Utility of Green Fluorescent Nucleic Acid Dyes and Aluminum Oxide Membrane Filters for Rapid Epifluorescence Enumeration of Soil and Sediment Bacteria

High background fluorescence and unspecific staining hampered the epifluorescence enumeration of bacteria in 45% of the tested soil and sediment samples with 4′,6-diamidino-2-phenylindole (DAPI) and polycarbonate membrane filters. These problems of the determination of total cell counts can be circumvented by using green fluorescent high-affinity nucleic acid dyes and aluminum oxide membrane filters. Due to the bright staining of cells, we recommend SYBR Green II as dye.     

Viruses of Entamoeba histolytica IV. Studies on the Nucleic Acids of the Filamentous and Polyhedral Viruses

The nucleic acids of two amoebal viruses were studied by several independent methods. The filamentous virus, V(ABRM), was shown to be inhibited by bromodeoxyuridine, iododeoxyuridine, and cytosine arabinoside. With acridine orange staining, V(ABRM) inclusions appeared greenish-yellow, indicating that these contained double-stranded nucleic acid. The polyhedral virus, V(301), was also inhibited by bromodeoxyuridine, iododeoxyuridine, and cytosine arabinoside. In addition, nucleic acid hybridization showed that a new DNA species was synthesized in infected amoebal cultures. The intracellular localization of this new DNA was consistent with previous electron microscope studies of the cytoplasmic maturation of V(301).     

Synthesis and Nucleic Acids Binding Properties of Diastereomeric Aminoethylprolyl Peptide Nucleic Acids (aepPNA)

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the “cis-” homothymine aepPNA decamers with (2′R,4′R) and (2′S,4′S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2′R,4′S) and (2′S,4′R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2′R,4′R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.     

Synthesis of Polyamide Nucleic Acids (Pnas), Pna /Dna-Chimeras and Phosphonic Ester Nucleic Acids (Phonas)

Abstract

The synthesis of polyamide nucleic acids (PNAs) and derivatives thereof by different synthetic routes is described. The first strategy makes use of 9-Fluorenylmethoxycarbonyl (Fmoc)/monomethoxytrityl (Mmt) protected building blocks, whereas the second approach involves the use of Mmt/acyl protected monomers, which allows the preparation of PNADNA chimera. Additionally, a block coupling strategy is presented for the synthesis of novel phosphonic ester nucleic acids (PHONAs).     

Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols,Matrix Effects,and Nucleic Acid Interference

Polyphosphate (polyP) is a ubiquitous biochemical with many cellular functions and comprises an important environmental phosphorus pool. However, methodological challenges have hampered routine quantification of polyP in environmental samples. We tested 15 protocols to extract inorganic polyphosphate from natural marine samples and cultured cyanobacteria for fluorometric quantification with 4′,6-diamidino-2-phenylindole (DAPI) without prior purification. A combination of brief boiling and digestion with proteinase K was superior to all other protocols, including other enzymatic digestions and neutral or alkaline leaches. However, three successive extractions were required to extract all polyP. Standard addition revealed matrix effects that differed between sample types, causing polyP to be over- or underestimated by up to 50% in the samples tested here. Although previous studies judged that the presence of DNA would not complicate fluorometric quantification of polyP with DAPI, we show that RNA can cause significant interference at the wavelengths used to measure polyP. Importantly, treating samples with DNase and RNase before proteinase K digestion reduced fluorescence by up to 57%. We measured particulate polyP along a North Pacific coastal-to-open ocean transect and show that particulate polyP concentrations increased toward the open ocean. While our final method is optimized for marine particulate matter, different environmental sample types may need to be assessed for matrix effects, extraction efficiency, and nucleic acid interference.     

一株乳酸菌胞外多糖产生的影响因素及其提取

应用苯酚一硫酸法对乳酸菌胞外多糖产生的影响因素进行了研究,表明该菌株在培养温度为30℃,培养时间为4048h,pH值降到4时,胞外多糖的产量最大。葡萄糖是乳酸菌产生多糖的良好碳源。在对乳酸菌的培养物进行离心、透析、脱蛋白、脱色,最后用乙醇沉淀,得到粗品多糖,粗品多糖至少含有两种分子量和含量相差很大的多糖。经过SephadexG-200凝胶柱得到多糖精品EPS—Ⅱ,薄层层析结果显示其为一纯化的样品。     

Nucleic Acids Electrotransfer-Based Gene Therapy (Electrogenetherapy): Past,Current, and Future

