Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Ca2+位于H2O2上游参与H2S诱导的拟南芥气孔关闭过程

以拟南芥为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,研究了Ca2+在硫化氢(H2S)诱导拟南芥气孔关闭过程中的作用及其与过氧化氢(H2O2)的关系。结果表明： H2S诱导气孔关闭, Ca2+螯合剂EGTA和质膜Ca2+通道阻断剂硝苯地平(Nif)能不同程度抑制H2S诱导的气孔关闭,而内质网钙泵阻断剂毒胡萝卜素(Thaps)对H2S的作用无显著影响。由此推测, Ca2+参与调节H2S诱导的拟南芥气孔关闭过程,且胞质中Ca2+来源于胞外Ca2+的内流。另外, H2S诱导拟南芥叶片NADPH氧化酶基因AtRBOHD和AtRBOHF以及细胞壁过氧化物酶基因AtPRX34表达增强,促进叶片和保卫细胞中H2O2积累, EGTA对此起抑制作用,而外源CaCl2处理上调AtRBOHD、AtRBOHF和AtPRX34的表达。表明Ca2+可能位于H2O2上游参与H2S诱导的拟南芥气孔关闭过程。     

Detoxification of SO2 in conifers differing in SO2-tolerance

Contents of organic sulfur, sulfate and the inorganic cations K⁺, Ca²⁺, Mg²⁺, Mn²⁺ and Na⁺ were compared in needles of three conifer species differing in tolerance to chronic SO₂ immissions. Sulfate and organic sulfur compounds were also measured in bark and wood. Field material was collected from Norway Spruce (Picea abies (L.) Karst.), Colorado Spruce (Picea pungens Engelm.) and Scots Pine (Pinus sylvestris L.) at sites where the SO₂ concentration in air was high, and at another site where it was low. In general, sulfate contents were higher but cation contents lower at the sites where SO₂ concentrations were high than where they were low. Up to 114mmol · (kg DW)^–1 sulfate was measured in fouryear-old needles of Norway Spruce from the Erzgebirge (annual mean of SO₂ in air 32 nl · 1^–1). Sulfate accumulation in this SO₂-sensitive conifer increased with SO₂ concentration in ambient air and with needle age, indicating that the main part of the sulfate resulted from the oxidative detoxification of SO₂. Loss of inorganic cations from ageing needles was reduced, or cation levels even increased, with increasing needle age, while sulfate accumulated. Apparently, cations served as counter-ions for sulfate, which is sequestered in the vacuoles. Individual trees differed in regard to the nature of cations which accumulated with sulfate. Calcium, potassium and magnesium were the dominating cations. Sodium levels were very low. Needles of the SO₂-tolerant conifers Colorado Spruce and Scots Pine growing next to Norway Spruce in the Erzgebirge did not accumulate, or accumulated less, sulfate with increasing needle age as compared to needles of Norway Spruce. However, somewhat more sulfate was found in the bark of the SO₂-tolerant species than in the bark of Norway Spruce. Scots Pine contained distinctly more sulfate in the wood than the other conifers. Since accumulation of organic sulfur compounds could not be observed with increasing needle age, or in bark and wood, reduction does not appear to play a major role in the detoxification of SO₂ by the investigated species. Physiological mechanisms permitting Colorado Spruce and Scots Pine to avoid the sulfate accumulation in the needles and the accompanying sequestration of cations that are observed in neighbouring Norway Spruce are discussed on the basis of the obtained data.Abbreviations S_org organic sulfur compounds Died June 10, 1991, aged 29, in a traffic accident. He initiated this work.This work was supported by the Sonderforschungsbereich 251 of the University of Würzburg and by the Projektgruppe Bayern zur Erforschung der Wirkung von Umweltschadstoffen (PBWU). The authors with to thank Prof. Dr. W Kaiser and Prof. Dr. W. Urbach (both Julius-von-Sachs-Institut, University of Würzburg, Germany) for HPLC-analysis and ICP-analysis.     

Conditioning in 2 x 2 tables

Summary .  Two-by-two tables arise in a number of diverse settings in biomedical research, including analysis of data from a clinical trial with a binary outcome and gating methods in flow cytometry to separate antigen-specific immune responses from general immune responses. These applications offer interesting challenges concerning what we should really be conditioning on—the total number of events, the number of events in the control condition, etc. We give several biostatistics examples to illustrate the complexities of analyzing what appear to be simple data.     

