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      Volume 39, Issue 5

      December 2014,   pages  739-944

    • Clipboard: Restraining the enhancers from straying

      Rakesh K Mishra

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    • What history tells us XXXV. Enhancers: Their existence and characteristics have raised puzzling issues since their discovery

      Michel Morange

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    • Phosphorylation of DegU is essential for activation of amyE expression in Bacillus subtilis

      Monica Gupta K Krishnamurthy Rao

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      Alpha (𝛼)-amylase (amyE) is one of the major exo-enzymes secreted by Bacillus subtilis during the post-exponential phase. The DegS-DegU two-component system regulates expression of majority of post-exponentially expressed genes in B. subtilis. It has been demonstrated that varying levels of the phosphorylated form of DegU (DegU-P) control different cellular processes. Exo-protease production is observed when effective concentration of DegU-P rises in the cell, whereas swarming motility is favoured at very low amounts of DegU-P. In this study we show that like other exo-proteases, expression of amyE is positively regulated by increase in DegU-P levels in the cell. We also demonstrate that residues at the DNA-binding helix-turn-helix (HTH) motif of DegU are necessary for the amyE expression. This observation is further reinforced by demonstrating the direct interaction of DegU on amyE promoter.

    • Variation in ion leakage parameters of two wheat genotypes with different Rht-B1 alleles in response to drought

      Konstantina V Kocheva Svetlana P Landjeva Georgi I Georgiev

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      The reaction to soil drying was evaluated in two Triticum aestivum near-isogenic lines carrying different alleles of the height-reducing gene Rht-B1 based on an improved method for assessment of electrolyte leakage. The two lines were previously shown to differ in their physiological responses to induced water deficit stress. Drought was imposed for 6 days on 10-day-old seedlings. Ion efflux from leaves was measured conductometrically in multiple time points during the 24 h incubation period, and the obtained biphasic kinetics was interpreted according to a previously developed theoretical model proposing different leakage rates through the apoplast and the symplast. Most of the model parameters were able to properly differentiate the two closely related genotypes. The mutant Rht-B1c displayed lower and slower electrolyte leakage in comparison with the wild-type Rht-B1a. It was speculated that the Rht genes expressing defective DELLA proteins might be involved in water stress response through modulation of cell wall stiffness, which influences its capacity for ions retention, and also by their contribution to ROS detoxification, thus indirectly stabilizing cellular membranes. The presented analytical approach relating processes of ion and water flow in and out of the cell could be used for characterization of membrane and cell wall properties of different genotypes under normal and stress conditions.

    • Quantum chemical investigations of AlN-doped C60 for use as a nano-biosensor in detection of mispairing between DNA bases

      Shamoon Ahmad Siddiqui Nadir Bouarissa Tabish Rasheed A Al-Hajry

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      Quantum chemical calculations were carried out to study the electronic structure and stability of adenine–thymine and the rare tautomer of adenine–thymine base pairs along with their Cu2+ complexes and their interactions with AlN-modified fullerene (C58AlN) using Density Functional Theory (B3LYP method). Since, these two forms of base pairs and their Cu2+ complexes have almost similar electronic structures, their chemical differentiation is an extremely difficult task. In this investigation, we have observed that AlN-doped C60 could be used as a potentially viable nanoscale sensor to detect these two base pairs as well as their Cu2+ complexes.

    • Identification and in silico analysis of helical lipid binding regions in proteins belonging to the amphitropic protein family

      Rob C A Keller

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      The role of protein–lipid interactions is increasingly recognized to be of importance in numerous biological processes. Bioinformatics is being increasingly used as a helpful tool in studying protein–lipid interactions. Especially recently developed approaches recognizing lipid binding regions in proteins can be implemented. In this study one of those bioinformatics approaches specialized in identifying lipid binding helical regions in proteins is expanded. The approach is explored further by features which can be easily obtained manually. Some interesting examples of members of the amphitropic protein family have been investigated in order to demonstrate the additional features of this bioinformatics approach. The results in this study seem to indicate interesting characteristics of amphitropic proteins and provide insight into the mechanistic functioning and overall understanding of this intriguing class of proteins. Additionally, the results demonstrate that the presented bioinformatics approach might be either an interesting starting point in protein–lipid interactions studies or a good tool for selecting new focus points for more detailed experimental research of proteins with known overall protein–lipid binding abilities.

