Synthesis and Incorporation of Pyrrole Carboxamide Nucleoside Triphosphates by DNA Polymerases

Abstract

We have synthesised and examined the enzymatic incorporation properties of the 5′-triphosphates of 2′-deoxyribosyl pyrrole 3-monocarboxamide (dMTP) and 2′-deoxyribosyl pyrrole 3,4-dicarboxamide (dDTP). These analogues we had hoped would behave as ambivalent base analogues in that they can present two alternative hydrogen-bonding faces either by rotation about the carboxamide group or about the glycosidic bond. The two pyrrole derivatives, dMTP and dDTP, exhibit a preference for incorporation with Klenow polymerase. They are preferentially incorporated as either A or C.     

A Protocol for Extraction of High-quality RNA and DNA from Peanut Plant Tissues

Peanuts are an increasingly important global food source. However, until recently the lack of effective protocols for the extraction of nucleic acids has made molecular studies of peanut development and maturation difficult. Here, we describe a method to isolate high-quality RNA and DNA from peanut tissue and have successfully applied this method to peanut plant roots, stems, leaves, flowers, and seeds. Spectrophotometric analysis showed that the average yields of total RNA from 100 mg of peanut materials ranged from 24.52 to 74.6 μg, and those of genomic DNA from the same tissues ranged from 23.47 to 57.68 μg. Using this protocol, we obtained OD260/280 values between 1.9 and 2.0 and isolated RNA which could be reverse transcribed in a manner suitable for RT-qPCR and expression analysis. In addition, genomic DNA isolated using this method produced reliable restriction enzyme digestion patterns and could be used for Southern blot hybridization.     

Uptake and Transport of Fusicoccin in Higher Plant Tissues

The uptake and release of ³H-labelled fusicoccin (FC) fed topea internode segments and the transport of [³H]FC in wholeplants and in excised parts of plants of pea, maize, and pumpkinhave been investigated. FC uptake showed three phases apparentlycorresponding to the penetration of FC: (1) into the free space;(2) into the cytoplasm; (3) into the vacuole. The first phase is a little thermosensitive and apparently includesadsorption binding to some free space component (cell surface?).The second and third phases are highly temperaturesensitiveand in part energy-dependent. However, no accumulation of FCagainst a concentration gradient is observed at high externalFC concentration. Accumulation at low concentration seems dueto the formation of a poorly exchangeable complex with somecell structure. The efflux of the FC penetrated within the cells is limitedby some highly temperature-sensitive process. A small fractionof the accumulated FC tends to be retained in the tissue, eitherbecause sequestered in some compartment or because bound tosome cellular component. FC transport in whole plants as well as in isolated plant partsseems to depend mainly on simple diffusion and on transportby mass flow in the xylem. Long distance transport of the toxinin pumpkin plants mainly occurs in the xylem, while the slowmovement in non-vascular tissues seems to depend on diffusioncombined with mass flow in the free space, and does not seemheavily influenced by metabolic factors.     

酸性α-淀粉酶的分离纯化与酶学性质研究

纯化了枯草芽胞杆菌xm-1菌株酸性α-淀粉酶,并对其酶学性质进行了研究。通过硫酸铵沉淀和Sephadex G-75凝胶层析将酸性α-淀粉酶粗酶液纯化了32.5倍,活力回收率为10.0%。酶性质测定结果表明,该酸性α-淀粉酶分子量约为60kD,最适反应温度为45℃、最适作用pH5.0,该酶在pH3.4-6.0下稳定,高温耐受性差。Cu2+、Zn2+、EDTA对酶有不同程度的抑制作用,Ca2+和Mn2+对酶具有较强的激活作用。     

Non-Enzymatic Reduction of Nitrite by Means of Ascorbic Acid in Citrus and Other Higher Plant Tissues

It has been proved that the nitrite reduction in the leaves and other plant tissues of citrus and other green plants is partly or mainly a non-enzymatic chemical process, and a heat-stable factor present in these tissues is responsible for this reduction. It is suggested that ascorbic acid plays a major role in this chemical reaction since the reduction is inhibited by ascorbic acid oxidase. A significant association was also found between the ascorbic acid content and the nitrite reduction capacity of citrus leaves. Evidence has been presented that this non-enzymatic chemical reduction of nitrite occurs also in vivo as undetached citrus leaves on branches placed in NaNO₂ solution have shown diminution of their ascorbic acid content along with the absorption of nitrite. Stronger accumulation of nitrite in these leaf tissues was observed under dark conditions, apparently due to the inhibition of the biosynthesis of the ascorbic acid.     

