Compartmentation of Zinc in Roots and Leaves of the Zinc Hyperaccumulator Thlaspi caerulescens J & C Presl

Thlaspi caerulescens is a metallophyte that is able to hyperaccumulate Zn. In the present study the subcellular compartmentation of Zn was investigated in roots and leaves of this species by means of X-ray microanalysis. Leaves accumulated higher average Zn concentrations than roots. In roots of plants exposed to 10 μM Zn, Zn concentrations in the apoplast were similar to those in vacuoles, while in plants treated with 100 μM Zn considerably higher Zn concentrations were detected in vacuoles than in the apoplast. In epidermal and sub-epidermal cells of leaves of plants from both treatments, Zn mainly accumulated in vacuoles and, to a lesser extent, in the apoplast. In vacuoles from plants exposed to 100 μM Zn, high Zn concentrations were associated with variable amounts of P, Ca and K. In leaves, the highest Zn concentrations (13,600 μg g^?1 d.m.) were found in globular crystals present in many vacuoles of epidermal and subepidermal cells. Smaller deposits with a variable Zn concentration between 1,000 and 18,300 μg g^?1 d.m. were observed in the epidermal and subepidermal cells of roots. Both the high Zn/P element ratios found in the crystals and the absence of Mg indicate that, in contrast to other plant species, myo-inositol hexaphosphate (phytate) is not the main storage form for Zn in Thlaspi caerulescens.     

Protein partitioning in two-phase aqueous polymer systems

Theories of protein partitioning in two-phase polymer systems which account for the effects of different aspects of system composition-such as the choice of materials, protein size, polymer molecular weight, polymer concentration, salt concentration, and affinity ligands-are reviewed. Although the present models provide some information about specific aspects of partitioning, a comprehensive and fundamental theory which can be used to predict protein partitioning behavior has not yet been developed. Some recommendations for future work are given.     

Identification of a signaling network in lateral nucleus of amygdala important for inhibiting memory specifically related to learned fear

We identified the Grp gene, encoding gastrin-releasing peptide, as being highly expressed both in the lateral nucleus of the amygdala, the nucleus where associations for Pavlovian learned fear are formed, and in the regions that convey fearful auditory information to the lateral nucleus. Moreover, we found that GRP receptor (GRPR) is expressed in GABAergic interneurons of the lateral nucleus. GRP excites these interneurons and increases their inhibition of principal neurons. GRPR-deficient mice showed decreased inhibition of principal neurons by the interneurons, enhanced long-term potentiation (LTP), and greater and more persistent long-term fear memory. By contrast, these mice performed normally in hippocampus-dependent Morris maze. These experiments provide genetic evidence that GRP and its neural circuitry operate as a negative feedback regulating fear and establish a causal relationship between Grpr gene expression, LTP, and amygdala-dependent memory for fear.     

The different components of a multisubunit cell number-counting factor have both unique and overlapping functions

Dictyostelium aggregation streams break up into groups of 10(3) to 2 x 10(4) cells. The cells sense the number of cells in a stream or group by the level of a secreted counting factor (CF). CF is a complex of at least 5 polypeptides. When the gene encoding countin (one of the CF polypeptides) was disrupted, the cells could not sense each other's presence, resulting in non-breaking streams that coalesced into abnormally large groups. To understand the function of the components of CF, we have isolated cDNA sequences encoding a second component of CF, CF50. CF50 is 30% identical to lysozyme (but has very little lysozyme activity) and contains distinctive serine-glycine motifs. Transformants with a disrupted cf50 gene, like countin(-) cells, form abnormally large groups. Addition of recombinant CF50 protein to developing cf50(-) cells rescues their phenotype by decreasing group size. Abnormalities seen in aggregating countin(-) cells (such as high cell-cell adhesion and low motility) are also observed in the cf50(-) cells. Western blot analysis of conditioned medium sieve column fractions showed that the CF50 protein is present in the same fraction as the 450 kDa CF complex. In the absence of CF50, secreted countin is degraded, suggesting that one function of CF50 may be to protect countin from degradation. However, unlike countin(-) cells, cf50(-) cells differentiate into an abnormally high percentage of cells expressing SP70 (a marker expressed in a subset of prespore cells), and this difference can be rescued by exposing cells to recombinant CF50. These observations indicate that unlike other known multisubunit factors, CF contains subunits with both overlapping and unique properties.     