    • Immobilization and characterization of inulinase from Ulocladium atrum on nonwoven fabrics

      Tarek M Mohamed Soad M Abu El-Souod Ehab M Ali Mohammed O El-Badry Mai M El-Keiy Aly Sayed Aly

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      Ulocladium atrum inulinase was immobilized on different composite membranes composed of chitosan/nonwoven fabrics. Km values of free and immobilized U. atrum inulinase on different composite membranes were calculated. The enzyme had optimum pH at 5.6 for free and immobilized U. atrum inulinase on polyester nonwoven fabric coated with 3% chitosan solution (PPNWF3), but optimum pH was 5 for immobilized U. atrum inulinase on polyester and polypropylene nonwoven fabrics coated with 1% chitosan solution. The enzyme had optimum temperature at 40°C for immobilized enzyme on each of polyester and polypropylene composite membranes coated with 1% chitosan, while it was 50°C for free and immobilized enzyme on polypropylene nonwoven fabric coated with 3% chitosan solution. Free U. atrum inulinase was stable at 40°C but thermal stability of the immobilized enzyme was detected up to 60°C. Reusability of immobilized enzyme was from 38 to 42 cycles of reuse; after this, the immobilized enzyme lost its activity completely. In conclusion, immobilized U. atrum inulinase was considerably more stable than the free enzyme, and could be stored for extended periods.

    • LNA-modified isothermal oligonucleotide microarray for differentiating bacilli of similar origin

      Jing Yan Ying Yuan Runqing Mu Hong Shang Yifu Guan

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      Oligonucleotide microarray has been one of the most powerful tools in the ‘Post-Genome Era’ for its high sensitivity, high throughput and parallel processing capability. To achieve high detection specificity, we fabricated an isothermal microarray using locked nucleic acid (LNA)-modified oligonucleotide probes, since LNA has demonstrated the advanced ability to enhance the binding affinity toward their complementary nucleotides. After designing the nucleotide sequences of these oligonucleotide probes for gram-positive bacilli of similar origin (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of these oligonucleotide probes by modifying some nucleotides using LNA. Furthermore, we optimized the experimental procedures of hydrating microarray slides, blocking side surface as well as labelling the PCR products. Experimental results revealed that KOD Dash DNA polymerase could efficiently incorporate Cy3-dCTP into the PCR products, and the LNA-isothermal oligonucleotide microarray were able to distinguish the bacilli of similar origin with a high degree of accuracy and specificity under the optimized experimental condition.

    • Isolation of a novel uric-acid-degrading microbe Comamonas sp. BT UA and rapid biosensing of uric acid from extracted uricase enzyme

      Tanushree Ghosh Priyabrata Sarkar

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      Uric-acid-utilizing soil bacteria were isolated, and 16s rRNA sequence was studied for strain identification. The most prominent uricase-producing bacterium was identified as Comamonas sp BT UA. Crude enzyme was extracted, freeze-dried and its Km and Vmax were determined as 40 𝜇M and 0.047 𝜇M min−1ml−1 using Line-weaver Burke plot. An activity of 80 U/mg of total protein was observed when cultured at 37°C for 84 h at pH 7. The purified enzyme was used to measure uric acid by spectrophotometric method and electrochemical biosensor. In the biosensing system the enzyme was immobilized on the platinum electrode with a biodegradable glutaraldehyde-crosslinked gelatin film having a swelling percentage of 109±3.08, and response was observed by amperometry applying fixed potential. The electrochemical process as obtained by the anodic peak current and scan rate relationship was further configured by electrochemical impedance spectroscopy (EIS). The polymer matrix on the working electrode gave capacitive response for the electrode–electrolyte interaction. The sensitivity of the biosensor was measured as 6.93 𝜇A𝜇M−1 with a sensor affinity [𝐾m(app)] of 50 𝜇M and 95% reproducibility after 50 measurements. The spectrophotometric method could be used in the range of 6–1000 𝜇M, whereas the biosensor generated linear response in the 1.5–1000 𝜇M range with a response time of 24 s and limit of detection of 0.56 𝜇M. Uric acid was estimated in human blood samples by the biosensor and satisfactory results were obtained.

