Adenine Content Research Articles

Analysis of the nucleotide sequence of a cryptic plasmid from Yersinia pestis strains in the Central-Caucasian high-mountain plague focus

An analysis of a 5.4-kbp cryptic plasmid detected in the course of whole-genome sequencing of the Yersinia pestis medieval biovar isolated in the Russian Central Caucasian high-mountain plague focus was performed. The identification of the nucleotide sequence of this cryptic plasmid and its structural and functional analysis revealed that it contained eight open reading frames, among which the following genes were identified: the rep gene of a replication protein, the virB6 gene of a type-IV secretion system inner membrane protein, the virB5gene of the type-IV secretion system minor pilin, and a number of genes probably associated with secretion and transport. A general analysis of the pCKF plasmid DNA showed that the adenine content was 28.34%, the cytosine content was 20.5%, the guanine content was 17.87%, and that of thymine was 33.28%, while the total G+C content appeared to be 38.38%. The G+C content of the chromosome of the Y pestis strain C-627 is 47.6%, which indicates that the pCKF plasmid was obtained from a microorganism equally-phylogenetically distant from the Yersinia bacteria andany other bacteria from the Enterobacteriaceae family. A comparison of the amino acid sequences.of hypothetical proteins encoded by pCKF plasmid with analogous proteins encoded by other bacteria was carried out. The possible contribution of the pCKF plasmid to the maintenance of the most ancient known phylogenetic line of Y. pestis medieval biovar, 2.MEDO, was discussed.

Influence of medium and elicitors on the production of cocaine, amino acids and phytohormones by Erythroxylum coca calli

Erythroxylum coca (Erythroxylaceae) is the source of the tropane alkaloid cocaine. Several lines of evidence suggest that tropane alkaloid biosynthesis in E. coca differs from that in solanaceous species, but there are many gaps in our understanding of the pathways in both groups. The development of an E. coca cell culture that produces cocaine could provide a reproducible model system for discovering novel biosynthetic genes and study pathway regulation. Calli cultures were successfully established from young leaf explants on three different media: Anderson’s Rhododendron, Gamborg B5, and modified Murashige-Tucker, all supplemented with growth regulators: 2,4-D (0.6 mg L−1), indole butyric acid (0.06 mg L−1), and benzylaminopurine (0.5 mg L−1). All accumulated cocaine and cinnamoylcocaine at levels of 0.05–0.5 nmol per gram dry weight, as determined by LC–MS, several orders of magnitude below the concentration found in the intact plant. Anderson’s Rhododendron medium supported the highest level of tropane alkaloid production, as well as the highest level of the amino acids arginine, glutamate, proline and phenylalanine, all thought to be precursors of cocaine, but contained generally lower levels of hydroxycinnamate-quinate esters, such as chlorogenic acid. These differences may be ascribed to its relatively low content of nitrate or salts, or its high content of adenine. Addition of 100 μM salicylic acid or coronalon, an analog of the bioactive jasmonic acid-isoleucine conjugate, did not result in any increase in tropane alkaloid production. These E. coca calli could provide valuable material for studies on tropane alkaloid biosynthesis and regulation.

Computational analysis of amino acids and their sidechain analogs in crowded solutions of RNA nucleobases with implications for the mRNA-protein complementarity hypothesis.

Many critical processes in the cell involve direct binding between RNAs and proteins, making it imperative to fully understand the physicochemical principles behind such interactions at the atomistic level. Here, we use molecular dynamics simulations and 15 μs of sampling to study the behavior of amino acids and amino acid sidechain analogs in high-concentration aqueous solutions of standard RNA nucleobases. Structural and energetic analysis of simulated systems allows us to derive interaction propensity scales for different amino acid/nucleobase combinations. The derived scales closely match and greatly extend the available experimental data, providing a comprehensive foundation for studying RNA–protein interactions in different contexts. By using these scales, we demonstrate a statistically significant connection between nucleobase composition of human mRNA coding sequences and nucleobase interaction propensities of their cognate protein sequences. For example, pyrimidine density profiles of mRNAs match uracil-propensity profiles of their cognate proteins with a median Pearson correlation coefficient of R = −0.70. Our results provide support for the recently proposed hypotheses that mRNAs and their cognate proteins may be physicochemically complementary to each other and bind, especially if unstructured, with the complementarity level being negatively influenced by mRNA adenine content. Finally, we utilize the derived scales to refine the complementarity hypothesis and closely examine its physicochemical underpinnings.

