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PubMed2002

Sulfur sparing in the yeast proteome in response to sulfur demand.

Genome-wide studies have recently revealed the unexpected complexity of the genetic response to apparently simple physiological changes. Here, we show that when yeast cells are exposed to Cd(2+), most of the sulfur assimilated by the cells is converted into glutathione, a thiol-metabolite essential for detoxification. Cells adapt to this vital metabolite requirement by modifying globally their proteome to reduce the production of abundant sulfur-rich proteins. In particular, some abundant glycolytic enzymes are replaced by sulfur-depleted isozymes. This global change in protein expression allows an overall sulfur amino acid saving of 30%. This proteomic adaptation is essentially regulated at the mRNA level. The main transcriptional activator of the sulfate assimilation pathway, Met4p, plays an essential role in this sulfur-sparing response.

Adaptation, Physiological↗
PubMed1976

Acetaldehyde: an intermediate in the formation of ethanol from glucose by lactic acid bacteria.

Group N streptococci formed acetaldehyde and ethanol from glucose. As the enzymes aldehyde dehydrogenase, phosphotransacetylase and acetate kinase were present this would enable these organisms to reduce acetyl-CoA to acetaldehyde and convert acetyl-CoA to acetyl phosphate and acetate. A pentose phosphate pathway which converted ribose-5-phosphate to glyceraldehyde-3-phosphate was also present. Acetaldehyde could not be formed via the hexose monophosphate shunt or by direct decarboxylation of pyruvate, as the enzymes phosphoketolase and alpha-carboxylase were absent. Phosphoketolase activity was induced in Streptococcus lactis subsp. diacetylactis after growth on D-xylose. Group N streptococci also contained an NAD-dependent alcohol dehydrogenase which reduced acetaldehyde to ethanol while both NAD- and NADP-dependent alcohol dehydrogenase activities were found in Leuconostoc cremoris.

Acetaldehyde↗
PubMed1982

Evidence for the regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex by a phosphorylation/dephosphorylation mechanism.

The regulation of the branched chain alpha-keto acid dehydrogenase complex by covalent modification was investigated in rat liver mitochondria. Depletion of intramitochondrial calcium and magnesium caused an inactivation of the branched chain alpha-keto acid dehydrogenase complex. Following inactivation of the branched chain complex, addition of calcium or magnesium ions separately to incubations of mitochondria only partially reactivated the enzyme complex. However, simultaneous addition of calcium and magnesium activated the branched chain enzyme complex rapidly and nearly completely. Mitochondrial incubations were performed in the presence of [32P]phosphate under conditions known to activate or to inactivate the branched chain alpha-keto acid dehydrogenase complex. Evidence demonstrating that [32P]-phosphate was incorporated into two major protein bands separated in sodium dodecyl sulfate-polyacrylamide gels of the mitochondrial incubations is presented. Migration of the labeled mitochondrial protein bands in the gel system corresponded exactly to the migration of the alpha subunit of the purified heart-derived pyruvate dehydrogenase (decarboxylase, E1) and the alpha subunit of the purified kidney-derived branched chain alpha-keto acid dehydrogenase (decarboxylase, E1). Furthermore, when the measured activity of the branched chain complex was minimized, the amount of [32P]phosphate incorporated into the alpha chain of the branched chain enzyme was maximal. Conversely, incubation conditions which activated maximally the enzyme complex minimized the [32P]phosphate incorporation into the alpha subunit of the branched chain dehydrogenase.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1984

Determination of dissociation constants for enzyme-reactant complexes for NAD-malic enzyme by modulation of the thiol inactivation rate.

Incubation of NAD-malic enzyme from Ascaris suum with the sulfhydryl reagents N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), or 4,4'-dithiodipyridine (4-PDS) results in rapid and complete loss of malate oxidative decarboxylase and pyruvate reductive carboxylase activities. With DTNB, this loss of activity occurs concomitantly with the modification of about 1 thiol group per subunit. The majority of the activity is lost when 0.5 thiol per subunit is modified, indicative of possible half-site reactivity with DTNB. Complete restoration of activity follows addition of dithiothreitol to enzyme inactivated by DTNB and 4-PDS but not with NEM. With the DTNB-inactivated enzyme, replacement of the thionitrobenzoate moiety with cyanide restores activity. The presence of a divalent metal ion (Mg2+ or Mn2+) results in enhancement of the inactivation rate with all sulfhydryl reagents. However, malate alone or competitors of malate provide protection which is more effective in the presence of Mg2+, while NAD provides only about 25% protection. Thus, the Ascaris suum NAD-malic enzyme has a thiol group probably located in or near the malate binding site, which is not essential for enzyme activity. The changes in the rate of inactivation in the presence of reactants were used to determine the dissociation constants for enzyme-reactant complexes. These data suggest that all three possible binary and all three possible ternary complexes form. The binding of malate to free enzyme exhibits negative cooperativity, which is eliminated by the presence of either NAD or Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

