Mechanisms of resistance in the rice cultivar Manikpukha to the rice stem nematode Ditylenchus angustus

The incompatible interaction between the rice cultivar Manikpukha and the rice stem nematode Ditylenchus angustus has been reported recently. This research focuses on the underlying mechanisms of resistance in Manikpukha. Invasion, post‐infection development and reproduction of D. angustus were compared in compatible and incompatible interactions to identify the stage in which resistance occurs. The results indicate that resistance in Manikpukha is associated with reduced development and reproduction, implying that resistance acts post‐invasion. We studied the possible involvement of three classical defence hormones, salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), in response to infection in a compatible interaction using biosynthesis/signalling‐deficient transgenic rice lines. All three hormones appear to have an influence on the basal defence of Nipponbare against the stem nematode. Although hormone application increases basal defences, expression studies and hormone analyses after nematode infection in Manikpukha did not show a clear involvement of the hormone defense pathways for SA, ET and JA. However, it seems that OsPAL1 plays a pivotal role in resistance, indicating that the phenylpropanoid pathway and its products might be key players in the incompatible interaction. Lignin measurement showed that, although basal levels are similar, Manikpukha had a significantly higher lignin content on nematode infection, whereas it was decreased in the susceptible cultivar. The results presented here show that SA, ET and JA are involved in basal defences, but the resistance of Manikpukha against D. angustus probably relies on products of the phenylpropanoid pathway.     

Quality prediction of cell substrate using gene expression profiling

Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of the products that they produce, such as viral vectors, recombinant proteins, or vaccines. Currently there is no effective technique available to monitor global quality of cells in cell culture. Here we describe a new method using gene expression profiling by microarray to predict the quality of cell substrates. Human embryonic kidney 293 cells are a commonly used cell substrate in the production of biological products. We demonstrate that the yield of adenoviral vectors was lower in over-confluent 293 cells, compared to 40 or 90% confluent cells. Total RNA derived from these cells of different confluence states was reverse transcribed, labeled, and used to hybridize 10K cDNA arrays to determine biomarkers for confluence states. Phenotype scatter-plot analysis and cluster analysis were used for class discovery. Based on this approach, we identified genes that were either up-regulated or down-modulated in response to different cell confluence states. By multivariate predictive models we identified a set of 37 genes that were either down-regulated or up-regulated compared to 90% confluent cells as a predictor of cell confluence and quality of 293 cell cultures. The predictive accuracy of these models was assessed by the leave-one-out cross-validation method. The expression of selected gene predictors was validated by quantitative PCR analysis. Our results demonstrate that gene expression profiling can assess the quality of cell substrates prior to large-scale production of a biological product.     

Effect of process parameters on 3-hydroxypropionic acid production from glycerol using a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8) and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2; thermostability, inactive at 55°C for 30 min). k _cat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K _m became twice of that of the wild type.     

Synthesis, microbicidal activity, and solution structure of the dodecapeptide from bovine neutrophils

The dodecapepetide sequence R-L-C-R-I-V-V-I-R-V-C-R with a disulfide bridge between the cysteine residues found in bovine neutrophils was synthesized by solid-phase procedures. Its antimicrobial activity against oral microorganisms such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus gordonii was examined, and its structural features were examined by CD and determined by two-dimensional (2D) nmr. The strains P. gingivalis (W50 and 381), A. actinomycetemcomitans (Y4 and 67), S. gordonii (DL1), and S. mutans (GS5) are found to be highly sensitive to this peptide at 2-2.5 microM concentrations, suggesting that the dodecapeptide is a potent antibiotic for oral pathogens. The weak negative n-sigma* band observed at approximately 265-270 nm in the CD spectra of this peptide provides evidence for the presence of a disulfide bridge. The negative n-pi* band at approximately 200 nm and the positive pi-pi* band at 185 nm suggest a folded structure for this peptide. The negative n-pi* shifts from 200 to 206 nm with an increase in intensity in dipalmitoylphosphotidylcholine vesicles, suggesting that the peptide might associate to form higher order aggregates in lipid medium. The assignment of backbone and side-chain proton resonances has been accomplished by the combined analysis of 2D total correlated and nuclear Overhauser effect spectroscopy. The temperature dependence of amide NH chemical shifts and (1)H-(2)H exchange effect on amide NH resonances indicate the involvement of amide NH groups of Cys3, Ile5, Ile8, Val10, and Arg12 in intramolecular hydrogen bonding. The coupling constant (J(NH-C(alpha)H)) values, the set of medium-, short-, and long-range nuclear Overhauser effects, and the results of restrained structure calculation using the distance geometry algorithm for nmr applications provide evidence for a folded, loop-like structure with a type I (III) beta-turn involving Ile5, Val6, Val7, and Ile8, and two antiparallel beta-strands involving the N-terminal Arg1, Leu2, Cys3, and Val4 and the C-terminal Arg9, Val10, Cys11, and Arg12 residues. The structure of the dodecapeptide mimics the amphiphilic structure of large 30-35 residue defensins and the peptide appears to exhibit similar antimicrobial potency.     