Simultaneous determination of Gastrodin and eight nucleosides and nucleobases in Tibet cultured Gastrodia elata by HPLC method

This study aims to establish an HPLC method for simultaneous determination of gastrodin and eight nucleosides and nucleobases components in Gastrodia elata. The separation was carried out on an Agilent Zorbax Bonus-RP (4.6 mm x 250 mm, 5 μm) column with a methanol-(0.04% acetic acid) water solution gradient elution program at a flow rate of 1.0 mL x min(-1). The column temperature was 36 degrees C, and the detection wavelength was 254 nm. The volume of injection was 20 μL. The nine components including gastrodin, cytosine, uracil, cytosine, adenine, thymine, uridine, guanosine and adenosine were well separated. The calibration curve was well linear in the range of 2.04-262.00 mg x L(-1), 0.20-24.67 mg x L(-1), 0.18-23.75 mg x L(-1), 0.20-25.83 mg x L(-1), 0.20-26.67 mg x L(-1), 0.16-20.00 mg x L(-1), 0.22-27.71 mg x L(-1), 0.20-24.29 mg x L(-1), 0.24-30.58 mg x L(-1), respectively, and the correlation coefficient was between 0.998 9-0.999 9. The average recovery of gastrodin and eight nucleosides and nucleobases were 96.4%-99.6%, RSD less than 2.7% (n = 6). The contents of gastrodin in all the seven Tibet cultured Gastrodia elata samples were over 2 mg x g(-1). Further, all samples contain higher contents of adenosine, guanosine, uridine and cytidine compared to low contents of cytosine, uracil, adenine and thymine. The established method is accurate, reproducible and suitable for the determination of gastrodin and eight nucleosides and nucleobases comppnents in Gastrodia elata.

Modification-free electrochemical approach for sensitive monitoring of purine DNA bases: Simultaneous determination of guanine and adenine in biological samples using boron-doped diamond electrode

A simple and modification-free electrochemical sensor for sensitive determination of purine DNA bases based on the use of bare and electrochemically un-pretreated boron-doped diamond electrodes is proposed. Cyclic voltammetry showed that guanine and adenine provided well defined irreversible oxidation peaks at +1.15 and +1.35V vs. Ag/AgCl/3M KCl, respectively, in Britton–Robinson buffer solution at pH 6 with sufficient potential separation. Both analytes provided a linear relation between peak current and concentration in the range from 0.3 to 19μM when both were present in the solution. For individual analytes in the absence of the other, the corresponding ranges for guanine and adenine were from 0.21 to 23μM and from 0.12 to 25μM, respectively. The limits of detection for guanine and adenine were 158 and 67nM, respectively, for simultaneous determinations in differential pulse mode, and 37 and 19nM, respectively, for individual quantifications. The proposed method was successfully applied for the assessment of guanine and adenine content in spiked human urine samples with satisfactory results. The analysis of native, thermally and acid denatured DNA samples from fish sperm and acid denatured DNA samples from human placenta yielded two oxidation peaks corresponding to guanine and adenine residues with (G+C)/(A+T) values close to those reported in literature. In this way, boron-doped diamond represents a modification-free electrochemical alternative to widely used modified electrodes for sensitive monitoring of purine DNA bases.