Casein kinase-2 phosphorylates serine-2 in the beta-subunit of initiation factor-2

We have previously presented evidence which suggests that casein kinase-2 phosphorylates a serine residue near the N-terminus of the beta-subunit of the initiation factor eIF-2 (Clark, S.J. et al. Biochim. Biophys. Acta 968, 211-219). We now report further data which confirm that it is serine-2 which is phosphorylated by casein kinase-2. This data includes (1) the electrophoretic mobilities of the phosphopeptides produced by different cleavage techniques, (2) the amino acid composition of the principal phosphopeptide generated by treatment with cyanogen bromide and (3) the resistance of this phosphopeptide to Edman degradation.     

Sulfenylation of ENOLASE2 facilitates H2O2-conferred freezing tolerance in Arabidopsis

1. Download : Download high-res image (155KB)
2. Download : Download full-size image

     

The essential role of H2O2 in the regulation of intracellular Ca2+ by epidermal growth factor in rat-2 fibroblasts

We have investigated a new mechanism by which epidermal growth factor (EGF) increases intracellular Ca(2+) ([Ca(2+)](i)) in Rat-2 fibroblasts. EGF induced a transient increase of [Ca(2+)](i), and sustained Ca(2+) increase disappeared in the absence of extracellular Ca(2+). However, EGF had no effect on the formation of inositol phosphates. Expression of N17Rac or scrape-loading of C3 transferase blocked the elevation of [Ca(2+)](i) by EGF, but not by lysophosphatidic acid (LPA). EGF increased intracellular H(2)O(2), with a maximal increase at 5 min, which was blocked by catalase, scrape-loading of C3 transferase, or expression of N17Rac. H(2)O(2) scavengers, catalase and N-acetyl-L-cysteine, also blocked the Ca(2+) response to EGF, but not to LPA. In the presence of EGTA, preincubation with EGF completely inhibited subsequent Ca(2+) response to extracellular H(2)O(2) and vice versa. Incubation with EGF or phosphatidic acid abolished subsequent elevation of [Ca(2+)](i) by phosphatidic acid or EGF, respectively. Furthermore, preincubation with LPA inhibited the subsequent Ca(2+) response to EGF, but not vice versa. These results suggested that intracellular H(2)O(2) regulated by Rac and RhoA, but not inositol phosphates, was responsible for the EGF-stimulated elevation of [Ca(2+)](i). It was also suggested that EGF cross talked with LPA in the regulation of [Ca(2+)](i) by producing intracellular H(2)O(2).     

2-amino-2-deoxy-D-erythrose in Agaricus bisporus

2-Amino-2-deoxy-D-erythrose was isolated from the cell wall of the fruit body of Agaricus bisporus. The structure of the amino sugar was determined by mass spectrography and 1H-NMR spectrography of its acetylated derivative and by paper chromatographic comparisons with authentic 2-amino-2-deoxy-D-erythrose. This amino sugar is a component of the glycoprotein fraction from the cell wall. Its content in the glycoprotein increased markedly, especially during the ripening stage of the fruit body.     

Catalase-dependent measurement of H2O2 in intact mitochondria

Mitochondria generate potentially damaging amounts of superoxide and H2O2 during oxidative metabolism. Although many assays are available to measure mitochondrial H2O2 generation, most detect H2O2 that has escaped the organelle. To measure H2O2 within mitochondria that contain catalase, we have developed an assay based on the ability of H2O2 to inhibit catalase in the presence of 3-amino-1,2,4-triazole. The assay is simple to perform, does not require expensive instrumentation, and is specific for H2O2. Results from this assay show that H2O2 generation in rat heart mitochondria reflects the activity of the electron transport chain. Further, liver mitochondria prepared from selenium-deficient rats have increased succinate-stimulated rates of H2O2 generation. This indicates that mitochondrial selenoenzymes are important for H2O2 removal. It also demonstrates the utility of this assay in measuring H2O2 release from mitochondria that do not contain catalase. The assay should be useful for study of both superoxide-dependent H2O2 generation in situ, and the role of endogenous mitochondrial catalase in H2O2 removal.     

Identification of 2Fe-2S cysteine ligands in putidaredoxin

The iron-sulfur center of putidaredoxin is coordinated by four cysteine sulfhydrals. In order to determine which of the six cysteine residues in the protein coordinate the Fe-S center, we have individually mutated cysteine residues 73, 85 and 86 into serines. Of these mutant proteins, only C85S and C73S express holo-protein as evidence by SDS-PAGE and EPR spectroscopy. This leads us to the conclusion that residues 39,45,48, and 86 are the cysteines that coordinate the iron-sulfur center in putidaredoxin.     