About 25 years after the publication of the first report on gene transfer in vitro in cultured cells by the means of electric pulses delivery, reversible cell electroporation for gene transfer and gene therapy (DNA electrotransfer) is at a cross in its development. Present knowledge on the effects of cell exposure to appropriate electric field pulses, particularly at the level of the cell membrane, is reported here. The importance of the models of electric field distribution in tissues and of the correct choice of electrodes and applied voltages is highlighted. The mechanisms involved in DNA electrotransfer, which include cell electropermeabilization and DNA electrophoresis, are also surveyed. This knowledge has allowed developing new nucleic acids electrotransfer conditions using combinations of permeabilizing pulses of high voltage and short duration, and of electrophoretic pulses of low voltage and long duration, which are very efficient and safer. Feasibility of electric pulses delivery for gene transfer in humans is discussed taking into account that electric pulses delivery is already regularly used for localized drug delivery in the treatment of cutaneous and subcutaneous solid tumors by electrochemotherapy. Because recent technological developments made DNA electrotransfer more and more efficient and safer, this non-viral gene therapy approach is now ready to reach the clinical stage. A good understanding of DNA electrotransfer principles and the respect of safe procedures will be key elements for a successful future transfer DNA electrotransfer into the clinics.     

Chromatographic Separation, and Characteristics of Nucleic Acids from HeLa Cells

The application of the phenol-duponol method to extraction of nucleic acids from HeLa cells is described. Chromatography of the phenol extract on an esterified bovine serum albumin column with a salt gradient of sodium chloride gives separation of soluble RNA, DNA, and two different high molecular RNA fractions. Ultracentrifugation of the DNA eluted from the column gives a sedimentation coefficient (s₂₀^o,w) of 38, which agrees with ultracentrifugation data on the phenol extract. The eluted RNA appears polydisperse at low ionic strength, but at high ionic strength and after alcohol precipitation two fractions with the sedimentation coefficients of 16 and 25 to 29, respectively, were obtained.     

xylo-Configured Oligonucleotides (XNA,Xylo Nucleic Acids): Synthesis and Hybridization Studies

Abstract

We report synthesis and high-affinity hybridization of fully modified home-thymine 2′-deoxy and 2′-deoxy-2′-fluoro xylo nucleic acids.     

The Uptake and Utilization of Bacteria, Amino Acids and Nucleic Acid Components by the Rumen Ciliate Eudiplodinium maggii

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii grown in vitro and incubated anaerobically engulfed all the bacteria tested except for Bacteroides ruminicola and Klebsiella aerogenes. There was considerable variation (160–9100 bacteria/h/protozoon at an external concentration of 10¹⁰ bacteria/ml) in the rate at which the bacteria were engulfed, but Eu. maggii showed some preference for bacteria of rumen origin. Some of the bacteria were digested with the release of soluble materials into the medium. Free amino acids were incorporated from an 0.1 mM solution at rates of 0.13 to 0.84 pmol/h/protozoon. Evidence is presented that Eu. maggii could obtain half the amino acids required for growth by the engulfment and digestion of bacteria and half by the uptake of free amino acids. Eudiplodinium maggii incorporated uridine 5' monophosphate and also hydrolysed this to uridine and then to uracil which was reduced to dihydrouracil. These products all appeared in the medium. Ribose was incorporated by the protozoon and appeared as glucose in protozoal and bacterial polysaccharide; none was incorporated as such into protozoal nucleic acid.     

The Effects of Borate Minerals on the Synthesis of Nucleic Acid Bases,Amino Acids and Biogenic Carboxylic Acids from Formamide

The thermal condensation of formamide in the presence of mineral borates is reported. The products afforded are precursors of nucleic acids, amino acids derivatives and carboxylic acids. The efficiency and the selectivity of the reaction was studied in relation to the elemental composition of the 18 minerals analyzed. The possibility of synthesizing at the same time building blocks of both genetic and metabolic apparatuses, along with the production of amino acids, highlights the interest of the formamide/borate system in prebiotic chemistry.     

Meeting report: Fourth international meeting on G-quadruplex Nucleic Acids (Singapore,July 1–4, 2013)

The fourth international meeting on G-quadruplex Nucleic Acids was held in the Nanyang Technological University (NTU) of Singapore. Over 150 participants gathered from more than 25 countries. Over 40 talks and 100 posters summarized our current knowledge of these unusual DNA and RNA structures.     

Formation of Short-Chain Fatty Acids from H(2) and CO(2) by a Mixed Culture of Bacteria

The biological utilization of CO(2) and H(2) for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37 degrees C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Nitric Oxide (NO)-Induced Death of Gram-Negative Bacteria from a Light-Controlled NO-Releasing Platform

A NO-delivery platform has been fabricated from polydimethylsiloxane (PDMS) and Pluronic(?) F127 gel that contains the light-sensitive NO donor, [Mn(PaPy(3) )(NO)]ClO(4) . The material was assembled layer-by-layer. First, a thin PDMS membrane was cast. It was then layered with cold 25% (w/v) Pluronic(?) F127 gel mixed with [Mn(PaPy(3) )(NO)]ClO(4) . Finally, it was covered with a thick layer (nearly impermeable to NO) of PDMS (=polydimethoxysiloxane) to allow release of NO only from the thinner side upon exposure to light. Light-induced NO release from this layered material has been confirmed via NO-specific electrode and by a modified soft Griess-agar assay. Incorporation of ca. 8?mg/g of [Mn(PaPy(3) )(NO)]ClO(4) in the Pluronic gel layer affords a material that drastically reduces the microbial loads of Acinetobacter baumannii and Pseudomonas aeruginosa via the antibiotic effects of the photoreleased NO. Application of this flexible layered NO-donating composite as bandage material has been proposed.     