秦岭细鳞鲑(Brachymystax lenok tsinlingensis)群体遗传结构分析

PDF HTML阅读 XML下载 导出引用 引用提醒 秦岭细鳞鲑群体遗传结构 DOI: 10.5846/stxb201301050033 作者: 作者单位: 甘肃省水产研究所/甘肃省冷水性鱼类种质资源与遗传育种重点实验室 兰州 730030,甘肃省水产研究所/甘肃省冷水性鱼类种质资源与遗传育种重点实验室 兰州 730030,甘肃省水产研究所/甘肃省冷水性鱼类种质资源与遗传育种重点实验室 兰州 730030,甘肃省水产研究所/甘肃省冷水性鱼类种质资源与遗传育种重点实验室 兰州 730030,甘肃省水产研究所/甘肃省冷水性鱼类种质资源与遗传育种重点实验室 兰州 730030,甘肃省水产研究所/甘肃省冷水性鱼类种质资源与遗传育种重点实验室 兰州 730030 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金（31160529）；甘肃省自然科学基金（1208RJYA033） Population genetic structure of Brachymystax lenok tsinlingensis as inferred from mtDNA D-loop sequence Author: Affiliation: Gansu Key Laboratory of Cold Water Fishes Germplasm Resources and Genetics Breeding/Gansu Fishers Research Institute,Gansu Key Laboratory of Cold Water Fishes Germplasm Resources and Genetics Breeding/Gansu Fishers Research Institute,Gansu Key Laboratory of Cold Water Fishes Germplasm Resources and Genetics Breeding/Gansu Fishers Research Institute,Gansu Key Laboratory of Cold Water Fishes Germplasm Resources and Genetics Breeding/Gansu Fishers Research Institute,Gansu Key Laboratory of Cold Water Fishes Germplasm Resources and Genetics Breeding/Gansu Fishers Research Institute,Gansu Key Laboratory of Cold Water Fishes Germplasm Resources and Genetics Breeding/Gansu Fishers Research Institute Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:秦岭细鳞鲑（Brachymystax lenok tsinlingensis）是秦岭地区特有鱼类，近年来由于环境恶化及人类活动的加剧，已对其造成严重影响，种群处于濒危状态，因此研究秦岭细鳞鲑的群体遗传结构、演化历史、分布动态等对其进行有效保护具有重要意义。本研究用线粒体D-loop区序列对秦岭地区6个群体（n =112）进行了遗传结构和群体演化分析。D-loop区扩增出的891bp 序列在112个个体中，检测到42个变异位点，共26个单倍型；碱基序列总的单倍型多样度较高为0.883，核苷酸多样度为0.00799。AMOVA分析显示，60.05%的分子差异位于群体内，39.95%的分子差异位于群体间，Fst值统计检验表明，除那布大河群体与漳河群体和千河群体之间差异不显著之外，其余两两群体之间Fst值统计检验均为显著。系统树和单倍型网络图分析表明，6 个地理群体的单倍型按照渭河上游和渭河中游两个河段形成两个大的类群，且5个群体共享一个单倍型H2，表明这些群体具有相同的演化历史，为同一个祖先群体演化而来。中性检验和歧点分布显示，秦岭细鳞鲑种群大小保持相对稳定， 未经历明显的种群扩张。同时建议将渭河上游秦岭细鳞鲑群体作为一个整体进行重点保护。 Abstract:Brachymystax lenok tsinlingensis is one of an endemic fishes distributing in Qinling mountains scatteredly. Due to environmental depravation and overfishing, its population size was sharply decreased. To establish a scientific basis to conserve this species, studies on the population genetic structure and phylogeography are necessary. In this study, the population genetic variations and phylogeographical patterns of 112 Brachymystax lenok tsinlingensis collected from six sites of the Wei River basin were investigated. An 891bp control region partial sequence of mtDNA was amplified in each sample, and 42 variable sites were detected, including 39 parsim-info sites and 3 singleton sites, with 26 haplotypes. The contents of cytosine, thymine, adenine and guanine were 23.1%, 30.8%, 31.7%, and 14.5%, respectively. Total haplotype diversity and nucleotide diversity were 0.883 and 0.00799, respectively. The genetic diversity of the Malu River population was the highest among these six populations, while the population of Zhang River was the lowest with only one haplotype H2. Of the 26 haplotypes, H2 was detected in 35 samples (31%) from 5 populations except the Malu River population. The result of AMOVA showed that 39.95% molecular variation was among populations and 60.05% within populations. The pairwise fixation index (Fst) revealed significant differences except between the Zhang River population and the Nabuda River population. Phylogenetic tree with Hucho taimen as outgroup showed that three geographic populations from the middle reach of Wei River including 16 haplotypes formed a big branch, and the rest haplotypes formed several small branches. The minimum spanning network for Brachymystax lenok tsinlingensis showed that the haplotypes of six geographical populations formed two big groups: the upper reach group and the middle reach group of the Wei River. The haplotype H2 was inner branch haplotype, it tend to be more widely as the ancestors haplotype, thus Brachymystax lenok tsinlingensis may have originated in the haplotype H2. Mismatch distribution analysis map showed a peak type, and Tajima's D value (-0.18715) of neutrality test is negative, but no significant difference, which indicates that Brachymystax lenok tsinlingensis in this research hadn't experience population expansion. Maybe due to climate change, the remaining individuals do not adapt to the living environment, the population don't carry out expansion. The haplotype H2 derives to the other haplotype in the different environmental, Wei River was cut off by a large number of water-shore projects, the migratory route is blocked, it was distributed with dots along the Wei River basin, lacked gene exchange between among populations. Due to the low level of genetic variation of three populations in the Wei River upper reach, more attentions should be paid to protect Brachymystax lenok tsinlingensis as a whole population. 参考文献 相似文献 引证文献

