amorphous silicon and its alloys

Abstract: Thin films of amorphous silicon (a-Si:H) and its alloys prepared by the glow discharge decomposition of silane and appropriate gases, are presently incorporated into six commercial products and approximately twenty other applications have been proposed for these materials. This presentation will review the present applications and a number of those proposed for the future. Finally, some of the material and technological issues of current importance will be discussed.

Changes in muscle fiber conduction velocity, mean power frequency, and mean EMG voltage during prolonged submaximal contractions

Abstract: Average muscle fiber conduction velocity, mean power frequency, and mean EMG voltage have been measured in human vastus lateralis during prolonged isometric knee extensions at 10, 20, 30, and 40% of the maximum knee extension force. During contractions at 10 and 20% of maximum force, conduction velocity and mean power frequency rose as the contraction progressed, whereas the conduction velocity and mean power frequency fell at 30 and 40% of the maximum force. The mean EMG voltage rose during the contractions, with steeper increases for higher forces. It is argued that two principal factors influence the EMG during prolonged submaximal contractions: firstly, the fatigue of current active motor units, and, secondly, recruitment of fresh motor units. These factors act in opposition to muscle fiber conduction velocity. Recruitment gives an increase in average conduction velocity, whereas fatigue provokes a slowing in conduction velocity.

An overlap between osmotic and anaerobic stress responses: a potential role for DNA supercoiling in the coordinate regulation of gene expression

Abstract: The regulation of several genes in response to osmotic and anaerobic stress has been examined. We have demonstrated a clear overlap between these two regulatory signals. Thus, the osmotically induced proU and ompC genes require anaerobic growth for optimum induction while the anaerobically induced tppB gene is also regulated by osmolarity. Furthermore, normal expression of tppB and ompC requires the positive regulatory protein OmpR, yet this requirement can be partially, or even fully, overcome by altering the growth conditions. Finally, the pleiotropic, anaerobic regulatory locus, oxrC, is also shown to affect expression of the osmotically regulated proU gene. The oxrC mutation is shown to affect the level of negative supercoiling of plasmid DNA and its effects on gene expression can be explained as secondary consequences of altered DNA topology. We suggest that there is a class of 'stress-regulated' genes that are regulated by a common mechanism in response to different environmental signals. Furthermore, our data are consistent with the notion that this regulatory overlap is mediated by changes in DNA supercoiling in response to these environmental stresses.