    • Modulation of phagolysosome maturation by bacterial tlyA gene product

      Ekansh Mittal Santosh Kumar Aejazur Rahman Musti V Krishnasastry

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      The pathogenic traits of TlyA proteins of Mycobacterium tuberculosis are not known. Expressions of TlyA in bacteria that do not express endogenous TlyA adhere better to RAW264.7 macrophages and get phagocytosed efficiently. The internalized bacteria avoid acidification to the extent of > 65% in the case of both TlyA-expressing E. coli and M. smegmatis. Consistent with this observation, we have observed decreased co-localizaton of Lysosomal Membrane Associated Protein-1 (∼35%), Early Endosomal Antigen-1 (∼34%), Rab5 (∼30%) and Rab7 (∼35%) and enhanced colocalizaton of Rab14 (∼80%) on both TlyA-expressing bacteria as well as on TlyA-coated latex beads. These results suggest that the mycobacterial TlyA, in general, can modulate phagolysosome maturation pathway immediately after entry into macrophages, while other important molecules may aid the bacterium for long-term, intracellular survival at later point of time.

    • Effects of nitric oxide modulating activities on development of enteric nervous system mediated gut motility in chick embryo model

      Hossein-Ali Arab Samad Muhammadnejad Seyed-Muhammad Faghihi Hossein Hassanpour Ahad Muhammadnejad

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      The enteric nervous system (ENS) arises from the enteric neural crest-derived cells (ENCCs), and many molecules and biochemical processes may be involved in its development. This study examined the effects of modulating embryonic nitric oxide (NO) activity on the intestinal motility induced by ENS. One-hundred-and-twenty fertilized chicken eggs were assigned to three main groups and incubated at 37°C and 60% humidity. The eggs were treated with 𝑁𝐺-nitro-L-arginine methyl ester (L-NAME), sodium nitroprusside (SNP), L-arginine (L-Arg) or vehicle from days 3 (1st group), 7 (2nd group) and 10 (3rd group) of incubation and continued up to day 18. On day 19, the embryos were sacrificed, the jejunal and colorectal segments were taken and the intestinal motility was assessed using isolated organ system. The intestinal motility was recorded normally and following cholinergic, adrenergic and non-adrenergic non-cholinergic (NANC) stimulations. The ENS structure was assessed by immunohistochemistry (IHC) using glial fibrillary acidic protein (GFAP). Rhythmic intestinal contractions were seen in all treatment groups, but inhibition of NO in the L-NAME-treated embryos caused significant decrease (𝑝 < 0.01) in the frequency and amplitude of the contraction. The responsiveness to adrenergic, cholinergic and NANC stimulations was also significantly decreased (𝑝 <0.05). The GFAP expression was significantly (𝑝 < 0.05) reduced in the L-NAME-treated embryos. This study showed that the inhibition of NO caused a deficient development of the ENS, leading to a decrease in the frequency and amplitude of the intestinal contractions and reduced the responsiveness to adrenergic, cholinergic and NANC signalling.

    • Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus)

      Xianlei Wang Xungang Tan Pei-Jun Zhang Yuqing Zhang Peng Xu

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      During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5′-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-month-old flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.

    • Prooxidative effects of aspartame on antioxidant defense status in erythrocytes of rats

      Marko D Prokić Milica G Paunović Miloš M Matić Nataša Z Djordjević Branka I Ognjanović Andraš Š Štajn Zorica S Saičić

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      Since aspartame (L-aspartyl-L-phenylalanine methyl ester, ASP) is one of the most widely used artificial sweeteners, the aim of the present study was to investigate its effects on serum glucose and lipid levels as well as its effects on oxidative/antioxidative status in erythrocytes of rats. The experiment included two groups of animals: the control group was administered with water only, while the experimental group was orally administered with ASP (40 mg/kg b.w.) daily, for a period of six weeks. When compared with the control group, the group administrated with ASP indicated higher values of serum glucose, cholesterol and triglycerides. Significantly increased concentrations of superoxide anion (O2•−), hydrogen peroxide (H2O2), peroxynitrite (ОNОО) and lipid peroxides (LPO) were recorded in the erythrocytes of ASP treated group in comparison to the control group. In the course of chronic ASP administration, the following was observed: the concentration of reduced glutathione (GSH) and the activity of catalase (CAT) increased. Thus, these findings suggest that long-term consumption of ASP leads to hyperglycemia and hyperlipidemia, as well as to oxidative stress in erythrocytes.