Arxula adeninivoransrecombinant adenine deaminase and its application in the production of food with low purine content

Construction of a transgenic Arxula adeninivorans strain that produces a high concentration of adenine deaminase and investigation into the application of the enzyme in the production of food with low purine content. The A. adeninivorans AADA gene, encoding adenine deaminase, was expressed in this yeast under the control of the strong inducible nitrite reductase promoter using the Xplor(®) 2 transformation/expression platform. The recombinant enzyme was biochemically characterized and was found to have a pH range of 5.5-7.5 and temperature range of 34-46 °C with medium thermostability. A beef broth was treated with the purified enzyme resulting in the concentration of adenine decreasing from 70.4 to 0.4 mg l(-1). It was shown that the production of adenine deaminase by A. adeninivorans can be increased and that the recombinant adenine deaminase can be used to lower the adenine content in the food. Adenine deaminase is one component of an enzymatic system that can reduce the production of uric acid from food constituents. This study gives details on the expression, characterization and application of the enzyme and thus provides evidence that supports the further development of the system.

A novel model of adenine-induced tubulointerstitial nephropathy in mice

BackgroundIn vivo models of uremia are important tools to study numerous aspects of acute and chronic kidney disease. Mouse models are pivotal because most genetically engineered animal models are mice, which allow dissecting the impact of selected target genes in renal failure. Adenine-based protocols to induce renal failure are available in rats, but have not been adapted in mice due to their reluctance to consume adenine. In the current paper we developed a novel method for induction of renal failure through dietary delivery of adenine mixed in a casein-based diet.ResultsAfter an induction phase, a stable model of renal impairment was obtained (target urea range 80–100 mg/dL), mimicking several aspects of chronic kidney disease - mineral and bone disorder including secondary hyperparathyroidism, bone abnormalities and pathological elevation of FGF23. No deaths occurred and the level of uremia was adaptable through adjustments of the adenine content, providing significant advantages compared to existing models. In an 8-week proof-of-concept study, renal histology showed mainly a tubulointerstitial damage with infiltrating leukocytes, interstitial edema and widening of the Bownman's space. Fibrosis was present in most animals as defined by histology and gene expression changes of fibrosis markers. Parathyroid cell proliferation was markedly increased but without signs of glandular hypertrophy. Skeletal histology showed increased trabecular bone and bone marrow adiposity whereas bone biomarkers (CTX and PINP) suggested higher bone formation, but surprisingly, lower bone resorption and perturbations in mineral metabolism.ConclusionsWe present a novel, non-surgical method for induction of renal failure in mice. This is an important complement to existing uremic models for pathophysiological studies in acute and chronic kidney disease, especially in terms of tubulointerstitial lesions.