A Transient Study of Formation of Conjugates during TNT Metabolism by Plant Tissues

The formation of TNT-derived conjugates was investigated in hairy root tissue cultures of Catharanthus roseus and in aquatic plant systems of Myriophyllum aquaticum. The temporal profiles of four TNT-derived conjugates, TNT-1, 2A-1, TNT-2 and 4A-1, were determined over 3 to 16-day exposure durations. When axenic C. roseus roots were exposed separately to 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene, the array and levels of conjugates varied. Exposure of axenic roots to either 4-amino-2,6-dinitrotoluene or 2-amino-4,6-dinitrotoluene resulted in the formation of only 4A-1 and 2A-1, respectively, and not TNT-1 and TNT-2. However, amendment of previously unexposed roots with TNT produced all four conjugates. The conjugates were preferentially accumulated within the biomass phase of root cultures. Significantly, conjugates TNT-1 and TNT-2 were observed in the biomass phase of intact M. aquaticum plants exposed to TNT. The results clearly indicate the presence of common TNT transformation products in two diverse plants species and tissue type. The distribution of conjugates formed via monoamine derivatives of TNT, however, may be a function of several factors, including the starting xenobiotic type and/or level. Initial bulk rate constants for disappearance of 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were also determined. Their magnitude followed the order: TNT >> 4-A-2,6-DNT > 2-A-4,6-DNT.     

Study of the Morphological and Functional State of Higher Plant Tissues by Optical Coherence Microscopy and Optical Coherence Tomography

Comparative analysis of two optical methods—optical coherence tomography (OCT) and optical coherence microscopy (OCM)—was made for vital visualization of plant tissues in tomato (Lycopersicon esculentum Mill), spiderwort (Tradescantia pallida (Rose) D. Hunt), orach (Atriplex sp.), and leaves and seeds of medium starwort (Stellaria media L.). The obtained OCT- and OCM-images allowed the morphological and functional state of plant tissues to be assessed in vivo. A higher spatial resolution of the OCM method, as compared to OCT method, allowed plant morphological structures to be identified with greater confidence. The morphological and functional state of tissues can be monitored with a time resolution of 1–4 s in intact plants, without removing them from the habitat.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 628–634.Original Russian Text Copyright © 2005 by Kutis, Sapozhnikova, Kuranov, Kamenskii.     

Occurrence and Metabolism of Indoleacetaldehyde in Certain Higher Plant Tissues Under Aseptic Conditions

Qualitative and quantitative evidence based on thin-layer chromatography and the Avena curvature test respectively, are presented for the normal and natural occurrence of indoleacetic acid (IAA), indoleacetaldehyde (IAAld) and tryptophol in the etiolated shoots of Pisum sativum, grown, harvested and extracted under aseptic conditions. Non-aseptic pea shoots contain much more IAA and IAAld than aseptic ones. Extraneous contribution to the indole pool of pea plants grown under non-sterile atmosphere, other than by the inherent agency of the plant itself, is therefore not excluded. Indoleacetaldehyde metabolizing activity of the tissues and cell-free preparations of etiolated Avena seedlings, leading to the production of IAA and tryplophol, is unaffected by the antibiotics actinomycin and streptomycin over a wide range of concentrations. Formation of IAA from IAAld is suppressed by a small degree (10 to 15 %) by chloramphenicol. But this antibiotic does not influence the concurrent production of tryptophol. It is deduced that epiphytic bacteria play little role in the transformation of IAAld to IAA and tryptophol by Avena tissues.     

Purification and Characterization of a Ferredoxin-NADP Oxidoreductase-Like Enzyme from Radish Root Tissues

An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The K_m values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP⁺ in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP⁺ oxidoreductase. The effect of NaCl and MgCl₂ concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin-NADP⁺ oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues.     

利用芸薹属植物嵌合体对组织与器官细胞层起源的研究

运用两种同时具备表型和分子谱系标记的芸薹属植物(榨菜和紫甘蓝)合成的种间平周嵌合体材料对植物的组织、器官的细胞层谱系进行了追踪分析。研究结果发现:植物的茎、叶、花等器官一般由茎尖分生组织的L1、L2、L3三层谱系细胞共同发育而成,但在不同组织器官中各层的贡献量不同;L1和L2层共同参与了叶缘的发育;不定根由L3层单独发育而成;分子标记分析显示花粉起源于L2层,但有性杂交实验也发现了少量L1层起源的花粉。该文为研究植物组织与器官的细胞层起源提供了新方法。     

Methods of Staining Plant Tissues for Differentiating the Natural Plant Oils from Petroleum Spray Oils

Petroleum, spray oils in sections of plant tissue have been distinguished from the plant oils by staining the fresh sections in the following dye solution: To a saturated aqueous solution of Nile blue sulfate, 0.5% sulfuric acid is added and the mixture is boiled under a reflux condenser for 4 or 5 hours. It should be as nearly alkaline as possible without a change of color. A solution of 50% alcohol and 50% acetone is then saturated with oil red O. One part of the Nile blue sulfate solution is then added to two parts of the oil red O solution. Allow to settle over night and filter. Stain several hours. Rinse in water and mount in glycerin jelly. A short discussion of the merits of this method and the differentiation of the spray oils by means of indophenol blue are also given.     

