Involvement of phospholipases C and D in early response to SAR and ISR inducers in Brassica napus plants.

Phospholipid signaling is an important component in eukaryotic signal transduction pathways. In plants, it plays a key role in growth and development as well as in responses to environmental stresses, including pathogen attack. We investigated the involvement of both phospholipase C (PLC, EC 3.1.4.11) and D (PLD, EC 3.1.4.4) in early responses to the treatment of Brassica napus plants with the chemical inducers of systemic acquired resistance (SAR): salicylic acid (SA), benzothiadiazole (BTH), and with the inducer mediating the induced systemic resistance (ISR) pathway, methyl jasmonate (MeJA). Rapid activation (within 0.5-6 h treatment) of the in vitro activity level was found for phosphatidyl inositol 4,5 bisphosphate (PIP2)-specific PLC (PI-PLC) and three enzymatically different forms of PLD: conventional PLDalpha, PIP2-dependent PLD beta/gamma, and oleate-stimulated PLDdelta. The strongest response was found in case of cytosolic PIP2-dependent PLD beta/gamma after BTH treatment. PLDdelta was identified in B. napus leaves and was very rapidly activated after MeJA treatment with the highest degree of activation compared to the other PLD isoforms. Interestingly, an increase in the amount of protein was observed only for PLDgamma and/or delta after ISR induction, but later than the activation occurred. These results show that phospholipases are involved in very early processes leading to systemic responses in plants and that they are most probably initially first activated on post translational level.     

Zeptomole Electrochemical Detection of Metallothioneins

Background

Thiol-rich peptides and proteins possess a large number of biological activities and may serve as markers for numerous health problems including cancer. Metallothionein (MT), a small molecular mass protein rich in cysteine, may be considered as one of the promising tumour markers. The aim of this paper was to employ chronopotentiometric stripping analysis (CPSA) for highly sensitive detection of MT.

Methodology/Principal Findings

In this study, we used adsorptive transfer stripping technique coupled with CPSA for detection of cysteine, glutathione oxidized and reduced, phytochelatin, bovine serum albumin, and metallothionein. Under the optimal conditions, we were able to estimate detection limits down to tens of fg per ml. Further, this method was applied to detect metallothioneins in blood serum obtained from patients with breast cancer and in neuroblastoma cells resistant and sensitive to cisplatin in order to show the possible role of metallothioneins in carcinogenesis. It was found that MT level in blood serum was almost twice higher as compared to the level determined in healthy individuals.

Conclusions/Significance

This paper brings unique results on the application of ultra-sensitive electroanalytical method for metallothionein detection. The detection limit and other analytical parameters are the best among the parameters of other techniques. In spite of the fact that the paper is mainly focused on metallothionein, it is worth mentioning that successful detection of other biologically important molecules is possible by this method. Coupling of this method with simple isolation methods such as antibody-modified paramagnetic particles may be implemented to lab–on-chip instrument.     

Preparation of D-galactofuranosyl nitromethanes: a revision and a new approach

Sodium methoxide-promoted methanolysis of 7-deoxy-7-nitro-L-glycero-L-galacto-heptitol peracetate rapidly and nearly quantitatively accumulates 7-deoxy-6-O-methyl-7-nitro-L-glycero-L-galacto-heptitol. The prolonged treatment then provides 76% of D-galactofuranosyl nitromethanes and finally results in the equilibrium of 77% of β-D-galactopyranosyl nitromethane and 7-9% of three other tautomeric D-galactosyl nitromethanes. Thermal treatment of 7-deoxy-7-nitro-L-glycero-L-galacto-heptitol in boiling water peaks at a 58% content of D-galactofuranosyl nitromethanes and ends in a similar equilibrium mixture of four D-galactosyl tautomers. The relevant kinetic parameters of the latter transformation are determined by a curve fitting using the nonlinear least-squares Marquardt-Levenberg algorithm.     

Autophagy inhibition in early but not in later stages prevents 3T3-L1 differentiation: Effect on mitochondrial remodeling

Autophagy is essential for successful white adipocyte differentiation but the data regarding the timing and relevance of autophagy action during different phases of adipogenesis are limited.     

Preparation and Enantioselectivity Binding Studies of a New Chiral Cobalt(II)porphyrin‐Tröger's Base Conjugate

A new bis[cobalt(II)porphyrin]‐Tröger's base conjugate was studied as a potential receptor for methyl esters of several amino acids. The conjugate was prepared as racemate, and then resolved via preparative high‐performance liquid chromatography (HPLC) on a chiral column. The high affinity to lysine, histidine, and proline methyl esters was found by complexation studies followed by UV‐Vis spectroscopy. The studies of pure enantiomers, followed by UV‐Vis and electronic circular dichroism spectroscopy, revealed the highest enantioselectivity for lysine methyl ester. Chirality 26:361–367, 2014. © 2014 Wiley Periodicals, Inc.     

Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions

Semen contains relatively ill-defined regulatory components that likely aid fertilization, but which could also interfere with defense against infection. Each ejaculate contains trillions of exosomes, membrane-enclosed subcellular microvesicles, which have immunosuppressive effects on cells important in the genital mucosa. Exosomes in general are believed to mediate inter-cellular communication, possibly by transferring small RNA molecules. We found that seminal exosome (SE) preparations contain a substantial amount of RNA from 20 to 100 nucleotides (nts) in length. We sequenced 20–40 and 40–100 nt fractions of SE RNA separately from six semen donors. We found various classes of small non-coding RNA, including microRNA (21.7% of the RNA in the 20–40 nt fraction) as well as abundant Y RNAs and tRNAs present in both fractions. Specific RNAs were consistently present in all donors. For example, 10 (of ∼2600 known) microRNAs constituted over 40% of mature microRNA in SE. Additionally, tRNA fragments were strongly enriched for 5’-ends of 18–19 or 30–34 nts in length; such tRNA fragments repress translation. Thus, SE could potentially deliver regulatory signals to the recipient mucosa via transfer of small RNA molecules.     

Dual Effects of Ketoconazole cis-Enantiomers on CYP3A4 in Human Hepatocytes and HepG2 Cells

Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (−)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (−)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, K_i 0.92 µM for testosterone, IC₅₀ 1.46 µM, K_i 2.52 µM for midazolam; (−)-ketoconazole IC₅₀ 0.90 µM, K_i 0.17 µM for testosterone, IC₅₀ 1.04 µM, K_i 1.51 µM for midazolam).     

Rare species in communities of tropical insect herbivores: pondering the mystery of singletons

The host specificity, taxonomic composition and feeding guild of rare species were studied in communities of herbivorous insects in New Guinea. Leaf‐chewing and sap‐sucking insects (Orthoptera, Phasmatodea, Coleoptera, Lepidoptera and Hemiptera‐Auchenorrhyncha) were sampled from 30 species of trees and shrubs (15 spp. of Ficus, Moraceae, six spp. of Macaranga and nine species of other Euphorbiaceae) in a lowland rain forest. Feeding trials were performed with all leaf‐chewers in order to exclude transient species. Overall, the sampling produced 80 062 individuals of 1050 species. The species accumulation curve did not attain an asymptote, despite 950 person‐days of sampling. Rare species, defined as those found as single individuals, remained numerous even in large samples and after the exclusion of transient, non‐feeding species. There was no difference among plant species in the proportion of rare species in their herbivore communities, which was, on average, 45%. Likewise, various herbivore guilds and taxa had all very similar proportions of rare and common species. There was also no difference between rare and common species in their host specificity. Both highly specialised species and generalists, feeding on numerous plants, contributed to the singleton records on particular plant species. Predominantly, a species was rare on a particular host whilst more common on other, often related, host species, or relatively rare on numerous other host plants, so that its aggregate population was high. Both cases are an example of the “mass effect”, since it is probable that such rare species were dependent on a constant influx of immigrants from the other host plants. These other plants were found particularly often among congeneric plants, less so among confamilial plants from different genera and least frequently among plants from different families. There were also 278 very rare species, found as one individual on a single plant species only. Their host specificity could not be assessed; they might have been either very rare specialists, or species feeding also on other plants, those that were not studied. The former possibility is unlikely since monophagous species, collected as singletons at the present sampling effort, would have existed at an extremely low population density, less than 1 individual per 10 ha of the forest.     

NADH- and Ferredoxin-Dependent Glutamate Synthase in the Life Span of the Second Leaf of Wheat Plant under Conditions of Senescence Induced by Nitrogen Deficiency and Natural Senescence

The aim of this paper was to study, in the second leaf of wheat plants with a long ontogenesis (47 d), the activity of the enzyme which catalyzes the synthesis of glutamic acid. The activity of the NADH-dependent glutamate synthase prevailed in young tissues of not yet fully expanded second leaf at the stage of incomplete autotrophy (at this stage, organic carbon and nitrogen substances are transferred into the leaf). This form was completely inhibited by azaserine (1 mmol1⁻¹). The activity of the ferredoxin-dependent GOGAT increased with increasing leaf blade area, with its peak occuring at the time of maximum expansion of the assimilation area. Thus fd-GOGAT activity was characteristic for the photosynthetic fully autotrophic phase of leaf development which is materialized in completely mature leaf tissues. In plants grown in full-strength Knop’s nutrient solution containing nitrogen, fd-GOGAT was active till the early and medium senescence, whereas only to the early senescence in plants grown in a solution lacking nitrogen. No fd-GOGAT could be detected at the stage of late leaf senescence.