Analysis of the bioactive components from different growth stages of Fritillaria taipaiensis P. Y. Li

High-performance liquid chromatography (HPLC) coupled with an evaporative light scattering detector (ELSD) or a diode array detector (DAD) were utilized for the quantitative analysis of 4 alkaloids (peimisine, sipeimine, peimine and peiminine) and 9 nucleosides and nucleobases (uracil, uridine, adenosine, adenine, inosine, thymine, cytidine, guanosine and thymidine) from Fritillaria taipaiensis P. Y. Li that had been cultivated in the same field for 2–6 years. The content of peimisine, sipeimine, peimine, peiminine, uracil, thymine, adenine and inosine in plants cultivated for 2–4 years was significantly higher than that of plants cultivated for 5–6 years, while the content of cytidine, uridine, guanosine, thymidine and adenosine did not change over this period. This is the first evaluation of variation in the bioactive compounds in F. taipaiensis over its life cycle.

Enzyme Based Amperometric Biosensor for Adenine Determination

AbstractAn enzyme‐based amperometric biosensor was developed for the rapid determination of adenine in biological and food samples. Recombinant adenine deaminase (EC 3.5.4.2) from Schizosaccharomyces pombe was co‐immobilized on graphite electrode with xanthine oxidase (EC 1.17.3.2), peroxidase (EC 1.11.1.7) and an Os mediator. Optimal working conditions were pH 7.0 and −150 mV vs. Ag/AgCl. The novel method was characterized by a detection limit of 1 µM and a linear measuring range of 0.005–0.1 mM. Different beer and tea samples were analyzed by the newly elaborated adenine biosensor. The results obtained correlate with the adenine content measured by the reference HPLC method.

AT-rich region and repeated sequences – the essential elements of replication origins of bacterial replicons

Repeated sequences are commonly present in the sites for DNA replication initiation in bacterial, archaeal, and eukaryotic replicons. Those motifs are usually the binding places for replication initiation proteins or replication regulatory factors. In prokaryotic replication origins, the most abundant repeated sequences are DnaA boxes which are the binding sites for chromosomal replication initiation protein DnaA, iterons which bind plasmid or phage DNA replication initiators, defined motifs for site-specific DNA methylation, and 13-nucleotide-long motifs of a not too well-characterized function, which are present within a specific region of replication origin containing higher than average content of adenine and thymine residues. In this review, we specify methods allowing identification of a replication origin, basing on the localization of an AT-rich region and the arrangement of the origin's structural elements. We describe the regularity of the position and structure of the AT-rich regions in bacterial chromosomes and plasmids. The importance of 13-nucleotide-long repeats present at the AT-rich region, as well as other motifs overlapping them, was pointed out to be essential for DNA replication initiation including origin opening, helicase loading and replication complex assembly. We also summarize the role of AT-rich region repeated sequences for DNA replication regulation.

Adenine-containing codons enhance protein synthesis by promoting mRNA binding to ribosomal 30S subunits provided that specific tRNAs are not exhausted

Adenines downstream of the initiation codon promote protein synthesis; however, some adenine-containing codons (AGA, AGG and AUA) at early positions inhibit protein synthesis when cognate tRNA is exhausted. It has also been reported, although not convincingly, the presence of adenines enhancing mRNA binding to the ribosome. To understand these apparent inconsistencies we analyzed the effect of these codons in mRNA-ribosome binding strength, mRNA stability, the production of peptidyl-tRNA (pep-tRNA) and protein synthesis. Constructs harboring lacZ derivatives were obtained by site directed mutagenesis where tandems of GGG, AGG, AGA, ATA and AAA codons were inserted at codon positions 2–3 and 3–4. Codons containing more adenines, irrespective of being common or rare, (AAA, ATA and AGA) promoted a higher synthesis of β-galactosidase (β-gal) in comparison with those rich in guanines (GGG and AGG) in a wild type transcription-translation system. Full-length mRNAs were also detected when the adenine-rich constructs were expressed in wild type cells. Under conditions where the pool of tRNAs is readily exhausted (pep-tRNA hydrolase defective cells), the adenine-rich lacZ derivatives caused a stronger and general inhibition of protein synthesis and cell growth. With the exception of the ATA lacZ derivative, only plasmid constructs containing hungry codons generated pep-tRNA (AGA and to a lesser extent AGG) in Pth defective cells. Codons containing more adenines clearly promoted lacZ mRNA binding to 30S subunit. The GGG lacZ mRNA showed a moderate increase in binding when mRNA secondary structures were disrupted by heating mRNAs before the binding assay which agrees with the lacZ mRNA secondary structures predicted with MFOLD. Altogether, these results indicate that mRNA binding to ribosome plays a major role in the enhancement of translation by adenine-rich codons irrespective of codon usage. This effect is naturally expressed in wild type systems and depends on adenine content, in contrast to the inhibition caused after over-expressing the lacZ derivatives containing rare codons in Pth defective cells.