The transduction of the signal for grape bud dormancy breaking induced by hydrogen cyanamide may involve the SNF-like protein kinase GDBRPK.

Alterations in gene expression during early stages of dormancy release in grapevine buds were analyzed to facilitate the identification of gene products that may mediate the signal transduction of a dormancy-release signal, or derepression of meristematic activity. In the present report we describe the identification of GDBRPK, a transcript for an SNF-like protein kinase that is up-regulated upon chemical induction of dormancy release by hydrogen cyanamide (HC). Since SNF and SNF-like protein kinases are known as sensors of stress signals, we hypothesize that GDBRPK may be involved in the perception of a stress signal induced by HC. We also describe a simultaneous and remarkable induction of both PDC and ADH transcripts that was observed shortly after HC application, and was of a transient nature. These data may imply that HC application leads to a transient respiratory stress, which likely results in a temporary increase in the AMP/ATP ratio. Since AMP is known as a stress signal that is sensed by SNF-like kinases, we suggest that the SNF-like GDBRPK could serve as the sensor of this signal.

Alcohol Dehydrogenase↗
PubMed1998

Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2.

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with alpha(2-8) linkages. When the bacterium was grown at 37 degrees C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 microg/ml. Release of this polymer was strictly regulated by the growth temperature, and above 40 degrees no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential presence of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of Nacetyl-D-mannosamine and pyruvate with apparent Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, which catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km values of 2 mM and 0.5 mM, respectively, and finally a membrane-associated polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 250 microM.

Cell Division↗
PubMed2004

Performance of a newly developed integrant of Zymomonas mobilis for ethanol production on corn stover hydrolysate.

Efficient conversion of lignocellulosic biomass requires biocatalysts able to tolerate inhibitors produced by many pretreatment processes. Recombinant Zymomonas mobilis 8b, a recently developed integrant of Zymomonas mobilis 31821(pZB5), tolerated acetic acid up to 16 g l(-1) and achieved 82%-87% (w/w) ethanol yields from pure glucose/xylose solutions at pH 6 and temperatures of 30 degrees C and 37 degrees C. An ethanol yield of 85% (w/w) was achieved on glucose/xylose from hydrolysate produced by dilute sulfuric acid pretreatment of corn stover after an overliming' process was used to improve hydrolysate fermentability.

Acetates↗
PubMed1993

Electroporation and DNA-dependent cell death in murine macrophages.

The difficulty of transfecting primary macrophages and macrophage cell lines has meant that relatively few studies on regulation of gene expression have been performed in these cells. This study has optimized an electroporation procedure for the macrophage cell line RAW 264, but shows that introduction of DNA into the cytoplasm of primary macrophages by electroporation is toxic to the cells. It is proposed that this cell death may have a physiological role in defence against certain viral infections which result in accumulation of cytoplasmic DNA. RAW 264 cells were efficiently transfected by electroporation, but electroporated bone marrow derived macrophages (BMM) showed large scale cell death over a period of 12 h. Electroporation without DNA was not toxic and DNase treatment of samples before transfection prevented cell death. The toxicity of DNA was concentration-dependent and sequence-independent. Synthetic, genomic and plasmid DNA all caused cell death. This sensitivity to DNA seems to be distinct from the antiviral state induced by double-stranded RNA and may be part of an uncharacterized viral defence system.

Animals↗
PubMed2003

Potent mammalian cerebroprotection and neuronal cell death inhibition are afforded by a synthetic antioxidant analogue of marine invertebrate cell protectant ovothiols.