    • Uncoupling protein 2 gene (UCP2) 45-bp I/D polymorphism is associated with adiposity among Malaysian women

      Yee-How Say Zi-Lian Ban Yogambigai Arumugam Trishal Kaur Mee-Lay Tan Phee-Phee Chia Sook-Ha Fan

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      This study investigated the association of Uncoupling Protein 2 gene (UCP2) 45-bp I/D polymorphism with obesity and adiposity in 926 Malaysian subjects (416 males; 265 obese; 102/672/152 Malays/Chinese/Indians). The overall minor allele frequency (MAF) was 0.14, while MAFs according to Malay/Chinese/Indian were 0.17/0.12/0.21. The polymorphism was associated with ethnicity, obesity and overall adiposity (total body fat percentage, TBF), but not gender and central adiposity (waist–hip ratio, WHR). Gender- and ethnicity-stratified analysis revealed that within males, the polymorphism was not associated with ethnicity and anthropometric classes. However, within females, significantly more Indians, obese and those with high TBF carried I allele. Logistic regression analysis among females further showed the polymorphism was associated with obesity and overall adiposity; however, when adjusted for age and ethnicity, this association was abolished for obesity but remained significant for overall adiposity [Odds Ratio (OR) for ID genotype =2.02 (CI=1.18, 3.45; 𝑝=0.01); I allele =1.81 (CI=1.15, 2.84; 𝑝=0.01)]. Indeed, covariate analysis controlling for age and ethnicity also showed that those carrying ID genotype or I allele had significantly higher TBF than the rest. In conclusion, UCP2 45-bp I/D polymorphism is associated with overall adiposity among Malaysian women.

    • Interrelationships between Bacillus sp. CHEP5 and Bradyrhizobium sp. SEMIA6144 in the induced systemic resistance against Sclerotium rolfsii and symbiosis on peanut plants

      Marí­a Soledad Figueredo María Laura Tonellie Tania Taurian Jorge Angelini Fernando Ibañez Lucio Valetti Vanina Muñoz Marí­a Soledad Anzuay Liliana Ludueña Adriana Fabra

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      Plant-growth-promoting bacteria are often used to enhance crop yield and for biological control of phytopathogens. Bacillus sp. CHEP5 is a biocontrol agent that induces systemic resistance (ISR) in Arachis hypogaea L. (peanut) against Sclerotium rolfsii, the causal agent of root and stem wilt. In this work, the effect of the co-inoculation of Bacillus sp. CHEP5 and the peanut nodulating strain Bradyrhizobium sp. SEMIA 6144 was studied on induction of both systemic resistance and nodulation processes. Bradyrhizobium sp. SEMIA 6144 did not affect the ability of Bacillus sp. CHEP5 to protect peanut plants from S. rolfsii by ISR and the priming in challenged-plants, as evidenced by an increment in phenylalanine ammonia-lyase enzyme activity. Additionally, the capacity of Bradyrhizobium sp. SEMIA 6144 to induce nodule formation in pathogen-challenged plants was improved by the presence of Bacillus sp. CHEP5.

    • Isolation and molecular characterization of an ethylene response factor NtERF1-1 in Nicotiana tabacum cv. Xanthi

      Jun-Shan Gao Li Hu Peng Xie Yan Meng Yong-Ping Cai Yi Lin

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      Apetala2/Ethylene Response Factors (AP2/ERF) play important roles in regulating gene expression under abiotic and biotic stress in the plant kingdom. Here, we isolated a member of the AP2/ERF transcription factors, NtERF1-1, from Nicotiana tabcum cv. Xanthi NN carrying the N gene, which is resistant to Tobacco mosaic virus (TMV). NtERF1-1 encoded a putative protein of 229 amino acids with a predicted molecular mass of 24.58 kDa. Nucleotide sequence analysis showed that NtERF1-1 contained a conserved DNA-binding domain at the N-terminal. Comparison of amino acid sequences revealed that NtERF1-1 possessed high similarities to ERFs from diverse plants. Semi-quantitative and real-time quantitative RT-PCR analyses indicated that NtERF1-1 was up-regulated following TMV infection. In addition, we speculated that NtERF1-1 might participate in the signal transduction pathway of defence response inducted by the interaction between the 𝑁 gene and TMV.