2,8-Dihydroxyadenine Urolithiasis. Case Report and Literature Review

2,8-Dihydroxyadenine (DHA) urolithiasis is a rare type of urinary stone disease secondary to deficiency of adenine phosphoribosyltransferase (APRT) activity, a rare, inherited autosomal recessive disease with an incidental rate from 0.4 to 1.2%. The prevalence is higher among Japanese than other ethnic groups. APRT normally catalyzes the conversion of adenine to adenosine monophosphate and its deficiency results in 2,8-dihydroxyadenine (2,8-DHA) accumulation. This compound is extremely insoluble and its crystallization can lead to stone formation and renal failure. We report the case of 2,8-dihydroxyadenine (DHA) urolithiasis in a 52-year-old male patient. In December 2008 a 52-year-old Caucasian man was admitted to our hospital with sudden pain in the left lumbar region. Abdominal X-ray did not show any radiopaque urinary stone. I.V. pielography showed a radiolucent left lumbar ureteral (0.6 mm) and renal (1.5 cm) stone. After therapy with tamsulosin, the ureteral stone was excreted. Successful ESWL treatment was performed for renal stone. He presented a clinical history of several episodes of bilateral renal colic and two prior ESWL treatment for radiolucent stones. Chemolitholysis was never successful. RESULTS. Stone analysis by infrared spectroscopy and microscopic examination of urine reveal typical 2,8-DHA crystals. APRT deficiency was detected in the hemolysate of erythrocyte. Partial deficiency of APRT in the patient's relatives showed heterozygosity of the enzyme defect. Allopurinol therapy successfully prevented further stone formation. 20 months later the patient remains stone free. Two types of deficit are commonly distinguished, depending on the level of residual APRT activity. Type I is complete enzyme deficiency. Type II shows residual activity in cell lysates, but enzyme activity is not demonstrable in intact cells. About 78% of the Japanese patients belong to type II. The diagnosis of the disease is based on stone analysis by infrared spectroscopy or microscopic examination of urine, which may reveal typical 2,8-DHA crystals. Molecular approach can identify mutations, which are responsible of this inherited disease. Excessive water intake, restriction of foods with high adenine contents and administration of allopurinol are useful treatments. APRT deficiency is a rare disease but we can consider this pathology in case of recurrent radiolucent stones after chemolitolysis.

Adenine in Viral mRNAs Manipulate the Carbon in Proteins

Protein s function is determined by the nature of carbon along its sequence. This carbon along the protein is determined by the nucleotides in the gene. The adenine in viral genetic material and carbon distribution along its proteins are the focus of this study. The results reveal that the presence of higher adenine content in viral material add adequate number of large number of hydrophobic residues in its proteins. The natural way of adding different sequences during development and evolution is better understood based on this carbon distribution analysis. Appropriate mutation that changes in carbon content and distribution in viral proteins might improve the functionality of the protein.