Changes of Nucleic Acids in the Preparation Process of Cell Protein Isolates from Yeast (Saccharomyces cerevisiae)

Cell protein isolates were prepared from yeast (S. cerevisiae) by alkali-extraction followed by acid precipitation. The relationships between alkali-treatment and nucleic acid contents in cell protein isolates were examined.

The isolate which was precipitated at pH 4.5 following extraction with 0.20 n NaOH at 80°C contained small amounts (less than 1 % of the isolate) of nucleic acids. However, the content of nucleotides in the isolate which was precipitated at pH 4.5 following extraction with 0.20 n NaOH at 37°C was 9.13% of the product. Treatment by washing or dialysis of the isolate had little effect in removing the nucleotides in the isolate.

This finding was explained by the interaction of nucleotide to cell protein isolate. The binding energy was measured by Hummel’s method.     

Effect of Feeding Millet (Sorghum vulgarie) Protein on Growth,Nucleic Acids and Proteins of Liver and Plasma of Rats

Effect of feeding millet (Sorghum vulgarie) at 5, 10 and 15 per cent protein levels respectively for a period of six weeks to rats on their liver DNA, RNA and proteins of liver, its subcellular fractions and plasma has been studied, and results compared with rats fed casein at 10 per cent level. Both liver DNA and RNA of rats fed millet at 5 per cent protein level were significantly increased. Liver proteins (mg/l00 g body weight) of rats fed millet at 5 and 10 per cent protein level were significantly increased and plasma proteins decreased. Incorporation of leucine-I-¹⁴C into both liver and plasma proteins of rats fed millet was significantly higher than the control.     

Biotransformations of Bile Acids with Bacteria from Cayambe Slaughterhouse (Ecuador): Synthesis of Bendigoles

The biotransformations of cholic acid ( 1a ), deoxycholic acid ( 1b ), and hyodeoxycholic acid ( 1c ) to bendigoles and other metabolites with bacteria isolated from the rural slaughterhouse of Cayambe (Pichincha Province, Ecuador) were reported. The more active strains were characterized, and belong to the genera Pseudomonas and Rhodococcus. Various biotransformation products were obtained depending on bacteria and substrates. Cholic acid ( 1a ) afforded the 3‐oxo and 3‐oxo‐4‐ene derivatives 2a and 3a (45% and 45%, resp.) with P. mendocina ECS10, 3,12‐dioxo‐4‐ene derivative 4a (60%) with Rh. erythropolis ECS25, and 9,10‐secosteroid 6 (15%) with Rh. erythropolis ECS12. Bendigole F ( 5a ) was obtained in 20% with P. fragi ECS22. Deoxycholic acid ( 1b ) gave 3‐oxo derivative 2b with P. prosekii ECS1 and Rh. erythropolis ECS25 (20% and 61%, resp.), while 3‐oxo‐4‐ene derivative 3b was obtained with P. prosekii ECS1 and P. mendocina ECS10 (22% and 95%, resp.). Moreover, P. fragi ECS9 afforded bendigole A ( 8b ; 80%). Finally, P. mendocina ECS10 biotransformed hyodeoxycholic acid ( 1c ) to 3‐oxo derivative 2c (50%) and Rh. erythropolis ECS12 to 6α‐hydroxy‐3‐oxo‐23,24‐dinor‐5β‐cholan‐22‐oic acid ( 9c , 66%). Bendigole G ( 5c ; 13%) with P. prosekii ECS1 and bendigole H ( 8c ) with P. prosekii ECS1 and Rh. erythropolis ECS12 (20% and 16%, resp.) were obtained.     

顽拗性黄皮胚脱水过程中核酸、蛋白质代谢的变化

黄皮种子发育晚期,胚内核酸、蛋白质合成能力增强,而花生胚的核酸、蛋白质合成能力在发育晚期则呈下降趋势。黄皮胚的发育在达到生理成熟后维持着活跃的生理代谢并转入萌发状态;而花生胚的代谢活性逐步降低并转入生理静止状态。脱水处理引起生理成熟期黄皮胚核酸、蛋白质合成能力急剧下降,核酸水解酶活性增强。不同程度脱水的黄皮胚吸胀２４ｈ,核酸、蛋白质的合成能力随脱水程度的加深而降低;生物大分子代谢能力的变化是顽拗性