    • Evolution and expression analysis of the soybean glutamate decarboxylase gene family

      Tae Kyung Hyun Seung Hee Eom Xiao Han Ju-Sung Kim

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      Glutamate decarboxylase (GAD) is an enzyme that catalyses the conversion of L-glutamate into 𝛾-aminobutyric acid (GABA), which is a four-carbon non-protein amino acid present in all organisms. Although plant GAD plays important roles in GABA biosynthesis, our knowledge concerning GAD gene family members and their evolutionary relationship remains limited. Therefore, in this study, we have analysed the evolutionary mechanisms of soybean GAD genes and suggested that these genes expanded in the soybean genome partly due to segmental duplication events. The approximate dates of duplication events were calculated using the synonymous substitution rate, and we suggested that the segmental duplication of GAD genes in soybean originated 9.47 to 11.84 million years ago (Mya). In addition, all segmental duplication pairs (GmGAD1/3 and GmGAD2/4) are subject to purifying selection. Furthermore, GmGAD genes displayed differential expression either in their transcript abundance or in their expression patterns under abiotic stress conditions like salt, drought, and cold. The expression pattern of paralogous pairs suggested that they might have undergone neofunctionalization during the subsequent evolution process. Taken together, our results provide valuable information for the evolution of the GAD gene family and represent the basis for future research on the functional characterization of GAD genes in higher plants.

    • Caspase-like proteins: Acanthamoeba castellanii metacaspase and Dictyostelium discoideum paracaspase, what are their functions?

      Entsar Saheb Wendy Trzyna John Bush

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      Caspases are cysteine proteases that are important regulators of programmed cell death in animals. Two novel relatives to members of the caspase families metacaspases and paracaspase have been discovered. Metacaspase type-1 was identified in Acanthamoeba castellanii, an opportunistic protozoan parasite that causes severe diseases in humans. Paracaspase was found in the non-pathogenic protozoan Dictyostelium discoideum. Since their discovery in Acanthamoeba and Dictyostelium, metacaspases and paracaspases have remained poorly characterized. At present we do not have sufficient data about the molecular function of these caspase-like proteins or their role, if any, in programmed cell death. How these caspase proteins function at the molecular level is an important area of study that will provide insight into their potential for treatment therapies against Acanthamoeba infection and other similar parasitic protozoan. Additionally, finding the molecular functions of these caspase-like proteins will provide information concerning their role in more complex organisms.The aim of this article was to review recent discoveries about metacaspases and paracaspases as regulators of apoptotic and non-apoptotic processes.

    • Functional genomics of tomato: Opportunities and challenges in post-genome NGS era

      Rahul Kumar Ashima Khurana

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      The Tomato Genome Sequencing Project represented a landmark venture in the history of sequencing projects where both Sanger’s and next-generation sequencing (NGS) technologies were employed, and a highly accurate and one of the best assembled plant genomes along with a draft of the wild relative, Solanum pimpinellifolium, were released in 2012. However, the functional potential of the major portion of this newly generated resource is still undefined. The very first challenge before scientists working on tomato functional biology is to exploit this high-quality reference sequence for tapping of the wealth of genetic variants for improving agronomic traits in cultivated tomatoes. The sequence data generated recently by 150 Tomato Genome Consortium would further uncover the natural alleles present in different tomato genotypes. Therefore, we found it relevant to have a fresh outlook on tomato functional genomics in the context of application of NGS technologies in its post-genome sequencing phase. Herein, we provide an overview how NGS technologies vis-à-vis available reference sequence have assisted each other for their mutual improvement and how their combined use could further facilitate the development of other ‘omics’ tools, required to propel the Solanaceae research. Additionally, we highlight the challenges associated with the application of these cutting-edge technologies.

    • Antifreeze proteins enable plants to survive in freezing conditions

      Ravi Gupta Renu Deswal

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      Overwintering plants secrete antifreeze proteins (AFPs) to provide freezing tolerance. These proteins bind to and inhibit the growth of ice crystals that are formed in the apoplast during subzero temperatures. Antifreeze activity has been detected in more than 60 plants and AFPs have been purified from 15 of these, including gymnosperms, dicots and monocots. Biochemical characterization of plant antifreeze activity, as determined by the high ice recrystallization inhibition (IRI) activities and low thermal hysteresis (TH) of AFPs, showed that their main function is inhibition of ice crystal growth rather than the lowering of freezing temperatures. However, recent studies showed that antifreeze activity with higher TH also exists in plants. Calcium and hormones like ethylene and jasmonic acid have been shown to regulate plant antifreeze activity. Recent studies have shown that plant AFPs bind to both prism planes and basal planes of ice crystals by means of two flat ice binding sites. Plant AFPs have been postulated to evolve from the OsLRR-PSR gene nearly 36 million years ago. In this review, we present the current scenario of plant AFP research in order to understand the possible potential of plant AFPs in generation of freezing-tolerant crops.

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