Hzf Determines cell survival upon genotoxic stress by modulating p53 transactivation

A critical unresolved issue about the genotoxic stress response is how the resulting activation of the p53 tumor suppressor can lead either to cell-cycle arrest and DNA repair or to apoptosis. We show here that hematopoietic zinc finger (Hzf), a zinc-finger-containing p53 target gene, modulates p53 transactivation functions in an autoregulatory feedback loop. Hzf is induced by p53 and binds to its DNA-binding domain, resulting in preferential transactivation of proarrest p53 target genes over its proapoptotic target genes. Thus, p53 activation results in cell-cycle arrest in Hzf wild-type MEFs, while in Hzf(-/-) MEFs, apoptosis is induced. Exposure of Hzf null mice to ionizing radiation resulted in enhanced apoptosis in several organs, as compared to in wild-type mice. These findings provide novel insights into the regulation of p53 transactivation function and suggest that Hzf functions as a key player in regulating cell fate decisions in response to genotoxic stress.     

Selective recovery of precious metals by persimmon waste chemically modified with dimethylamine

Persimmon waste was chemically modified with dimethylamine (DMA) to obtain a tertiary-amine-type gel, named DMA persimmon waste gel (DMA-PW). It was found to be effective for the adsorption of Au(III), Pd(II), and Pt(IV) in hydrochloric acid medium. In contrast, base metals such as Cu(II), Zn(II), Fe(III), and Ni(II) were not practically adsorbed. The formation of ion pairs of the metal chloro complex anions with the protonated adsorption gels was proposed as the main adsorption process. The gel exhibited selectivity only for precious metals with a remarkably high capacity for Au(III), i.e., 5.63 mol/kg dry gel and comparable capacities, i.e., 0.42 and 0.28 mol/kg for Pd(II) and Pt(IV), respectively. According to the kinetic and electrochemical studies, the adsorption rate of Au(III) was greatly enhanced by the chemical modification. Also, its excellent adsorption characteristics for the precious metals were confirmed by adsorption and elution tests using a column packed with the DMA-PW gel.     

Anaerobic biotransformation of furfural to furfuryl alcohol by a methanogenic archaebacterium

The metabolic conversion of furfural by a methanogenic Archaea, Methanococcus sp., strain B was studied. The organism was grown on H₂–CO₂ in the presence of various concentrations of furfural. Furfural at higher concentrations, namely, 25 and 30 mM inhibited growth of this organism. At concentrations 5, 10, and 15 mM, no inhibition was observed. Furfural was completely (100%) metabolized at the concentration of 15 or <15 mM in the cultures within five days of incubation. The end product observed during furfural metabolism was furfuryl alcohol. An almost stoichiometric quantity of furfuryl alcohol was produced. This biotransformation is likely to be of value in the detoxification of furfural and its ultimate conversion to methane and CO₂ by the anaerobic process.     

Predicting stable functional peptides from the intergenic space of E. coli

Expression of synthetic proteins from intergenic regions of E. coli and their functional association was recently demonstrated (Dhar et al. in J Biol Eng 3:2, 2009. doi:10.1186/1754-1611-3-2). This gave birth to the question: if one can make ‘user-defined’ genes from non-coding genome—how big is the artificially translatable genome? (Dinger et al. in PLoS Comput Biol 4, 2008; Frith et al. in RNA Biol 3(1):40–48, 2006a; Frith et al. in PLoS Genet 2(4):e52, 2006b). To answer this question, we performed a bioinformatics study of all reported E. coli intergenic sequences, in search of novel peptides and proteins, unexpressed by nature. Overall, 2500 E. coli intergenic sequences were computationally translated into ‘protein sequence equivalents’ and matched against all known proteins. Sequences that did not show any resemblance were used for building a comprehensive profile in terms of their structure, function, localization, interactions, stability so on. A total of 362 protein sequences showed evidence of stable tertiary conformations encoded by the intergenic sequences of E. coli genome. Experimental studies are underway to confirm some of the key predictions. This study points to a vast untapped repository of functional molecules lying undiscovered in the non-expressed genome of various organisms.