Potassium chloride and rare earth elements improve plant growth and increase the frequency of the Agrobacterium tumefaciens-mediated plant transformation

Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors. Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases the frequency of both homologous recombination and plant transgenesis. Here we tested the influence of KCl and salts of rare earth elements, Ce and La on the efficiency of Agrobacterium-mediated plant transformation. We found that exposure to KCl, CeCl₃ and LaCl₃ leads to an increase in recombination frequency in Arabidopsis and tobacco. Plants grown in the presence of CeCl₃ and LaCl₃ had higher biomass, longer roots and greater root number. Analysis of transformation efficiency showed that exposure of tobacco plants to 50 mM KCl resulted in ~6.0-fold increase in the number of regenerated calli and transgenic plants as compared to control plants. Exposure to various concentrations of CeCl₃ showed a maximum increase of ~3.0-fold in both the number of calli and transgenic plants. Segregation analysis showed that exposure to KCl and cerium (III) chloride leads to more frequent integrations of the transgene at a single locus. Analysis of transgene intactness showed better preservation of right T-DNA border during transgene integration. Our data suggest that KCl and CeCl₃ can be effectively used to improve quantity and quality of transgene integrations.     

Description of Proyseria petterae n. sp., with an amended generic diagnosis and a review of the species of Proyseria Petter, 1959 and Stegophorus Wehr, 1934 (Nematoda: Acuariidae)

Proyseria decora (Dujardin, 1845) (the type-species of the genus Proyseria Petter, 1959) is redescribed on the basis of specimens from Alcedo atthis (L.) (Coraciiformes: Alcedinidae) from Iran. P. petterae n. sp. is described from Corythornis vintsioides (Eydoux & Gervais) (Alcedinidae) from Madagascar by light and scanning electron microscopy. Proyseria sp. from Alcedo euryzona Temminck from continental Malaysia is described on the basis of a single male specimen. Stegophorus alcedonis Puqin, Yanyin & Guocal, 1991 from A. atthis in China is transferred to the genus Proyseria as P. alcedonis n. comb. The generic diagnosis of Proyseria is amended. Review of the species of the genera Proyseria and Stegophorus Wehr, 1934 is presented.     

A New Genus of Fossil Sand Crab (Anomura: Albuneidae) from the Oligocene of Italy

The recently described fossil albuneid Paralbunea galantensis De Angeli and Marangon differs from the type of the genus Paralbunea Serène and is herein made the type species of a new genus, Harryhausenia , which is closely related to Zygopa Serène and Umali. A discussion is given on the relationships between the new genus and Zygopa , as well as between Zygopa and other albuneid taxa.     

First record of shape Saturnius papernai Overstreet, 1977 in the Black Sea,with a review of the genus shape Saturnius Manter, 1969 (Digenea,Bunocotylidae)

Saturnius papernai Overstreet, 1977 is recorded from the stomach of Mugil cephalus L. (Osteichthyes, Mugilidae) in the Black Sea (a new geographical record) off the southern part of the Bulgarian coast. The species is redescribed and figured on the basis of the Black Sea specimens. Some amendments to the generic diagnosis are proposed concerning the presence of a uterine seminal receptacle and a posterior (third) circular flange. It is proposed that the occurrence of S. papernai is a feature of one of the three shoals of M. cephalus in the Black Sea, i.e. the Balkan shoal. In a zoogeographical analysis, S. papernai is regarded as a Mediterranean immigrant, probably a characteristic element of only the South-Western Faunistic Region of the Black Sea. A review of the genus Saturnius Manter, 1969 is presented.     

Distribution of radiolabeled l-glutamate and d-aspartate from blood into peripheral tissues in naive rats: Significance for brain neuroprotection

Excess l-glutamate (glutamate) levels in brain interstitial and cerebrospinal fluids (ISF and CSF, respectively) are the hallmark of several neurodegenerative conditions such as stroke, traumatic brain injury or amyotrophic lateral sclerosis. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As in previous studies, we have established the role of blood glutamate levels in brain neuroprotection, we have now investigated the contribution of the peripheral organs to the homeostasis of glutamate in blood. We have administered naive rats with intravenous injections of either l-[1-¹⁴C] Glutamic acid (l-[1-¹⁴C] Glu), l-[G-³H] Glutamic acid (l-[G-³H] Glu) or d-[2,3-³H] Aspartic acid (d-[2,3-³H] Asp), a non-metabolized analog of glutamate, and have followed their distribution into peripheral organs. We have observed that the decay of the radioactivity associated with l-[1-¹⁴C] Glu and l-[G-³H] Glu was faster than that associated with glutamate non-metabolized analog, d-[2,3-³H] Asp. l-[1-¹⁴C] Glu was subjected in blood to a rapid decarboxylation with the loss of ¹⁴CO₂. The three major sequestrating organs, serving as depots for the eliminated glutamate and/or its metabolites were skeletal muscle, liver and gut, contributing together 92% or 87% of total l-[U-¹⁴C] Glu or d-[2,3-³H] Asp radioactivity capture. l-[U-¹⁴C] Glu and d-[2,3-³H] Asp showed a different organ sequestration pattern. We conclude that glutamate is rapidly eliminated from the blood into peripheral tissues, mainly in non-metabolized form. The liver plays a central role in glutamate metabolism and serves as an origin for glutamate metabolites that redistribute into skeletal muscle and gut. The findings of this study suggest now that pharmacological manipulations that reduce the liver glutamate release rate or cause a boosting of the skeletal muscle glutamate pumping rate are likely to cause brain neuroprotection.     

