Sterilization of Plants for Phytoremediation Studies by Bleach Treatment

Bleach treatment of plants was studied as a simple alternative to axenic tissue cultures for demonstrating phytodegradation of aqueous and gas-phase environmental contaminants. Parrotfeather (Myriophyllum aquaticum), spinach (Spinacia oleracea), and wheat (Triticum aestivum) were exposed to 0.525% NaC10 solutions for 15 s, then rinsed in deionized water. Plate counts indicated that 97 to 100% of viable bacteria were removed from parrotfeather and spinach. Transformation rates for 2,4,6-trinitrotoluene (TNT) by bleached and untreated parrotfeather were virtually identical. Similarly, treated and untreated spinach, wheat heads, and wheat leaves removed methyl bromide (MeBr) from air at the same rates. However, wheat root with attendant adhering soil was rendered inactive by bleach treatment. Parrotfeather roots examined by dissecting microscope and by electron microscope showed no significant damage caused by bleach treatment.     

Starvation promotes Dictyostelium development by relieving PufA inhibition of PKA translation through the YakA kinase pathway.

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.     

Synthesis of N,N-Dialkylaniline-2′-deoxyuridine Conjugates for DNA-Mediated Electron Transfer Studies

Abstract

Syntheses of two analogs of deoxyuridine with N,N-dialkylaniline chromophores are reported. 5-[3-(N-methylphenylamino)propanoyl]-2′-deoxyuridine (1) and 5-[2-(4-N,N-dimethylaminophenyl)ethyl)]-2′-deoxyuridine (2) are prepared by palladium-mediated coupling. Preparation of 2 was facilitated by in situ transient O⁴-trimethylsilyl protection during alkynylation which suppressed secondary cyclization of the coupling adduct.     

Lysosomal accumulation and recycling of lipopolysaccharide to the cell surface of murine macrophages, an in vitro and in vivo study.

In this study, we detailed in a time-dependent manner the trafficking, the recycling, and the structural fate of Brucella abortus LPS in murine peritoneal macrophages by immunofluorescence, ELISA, and biochemical analyses. The intracellular pathway of B. abortus LPS, a nonclassical endotoxin, was investigated both in vivo after LPS injection in the peritoneal cavity of mice and in vitro after LPS incubation with macrophages. We also followed LPS trafficking after infection of macrophages with B. abortus strain 19. After binding to the cell surface and internalization, Brucella LPS is routed from early endosomes to lysosomes with unusual slow kinetics. It accumulates there for at least 24 h. Later, LPS leaves lysosomes and reaches the macrophage cell surface. This recycling pathway is also observed for LPS released by Brucella S19 following in vitro infection. Indeed, by 72 h postinfection, bacteria are degraded by macrophages and LPS is located inside lysosomes dispersed at the cell periphery. From 72 h onward, LPS is gradually detected at the plasma membrane. In each case, the LPS present at the cell surface is found in large clusters with the O-chain facing the extracellular medium. Both the antigenicity and heterogenicity of the O-chain moiety are preserved during the intracellular trafficking. We demonstrate that LPS is not cleared by macrophages either in vitro or in vivo after 3 mo, exposing its immunogenic moiety toward the extracellular medium.     

Molecular identification of filterable bacteria and archaea in the water of acidic lakes of northern Russia

Wetland ecosystems are the natural centers of freshwater formation in northern Russia lowland landscapes. The humic acidic waters formed in bogs feed the numerous lakes of the northern regions. One milliliter of the water in these lakes contains up to 10⁴ ultrasmall microbial cells that pass through “bacterial” filters with a pore size of 0.22 μm. The vast majority of these cells do not grow on nutrient media and cannot be identified by routine cultivation-based approaches. Their identification was performed by analysis of clone libraries obtained by PCR amplification of archaeal and bacterial 16S rRNA genes from the fraction of cells collected from water filtrates of acidic lakes. Most of the obtained bacterial 16S rRNA gene sequences represented the class Betaproteobacteria and exhibited the highest homology of (94–99%) with 16S rRNA genes of representatives of the genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia. The archaeal 16S rRNA gene clone library comprised genes of Euryarchaeota representatives. One-third of these genes exhibited 97–99% homology to the 16S rRNA genes of taxonomically described organisms of the orders Methanobacteriales and Methanosarcinales. The rest of the cloned archaeal 16S rRNA genes were only distantly related (71–74% homology) to those in all earlier characterized archaea.     

Linoleic acid hydroperoxide favours hypochlorite- and myeloperoxidase-induced lipid peroxidation

Liposomes composed of soybean phosphatidylcholine were peroxidized using the reagent sodium hypochlorite or the myeloperoxidase-hydrogen peroxide-Cl^- system. Linoleic acid hydroperoxide previously prepared from linoleic acid by means of lipoxidase was incorporated into liposomes. The yield of thiobarbituric acid reactive substances (TBARS) continuously increased with higher amounts of hydroperoxide groups after the initiation of lipid peroxidation by hypochlorous acid producing systems. The accumulation of TBARS was inhibited by scavengers of free radicals such as butylated hydroxytoluene and by the scavengers of hypochlorous acid, taurine and methionine. Lipid peroxidation was also prevented by sodium azide or chloride free medium in the myeloperoxidase-hydrogen peroxide-Cl^- system. Here we show for the first time that the reaction of hypochlorous acid with a biologically relevant hydroperoxide yields free radicals able to cause further oxidation of lipid molecules.     

Subtle re-location of dendritic spine branches containing membrane with fast spiking mechanisms can alter synaptic efficacy

The potential physiological impact of morphological changes in the active dendritic spines, which are believed to be associated with altered synaptic efficacy, was investigated in a computer simulation study using the NEURON package [1]. A compartmental model of a simplified neuron was built, which included 30 complex spines (neck, head, and active zone) and accommodating AMPA-type synaptic inputs with alpha-function conductances. Hodgkin-Huxley type excitable membranes were inserted into the spine heads. It was shown that arranging spines in dense clusters, as opposed to a uniformly random spine distribution, has a negligible effect on the synaptic signal transfer (other model conditions, including synaptic input and spine density, remained unchanged). However, if a proportion (e.g., 3–20%) of the spines partly fuse with their neighbors forming branched spines, this could increase dramatically the cell response to the unchanged synaptic input. Results of this pilot study provide the basis for a more detailed investigation of the relationship between the spine arrangement and synaptic function, considering dual-component synaptic currents and mechanisms controlling ion fluxes in the dendritic compartments.     

Contents of polyamines during vernalization in wheat and the effect of zearalenone

The contents of endogenous free and conjugated polyamines, putrescine (Put) and spermidine (Spd), were determined during 9 week of vernalization (at 5 °C) in winter wheat seedlings cultivated on Murashige and Skoog media without (MS0) and with 2 mg dm⁻³ zearalenone (MSZEN). At the 4^th week of chilling treatment, which is sufficient to induce generative development in 30 % of plants, the marked increase in free and conjugated forms of Put and free Spd were observed. The presence of ZEN in medium significantly accelerated the vernalization. About 20 % of plants treated with ZEN flowered already after 2 weeks and 40 % after 3 weeks of chilling. Significantly higher content of free Put was determined in roots grown on MSZEN compared with MS0 during the first 5 weeks of vernalization with maximum at the 4^th week. After germination, a marked decrease in free Spd content was observed both in plants grown on MS0 and MSZEN. Application of ZEN significantly slowed down the Spd decline in leaves and roots during the first and second week of vernalization. The content of Spd and its conjugates decreased in vernalized plants after 1 week of cultivation at 20 °C.