Enzyme instability and proteolysis during the purification of an alcohol dehydrogenase from Drosophila melanogaster.

The alcohol dehydrogenase of the Drosophila melanogaster adhUF allele (alloenzyme with ultra-fast electrophoretic mobility) was unstable in crude or partially purified preparations. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that inactivation was porbably due to proteolytic degradation, and new method of purification of the enzyme was developed. After three steps, namely salmine sulphate precipitation, hydroxyapatite chromatography and Sephadex G-100 gel filtration, a 10-fold purified preparation was obtained. The enzyme produced was relatively stable compared with alcohol dehydrogenase purified by other methods, and was shown to be proteinase-free. The enzyme had a subunit mol.wt. of 24000 and had a single thiol residue per subunit available for titration with 5,5'-dithiobis-(2-nitrobenzoic acid). The amino acid composition and C-terminal amino acid sequence of the enzyme were determined. The substrate specificity of this alcohol dehydrogenase was also characterized. These results are discussed in relation to experiments on the evolutionary significance of thermostability at the adh locus.     

Analysis of the Components Released from Potato Tuber Tissues during Maceration by Pectolytic Enzymes

Endo-pectin lyase and endo-polygalacturonase of Aspergillus japonicus attack the middle lamella of plant tissue and cause tissue maceration. Galacturonides, neutral sugars, and proteins were released from potato tuber tissues during maceration by both purified enzymes. These three components accounted for 92% of the soluble products. The neutral sugars released were rhamnose, arabinose, and galactose with a molar ratio of 1:3:15. They were covalently linked to galacturonides. Over 85% of the galacturonides released by the enzymes were short chain products, which indicated that a large portion of the main chain of pectic substances is a homogalacturonan. The results of chromatography on columns of Sephadex G-100 and DEAE-cellulose suggested that a protein component may be attached to pectic substances. This protein did not contain hydroxyproline and, therefore, was different from the cell wall structural glycoprotein.     

植物组织RNA提取的难点及对策

酚类化合物、多糖、蛋白质和未知次级代谢产物是影响植物组织ＲＮＡ提取的几个主要干扰因素。本文对它们在植物ＲＮＡ提取过程中的干扰作用、现象以及消除它们的一些方法进行了综述。     

Investigation of Possible Participation of Nucleoside Transport Systems in the Postischemic Release of Purines and Pyrimidines from Cold Stored Liver

The aim of the study was to elucidate the role of nucleoside transport systems in the postischemic release of nucleosides and nucleobases accumulated by the rat liver during cold storage. Livers were preserved for 24 h in Euro-Collins (EC) or in a lactobionate-based solution (LBS) without exogenous adenosine. The rates of release of uric acid, xanthine, hypoxanthine, inosine, adenosine, uridine, and cytidine were monitored during early reperfusion. The greater part of the purines and pyrimidines (up to 80%) was lost in the first 2 min of reperfusion. After storage in EC, uric acid and xanthine formed more than 90% of the total purines released; nucleosides did not exceed 5% of the total. After storage in LBS, hypoxanthine formed more than 80% of purine efflux and the release of inosine and uridine was increased 5-10 times. These changes were shown to be due to the presence of allopurinol in LBS. Dipyridamole (an inhibitor of equilibrative nucleoside transporters) decreased the efflux of uric acid after storage in EC but residual release remained high. Dipyridamole exerted the most pronounced effect on the release of nucleosides (inosine and uridine) from livers stored in LBS. The use of sodium-free media for liver preservation and reperfusion did not alter the rates of purine and pyrimidine release. We conclude that equilibrative nucleoside transporters mediate the postischemic release of nucleosides and also, but to a less degree, of uric acid. Simple diffusion is an important factor in the release of nucleobases. Active Na(+)/nucleoside cotransport does not play an important role in early reperfusion.     

Photoreactivation of Transforming DNA by an Enzyme from Bakers' Yeast

Ultraviolet-inactivated Hemophilus influenzae transforming DNA recovers its activity when mixed with cell-free extracts of bakers' yeast and exposed to visible light. The active agent in the extract is not used up in the reaction, and purification has not separated it into more than one non-dialyzable component. It differs from the agent in Escherichia coli extract, which produces very similar photoreactivation, but which can be resolved into non-dialyzable and dialyzable components, the latter being used up during illumination. The yeast agent can be salted out of solution and recovered quantitatively; it is inactivated by crystalline trypsin and chymotrypsin and by brief heating at 60°C.—all facts suggesting that it is an enzyme for which ultraviolet lesions in the DNA serve as substrate. The kinetics of recovery are also consistent with such an assumption. This enzyme is unusual both because it is involved in a light-dependent reaction and because it has a non-destructive action on DNA outside an intact cell.     

VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.