Implicit strategies for neuroprotection in the adult brain include GABAA receptor activation, N-methyl-d-aspartate receptor and sodium voltage-gated channel inhibition. Ironically, these same targets may be harmful to the immature or developing brain. Protection has been demonstrated for both immature and mature brain with the use of a synthetic ovothiol analogue. The following beneficial effects have been demonstrated in mice: protection against audiogenic seizures, brain structures with clear-cut delineation of ibotenate-challenged white and grey matter lesions along with exceptional early and delayed protections, and potent cerebral cell death inhibition. The compound lacks both GABAergic activity and sodium channel blocker properties, which may help explain the lack of toxicity normally expressed in an immature brain utilizing these agents [J.W. Olney (2002) Neurotoxicology, 93, 1-10]. The oxidized form of the compound is virtually devoid of antioxidant activity. In vivo it exhibits cerebroprotective properties similar to those of reduced compounds endowed with antioxidant properties. This unexpected finding has prompted an extensive in vitro exploration of underlying molecular mechanisms that have led to the identification of several recycling mechanisms consistent with non rate-limiting conversion of oxidized to reduced compound forms. Taken as a whole, this work offers an unique combined in vitro and in vivo support that: (i). antioxidant therapy, here engineered from marine invertebrate egg protectants, may be a valuable strategy in protecting both mammalian adult and developing brain; and (ii). recycling (thiol-disulphide exchange) properties of the oxidized form of an antioxidant compound are as important as the antioxidant potential exhibited by a bioactive reduced antioxidant in certain neuroprotective processes.

Animals↗
PubMed1990

Relationships between the neuronal sodium/potassium pump and energy metabolism. Effects of K+, Na+, and adenosine triphosphate in isolated brain synaptosomes.

The relationships between Na/K pump activity and adenosine triphosphate (ATP) production were determined in isolated rat brain synaptosomes. The activity of the enzyme was modulated by altering [K+]e, [Na+]i, and [ATP]i while synaptosomal oxygen uptake and lactate production were measured simultaneously. KCl increased respiration and glycolysis with an apparent Km of about 1 mM which suggests that, at the [K+]e normally present in brain, 3.3-4 mM, the pump is near saturation with this cation. Depolarization with 6-40 mM KCl had negligible effect on ouabain-sensitive O2 uptake indicating that at the voltages involved the activity of the Na/K ATPase is largely independent of membrane potential. Increases in [Na+]i by addition of veratridine markedly enhanced glycoside-inhibitable respiration and lactate production. Calculations of the rates of ATP synthesis necessary to support the operation of the pump showed that greater than 90% of the energy was derived from oxidative phosphorylation. Consistent with this: (a) the ouabain-sensitive Rb/O2 ratio was close to 12 (i.e., Rb/ATP ratio of 2); (b) inhibition of mitochondrial ATP synthesis by Amytal resulted in a decrease in the glycoside-dependent rate of 86Rb uptake. Analyses of the mechanisms responsible for activation of the energy-producing pathways during enhanced Na and K movements indicate that glycolysis is predominantly stimulated by increase in activity of phosphofructokinase mediated via a rise in the concentrations of adenosine monophosphate [AMP] and inorganic phosphate [Pi] and a fall in the concentration of phosphocreatine [PCr]; the main moving force for the elevation in mitochondrial ATP generation is the decline in [ATP]/[ADP] [Pi] (or equivalent) and consequent readjustments in the ratio of the intramitochondrial pyridine nucleotides [( NAD]m/[NADH]m). Direct stimulation of pyruvate dehydrogenase by calcium appears to be of secondary importance. It is concluded that synaptosomal Na/K pump is fueled primarily by oxidative phosphorylation and that a fall in [ATP]/[ADP][Pi] is the chief factor responsible for increased energy production.

Adenosine Triphosphate↗
PubMed2001

Evidence of root zone hypoxia in Brassica rapa L. grown in microgravity.