Visualizing the principal component of 1H,15N-HSQC NMR spectral changes that reflect protein structural or functional properties: application to troponin C

Laboratories often repeatedly determine the structure of a given protein under a variety of conditions, mutations, modifications, or in a number of states. This approach can be cumbersome and tedious. Given then a database of structures, identifiers, and corresponding (1)H,(15)N-HSQC NMR spectra for homologous proteins, we investigated whether structural information could be ascertained for a new homolog solely from its (1)H,(15)N-HSQC NMR spectrum. We addressed this question with two different approaches. First, we used a semi-automated approach with the program, ORBplus. ORBplus looks for patterns in the chemical shifts and correlates these commonalities to the explicit property of interest. ORBplus ranks resonances based on consistency of the magnitude and direction of the chemical shifts within the database, and the chemical shift correlation of the unknown protein with the database. ORBplus visualizes the results by a histogram and a vector diagram, and provides residue specific predictions on structural similarities with the database. The second method we used was partial least squares (PLS), which is a multivariate statistical technique used to correlate response and predictor variables. We investigated the ability of these methods to predict the tertiary structure of the contractile regulatory protein troponin C. Troponin C undergoes a closed-to-open conformational change, which is coupled to its function in muscle. We found that both ORBplus and PLS were able to identify patterns in the (1)H,(15)N-HSQC NMR data from different states of troponin C that correlated to its conformation.     

Importance of DNA methylation in the inheritance of radiation-induced aberrant expression of microRNA

We studied microRNA gene expression in HeLa cells following exposure for 6 h and 8 days to Co⁶⁰ gamma rays at a dose of 4 Gy using an approach of large-scale parallel DNA sequencing. We identified 12 microRNAs with aberrant expression which were maintained in cell generations. The analysis of radiation-induced aberrant expression of pre-microRNAs made it possible to assess the importance of nuclear and cytoplasmic stages of microRNA biogenesis for preservation of its aberrant expression. On cell treatment by 5-azacytidine, aberrant expression was maintained only in two microRNAs: miR-21-3p and miR-422a, which demonstrated an increase in expression. Radiation-induced decrease in expression in ten examined microRNAs was dependent on DNA demethylation. At the same time, expression in a microRNA set, which demonstrated inheritable alteration of the expression after gamma-radiation exposure in the untreated cells, was not dependent or was weakly dependent on DNA methylation. The obtained results suggest that ionizing radiation induces aberrant DNA methylation, which affects inherited expression changes in microRNAs in cell generations after exposure to the mutagen.     

Intramolecular complementing mutations in tobacco mosaic virus movement protein confirm a role for microtubule association in viral RNA transport

The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MP(P81S)-GFP and MP(P81S;T104I;R167K)-GFP differ in subcellular distribution. MP(P81S)-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MP(P81S;T104I;R167K)-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.     

Structural insight into the molecular basis of polyextremophilicity of short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus

Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme – half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous “resilience” of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.     

Null Mutation in Puroindoline A is Prevalent in Indian Wheats: Puroindoline Genes are Located in The Distal Part of 5DS

PCR amplification and protein analysis of pinA and pinB, the two components of friabilin, a marker protein for grain softness, were carried out in one hundred varieties from India and forty varieties from Kansas State, USA. Glycine to serine change in pinB or null mutation in pinA (absence of gene) has been reported linked with grain hardness. Here we report that majority of Kansas State hard wheats possess glycine to serine mutation in pinB and few have null mutation in pinA. In contrast, majority of varieties released in India are hard and have null mutation in pinA.There are some exceptions where some hard wheats in India do not exhibit either null mutation in pinA or glycine to serine change in pinB.There might be some additional mutations that are to be characterised in elucidating the molecular basis of hardness for usage in genetic engineering. PinA and pinB genes have been assigned on the distal part of 5D short arm using deletion lines of Chinese spring wheat where hardness gene Ha is reported to be present.