Using principal component analysis to find correlations between loop-related and thermodynamic variables for G-quadruplex-forming sequences

The application of Principal Component Analysis (PCA) is proposed here as a simple means of revealing correlations between thermodynamic variables corresponding to folding equilibria of intramolecular G-quadruplexes and Watson–Crick duplexes, and the length of loops in the corresponding guanine-rich DNA sequences. To this end, two previously studied data sets were analyzed (Arora and Maiti, J . Phys. Chem. B. 2009 and Kumar and Maiti, Nucleic Acids. Res. 2008). All of the sequences considered shared the common structure 5’- GGG - loop1 - GGG - loop2 - GGG - loop3 - GGG -3’. PCA of these data sets supported a series of correlations between the variables studied. First, the association of loop length with thermodynamic stability and quadruplex structure was corroborated. Secondly, it is proposed that the addition of ethylene glycol produces a stronger stabilization on those sequences showing long loop1 and/or loop3. Thirdly, it is proposed that a low content of adenine in loop1 and/or loop3 will produce an increase in the stability of G-quadruplex and its related Watson–Crick duplex.

Effects of SAG12-ipt expression on cytokinin production, growth and senescence of creeping bentgrass (Agrostis stolonifera L.) under heat stress

The study was conducted to determine the effects of expression of a transgene encoding adenine isopentenyl transferase (ipt), which controls cytokinin synthesis, on growth and leaf senescence of creeping bentgrass (Agrostis stolonifera L.), subjected to heat stress. Creeping bentgrass (cv. Penncross) was transformed with ipt ligated to a senescence-activated promoter (SAG12). Eight SAG12-ipt transgenic lines exhibiting desirable turf quality and a transgenic control line (transformed with the empty vector) were evaluated for morphological and physiological changes under normal growth temperature (20°C) and after 14 days of heat stress (35°C) in growth chambers. Six of the SAG12-ipt lines developed more tillers than the control line during establishment under normal growth temperature of 20°C. Following 14 days of heat stress, four of the SAG12-ipt lines had increased 65–83% of roots and for all six SAG12-ipt lines root elongation continued, whereas root production ceased and total root length decreased for the control line. Root isopentenyl adenine (iPA) content increased 2.5–3.5 times in five of the SAG12-ipt lines, whereas in the control line iPA decreased 20% after 14 days at 35°C. Total zeatin riboside (ZR) content was maintained at the original level or increased in five of the SAG12-ipt lines, whereas in the control line ZR decreased under heat stress. Our results suggest expression of SAG12-ipt in creeping bentgrass stimulated tiller formation and root production, and delayed leaf senescence under heat stress, suggesting a role for cytokinins in regulating cool-season grass tolerance to heat stress.

Human Immunodeficiency Virus (HIV) Type 1 Proviral Hypermutation Correlates with CD4 Count in HIV-Infected Women from Kenya

APOBEC3G is an important innate immune molecule that causes human immunodeficiency virus type 1 (HIV-1) hypermutation, which can result in detrimental viral genome mutations. The Vif protein of wild-type HIV-1 counteracts APOBEC3G activity by targeting it for degradation and inhibiting its incorporation into viral particles. Additional APOBEC cytidine deaminases have been identified, such as APOBEC3F, which has a similar mode of action but different sequence specificity. A relationship between APOBEC3F/G and HIV disease progression has been proposed. During HIV-1 sequence analysis of the vpu/env region of 240 HIV-infected subjects from Nairobi, Kenya, 13 drastically hypermutated proviral sequences were identified. Sequences derived from plasma virus, however, lacked hypermutation, as did proviral vif. When correlates of disease progression were examined, subjects with hypermutated provirus were found to have significantly higher CD4 counts than the other subjects. Furthermore, hypermutation as estimated by elevated adenine content positively correlated with CD4 count for all 240 study subjects. The sequence context of the observed hypermutation was statistically associated with APOBEC3F/G activity. In contrast to previous studies, this study demonstrates that higher CD4 counts correlate with increased hypermutation in the absence of obvious mutations in the APOBEC inhibiting Vif protein. This strongly suggests that host factors, such as APOBEC3F/G, are playing a protective role in these patients, modulating viral hypermutation and host disease progression. These findings support the potential of targeting APOBEC3F/G for therapeutic purposes.