A series of experiments was conducted aboard the U.S. space shuttle and the Mir space station to evaluate microgravity-induced root zone hypoxia in rapid-cycling Brassica (Brassica rapa L.), using both root and foliar indicators of low-oxygen stress to the root zone. Root systems from two groups of plants 15 and 30 d after planting, grown in a phenolic foam nutrient delivery system on the shuttle (STS-87), were harvested and fixed for microscopy or frozen for enzyme assays immediately postflight or following a ground-based control. Activities of fermentative enzymes were measured as indicators of root zone hypoxia and metabolism. Following 16 d of microgravity, ADH (alcohol dehydrogenase) activity was increased in the spaceflight roots 47% and 475% in the 15-d-old and 30-d-old plants, respectively, relative to the ground control. Cytochemical localization showed ADH activity in only the root tips of the space-grown plants. Shoots from plants that were grown from seed in flight in a particulate medium on the Mir station were harvested at 13 d after planting and quick-frozen and stored in flight in a gaseous nitrogen freezer or chemically fixed in flight for subsequent microscopy. When compared to material from a high-fidelity ground control, concentrations of shoot sucrose and total soluble carbohydrate were significantly greater in the spaceflight treatment according to enzymatic carbohydrate analysis. Stereological analysis of micrographs of sections from leaf and cotyledon tissue fixed in flight and compared with ground controls indicated no changes in the volume of protoplast, cell wall, and intercellular space in parenchyma cells. Within the protoplasm, the volume occupied by starch was threefold higher in the spaceflight than in the ground control, with a concomitant decrease in vacuolar volume in the spaceflight treatment. Both induction of fermentative enzyme activity in roots and accumulation of carbohydrates in foliage have been repeatedly shown to occur in response to root zone oxygen deprivation. These results indicate that root zone hypoxia is a persistent challenge in spaceflight plant growth experiments and may be caused by microgravity-induced changes in fluid and gas distribution.

Alcohol Dehydrogenase↗
PubMed1978

Thiamin deficiency effects on rat leukocyte pyruvate decarboxylation rates.

Thiamin status usually is assessed by urinary excretion of thiamin or by exogenous thiamin pyrophosphate (TPP) stimulation of erythrocyte transketolase activity. Because of the possible great utility of a biologically and chemically sensitive alternative method for thiamin status assessment, studies were made of rat leukocyte pyruvate decarboxylation activity in thiamin deficiency. Pyruvate decarboxylation rates were determined by assaying 14CO2 produced by leukocytes from 1-14C-pyruvic acid in vitro. Reaction conditions were 5 mumoles pyruvic acid, 2.2 X 10(4) DPM 1-14C-pyruvic acid, leukocytes from 5 ml whole blood, 50 mumoles NaH2PO4, 5 mumoles MgSO4, and 1 mumole MnSO4 at pH 7.4 in 1 ml reaction volume at 25 C. Four weeks of thiamin deficiency decreased leukocyte pyruvate decarboxylation rates and markedly increased the TPP effect on this reaction. Dual weekly assays in the same rats showed that 21 days of thiamin deficiency significantly increased the TPP effect on leukocyte pyruvate decarboxylation rates. In contrast, the TPP effect on erythrocyte transketolase activity was significantly increased after only 7 days of thiamin deficiency. Erythrocyte transketolase is more sensitive than leukocyte pyruvate decarboxylation rate to early thiamin deficiency in rats.

Animals↗
PubMed1986

Effect of biotin deficiency and supplementation on lipid metabolism in rats: cholesterol and lipoproteins.

The effect of biotin deficiency and supplementation upon tissue cholesterol and serum lipoprotein profile in rats was investigated. Biotin-dependent carboxylases in liver of deficient animals were decreased to 12-27% of control activity. The brain carboxylase activities of deficient rats were reduced less than in liver. The total, free, and esterified cholesterol were decreased in the serum of deficient rats compared to those in control or supplemented rats. Deficient rats had increased serum low-density lipoprotein fractions and decreased very low-density lipoprotein fractions, but no change in the high-density lipoprotein fractions. No differences were found between groups in the cholesterol content of liver, cerebrum, or cerebellum. Pharmacologic doses of biotin had no effect on tissue cholesterol content or serum lipoprotein profile. These results suggest that the neurologic symptoms in biotin deficiency and in the inherited multiple carboxylase deficiencies are not due to alterations in the content of cholesterol or lipoproteins.

Acetyl-CoA Carboxylase↗
PubMed1992

Molecular cloning of a gene (cfp) encoding the cytoplasmic filament protein P59Nc and its genetic relationship to the snowflake locus of Neurospora crassa.