Metabolism and actions of 2-deoxy-2-fluoro-D-galactose in vivo

The synthetic D-galactose analog 2-deoxy-2-fluoro-D-galactose (dGalF) offers unique advantages for studies of the D-galactose pathway by non-invasive techniques using 19F-NMR spectroscopy or positron emission from the 18F-labeled compound. The metabolism of 2-deoxy-2-fluoro-D-galactose was studied in rodents using the unlabeled, the 18F-labeled, and the 14C-labeled D-galactose analog. Analyses for the metabolites of 2-deoxy-2-fluoro-D-galactose were performed by HPLC, enzymatic methods, and 19F-NMR spectroscopy in vivo and in vitro. The metabolism of 2-deoxy-2-fluoro-D-galactose was most active in the liver which took up the major part of the administered dose of the 14C-labeled D-galactose analog, but renal excretion was also pronounced. This was confirmed by in vivo scanning of the rat using the 18F-labeled sugar (1.5 microCi/g; 25 nmol/g) and examination by positron-emission tomography and gamma camera. The dose dependence of the levels of the hepatic metabolites of 2-deoxy-2-fluoro-D-galactose was investigated for doses between 25 nmol/g body mass and 1 mumols/g body mass. After 1 h, the major part of the acid-soluble uracil nucleotides consisted of UDP-2-deoxy-2-fluoro-D-hexoses when the dose was at least 0.1 mumols/g. With higher doses, 2-deoxy-2-fluoro-D-galactose 1-phosphate became the predominant initial metabolite. After a dose of 1 mumols/g 2-deoxy-2-fluoro-D-galactose 1-phosphate accumulated rapidly (5.3 +/- 0.4 mumols/g liver after 30 min) followed by the formation of UDP-2-deoxy-2-fluoro-D-galactose and UDP-2-deoxy-2-fluoro-D-glucose (0.7 +/- 0.1 mumols/g and 1.8 +/- 0.1 mumols/g, respectively, after 5 h). The diversion of uridylate, due to the accumulation of UDP-2-deoxy-2-fluoro-D-hexoses, was associated with a rapid depletion of hepatic UTP, UDP-glucose, and UDP-galactose. The UTP content was decreased to 11 +/- 6% of normal within 15 min after administration of 2-deoxy-2-fluoro-D-galactose at a dose of 1 mumols/g. The UTP-depleting action was minimal, however, at a dose of 25 nmols/g or less, indicating that interference in uridylate metabolism would be negligible at the doses required for positron-emission tomography of the liver using the 18F-labeled compound. At higher doses, the UTP deficiency induced by 2-deoxy-2-fluoro-D-galactose could be useful in the chemotherapy of D-galactose-metabolizing tumors such as hepatocellular carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radical-induced deamination of 2-amino-2-deoxy-d-glucose in aqueous solution

2-Amino-2-deoxy-d-glucose (10mM) as the free base (pH 8.5) and the hydrochloride (pH 5) were γ-irradiated in aqueous solution under deoxygenated (N₂O-saturated) and oxygenated [N₂O/O₂(4:1)-saturated] conditions. Ammonia and 18 nitrogen-free products were identified and their G-values determined. Mechanisms for the radical-induced deamination are proposed. The radicals centered at C-1, C-2, C-3, and the nitrogen are suggested as initiators in the deamination processes.     

Mechanistic studies of O2-resistant N2-fixation in N2-fixing Escherichia coli

Escherichia coli carrying the entire nif gene cluster from Klebsiella pneumoniae on a multicopy plasmid becomes more O2-resistant in a N-free medium as a result of the integration of the nif gene cluster into the chromosome and the loss of the plasmid (H.Iwahashi and J.Someya, Biochem. Biophys. Res. Comm. 1990, 168: 288–294). Our purpose is to characterize the physiological reason why the strain became O2-resistant by measuring the levels of nif proteins in cells under microaerobic conditions. The O2-resistant strain had a higher amount of NifH and a lower amount of NifL under microaerobic conditions (compared to that under anaerobic conditions), while the parent strain showed the opposite characteristics. Thus, the biochemical mechanism of the O2-resistant strain is attributed to the strain's ability to synthesize and maintain a high amount of NifH and a low amount of NifL under microaerobic conditions. © Rapid Science Ltd. 1998     

Fluxes of Ca2+, Sr2+ and Mg2+ in synaptosomes.

Synaptosomes isolated from sheep brain cortex accumulate Ca²⁺, Sr²⁺ and Mg²⁺ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca²⁺, 85 nmol of Mg²⁺ and 80 nmol of Sr²⁺. The loss of Ca²⁺ or Sr²⁺ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na⁺> K⁺ > Li⁺> choline, whereas for the loss of Mg²⁺ this order is different: K⁺ > Na⁺ > Li ～ choline. The efflux of Ca²⁺ or Sr²⁺ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg²⁺ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca²⁺ for Na⁺ in synaptosomes operates similarly for Sr²⁺, but not for Mg²⁺.