DNA Hydrolysis and Voltammetric Determination of Guanine and Adenine Using Different Electrodes

This work reports the development of a biosensor method for the label‐free detection of specific DNA sequences. In the initial phase, square wave voltammetry (SWV) was used in a comparative investigation into the electrochemical oxidation of purines (guanine and adenine) and DNA fragments at various electrode surfaces: carbon paste (CPE), glassy carbon electrode (GCE), and gold (AuE). Relative to the carbon electrodes, an approximate 4.0‐fold, 6.0‐fold, and 3.25‐fold increase in the anodic response was observed when guanine, adenine, and hydrolyzed DNA, respectively, were measured on the AuE. It was shown that the guanine and adenine bases could be successfully determined by use of SWV for a deoxyribonucleic acid sample following acid hydrolysis. This label‐free detection of hydrolyzed DNA on gold electrodes has significant advantages over methods using existing carbon electrode materials because of its higher sensitivity and the potential applicability of microfabrication techniques for the production of the requisite gold electrodes. In another phase of development, the times and conditions for DNA hydrolysis and purine release were investigated. It was shown that under optimal conditions, trace levels of the purine bases could be readily detected following 20 min of hydrolysis at room temperature. The proposed method can be used to estimate the guanine and adenine contents in DNA with in a linear range of 5–30 ng ml−1. Finally, when appropriate probe sequences were first adsorbed on the surface of the screen‐printed gold electrode (SPGE), this electrochemical biosensor could be used to specifically detect sequences from ss corona virus aviair following hybridization and hydrolysis reactions on the sensor surface. No enhancement of the voltammetric response was observed when the sensor was challenged with a non‐complementary DNA sequence.

Evaluation of preservation solutions by ESR-spectroscopy: Superior effects of University of Wisconsin over Histidine–Tryptophan–Ketoglutarate in reducing renal reactive oxygen species

Despite the causative role of oxidative stress in renal ischemia-reperfusion (I-R) injury effects of preservation solutions on reactive oxygen species (ROS) release have not been sufficiently evaluated. We compared the effects of most common solutions in kidney transplantation, University of Wisconsin (UW) and Histidine-Tryptophan-Ketoglutarate (HTK). ROS formation in isolated perfused rat kidney was detected by electron spin resonance spectroscopy using spin label 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Donor kidneys from Lewis rats were pretreated with saline (controls), in therapeutic groups, kidneys underwent 18 h of cold storage (CS) preserved by HTK or UW solution. Experimental protocol included a stabilization period followed by additional I-R. Kidneys preserved by HTK produced highest ROS values in the control period after CS, whereas levels in UW and control group did not vary significantly. A peak release induced by additional I-R was also significantly highest in HTK kidneys, and UW did not differ from controls. During reperfusion, levels in HTK exceeded control and UW values. Renal vascular resistance, caspase-3-activity, and tissue hydration were enhanced in HTK compared with UW group, whereas ATP concentration was less reduced in UW-preserved tissue. These data show the greater antioxidative potential of UW solution, which also attenuated organ impairment after CS in the early reperfusion period.

Physiological analysis of Methylobacterium extorquens AM1 grown in continuous and batch cultures

Chemostat cultures of Methylobacterium extorquens AM1 grown on methanol or succinate at a range of dilution rates were compared to batch cultures in terms of enzyme levels, poly-beta-hydroxybutyrate content, and intracellular concentrations of adenine and pyridine nucleotides. In both chemostat and batch cultures, enzymes specific to C1 metabolism were up-regulated during growth on methanol and down-regulated during growth on succinate, polyhydroxybutyrate levels were higher on succinate, intracellular ATP levels and the energy charge were higher during growth on methanol, while the pools of reducing equivalents were higher during growth on succinate. For most of the tested parameters, little alteration occurred in response to growth rate. Overall, we conclude that the chemostat cultivation conditions developed in this study roughly mimic the growth in batch cultures, but provide a better control over the culturing conditions and a better data reproducibility, which are important for integrative functional studies. This study provides baseline data for future work using chemostat cultures, defining key similarities and differences in the physiology compared to existing batch culture data.