P59Nc is a 59-kD polypeptide associated with 8-10-nm diameter cellular filaments in normal Neurospora crassa strains. Abnormally sized and shaped bundles of these structures are present in N. crassa strains carrying mutations at the locus sn (snowflake). By using molecular cloning and restriction fragment length polymorphism (RFLP) segregation analysis strategies we show here that sn is not the genetic locus of P59Nc. Several P59Nc cDNAs were cloned from a N. crassa lambda GT11 library after immunoscreening with specific polyclonal anti-P59Nc antibodies. Additional longer cDNAs were obtained from a N. crassa cDNA-lambda ZAP library. When used as probes in Southern blots of total DNA from wild-type strains, multicent-2 (a multiple mutant strain), and snowflake mutants, the P59Nc cDNAs revealed comparable patterns of hybridizing bands for all of the restriction enzymes tested. Analysis of segregation of BclI and ClaI RFLPs, detected in the genomic region of the P59Nc gene (locus cfp: cellular filament polypeptide), among a set of strains designed for RFLP mapping, or among selected progeny of crosses involving a snowflake parent, respectively, indicate that (i) there is in N. crassa a single cfp locus positioned on the right arm of linkage group VII between the locus for and the proximal breakpoint of the translocation T(VII----I)5936; (ii) the sn mutations in the centromere region of chromosome I do not represent translocations of cfp; and (iii) the snowflake mutants possesses a normal copy of the P59Nc gene on their chromosomes VII.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Long-term anoxia tolerance in leaves of Acorus calamus L. and Iris pseudacorus L.

Mature green leaves of Acorus calamus and Iris pseudacorus have been shown to survive at least 28 d of total anoxia in the dark during the growing season, increasing up to 75 d and 60 d in overwintering leaves in A. calamus and I. pseudacorus, respectively. During the period of anaerobic incubation the glycolytic rate is reduced, carbohydrate reserves are conserved and ethanol levels in the tissues reached an equilibrium. Prolonged anoxia significantly suppressed leaf capacity for respiration and photosynthesis. After 28 d of anoxia, respiratory capacity was reduced in A. calamus and I. pseudacorus by 80% and 90%, respectively. The photosynthetic capacity of leaves decreased by 83% in A. calamus and by 97% in I. pseudacorus after 28 d of anoxia. This reduction in photosynthetic capacity was accompanied by a modification of the chlorophyll fluorescence pattern indicating damage to the PSII reaction centre and subsequent electron transport. Chlorophyll content was only slightly reduced after 28 d under anoxia and darkness in A. calamus, whereas there was a 50% reduction in I. pseudacorus. On return to air A. calamus leaves that endured 28 d of anoxia recovered full photosynthetic activity within 7 d while those of I. pseudacorus had a lag phase of 3-10 d. This well-developed ability to endure prolonged periods of oxygen deprivation in both these species is associated with a down-regulation in metabolic activity in response to the imposition of anaerobiosis. It is suggested that when leaf damage eventually does take place in these species after protracted oxygen deprivation, it is anoxic rather than post-anoxic stress that is responsible.

Acorus↗
PubMed2002

Legume embryos develop in a hypoxic environment.

Specific morphological and biochemical characteristics of seeds can cause oxygen deficiency within maternal and embryonic tissues. In this study, optical sensors were used to measure O(2) profiles across developing seeds of Vicia faba and Pisum sativum and developmental and environmental modulations of internal O(2) levels were studied. In addition, the metabolic state of developing embryos was analysed by monitoring adenylate energy charge, adenylate nucleotides and the levels of nucleotide sugars. Within the seed coat O(2) concentration decreased sharply to approximately 3% towards the inner border. Lowest O(2) levels were detected within the endospermal cavity between the seed coat and embryo. It is probable that low seed coat permeability provides an hypoxic environment for legume embryo development. The O(2) concentration in embryonic tissue changed during development with the lowest levels in the early stages. Measured in darkness, the levels were below 3%, but increased upon illumination indicating that photosynthesis significantly contributes to internal O(2) levels. Only in very young embryos were ATP levels and energy charge low. Otherwise they were maintained at a constant higher value. ADP-glucose and UDP-glucose did not show large fluctuations. Throughout embryo development fermentative activity did not play a major role. Obviously, specific mechanisms prevent seed tissues from becoming anoxic during development. The possible role of low oxygen on seed metabolism and on the control of seed development in legumes is discussed.

Adenine Nucleotides↗