Induction of protective CD4+ T cell-mediated immunity by a Leishmania peptide delivered in recombinant influenza viruses

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4(+) Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK(158-173) CD4(+) peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK(158-173)-specific CD4(+) T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK(158-173) triggers LACK(158-173)-specific Th1-biased CD4(+) T cell responses within an appropriate cytokine milieu (IFN-γ, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2-4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-γ-producing CD4(+) T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK(158-173) led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-γ producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity.     

Using Magnetically Responsive Tea Waste to Remove Lead in Waters under Environmentally Relevant Conditions

We report the use of a simple yet highly effective magnetite-waste tea composite to remove lead(II) (Pb²⁺) ions from water. Magnetite-waste tea composites were dispersed in four different types of water–deionized (DI), artificial rainwater, artificial groundwater and artificial freshwater–that mimic actual environmental conditions. The water samples had varying initial concentrations (0.16–5.55 ppm) of Pb²⁺ ions and were mixed with the magnetite-waste tea composite for at least 24 hours to allow adsorption of the Pb²⁺ ions to reach equilibrium. The magnetite-waste tea composites were stable in all the water samples for at least 3 months and could be easily removed from the aqueous media via the use of permanent magnets. We detected no significant leaching of iron (Fe) ions into the water from the magnetite-waste tea composites. The percentage of Pb adsorbed onto the magnetite-waste tea composite ranged from ∼70% to 100%; the composites were as effective as activated carbon (AC) in removing the Pb²⁺ ions from water, depending on the initial Pb concentration. Our prepared magnetite-waste tea composites show promise as a green, inexpensive and highly effective sorbent for removal of Pb in water under environmentally realistic conditions.     

Biased mutations and microsatellite variation

Mutation bias is one of the forces that may constrain the variation at microsatellite loci. Here, we study the dynamics of population statistics and the genetic distance between two populations under multiple stepwise mutations with linear bias and random drift. Expressions are derived for these statistics as functions of time, as well as at mutation-drift equilibrium. Applying these expressions to published data on humans and chimpanzees, the regression coefficient of mutation bias on allele size was estimated to be at least between - 0.0064 and -0.013. The assumption of mutational bias produces larger estimates of divergence times than are obtained in its absence; in particular, the time of split between African and non-African human populations is estimated to be between 183,000 and 222,000 years, assuming one-step mutations and no selection. With multistep mutations, the divergence time is estimated to be lower.      

Design of surfactants suitable for protein extraction by reversed micelles

New surfactants have been synthesized for potential use in reversed micellar protein extraction operations. Preferential solubility of the surfactant in an aliphatic solvent such as hexane, heptane, or isooctane and the formation of reversed micelles accompanied with solubilization of significant quantities of water can be achieved by using strongly hydrophobic, twin alkyl chains as the hydrophobic moiety. Different surfactants having identical water-solubilizing capacities can have significantly different behavior in protein extractions, where extraction efficiency appears to be governed by the nature of the interfacial complex that forms between surfactants and proteins. Bulky surfactant chains provide a steric hindrance to the adsorption of the surfactant to the protein surface, thus inhibiting solvation of the protein/surfactant complex, and hence protein extraction. Under these conditions, a precipitate forms either in the bulk aqueous phase or at the interface. Surfactants that can form a close-packed complex with the protein are excellent protein-solubilizing agents. Dioleyl phosphoric acid (DOLPA) appears to be the best surfactant currently available for protein extraction. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 26-32, 1997.     

Identification of polypeptides associated with sarcolemmal vesicles enriched in orthogonal arrays

Electron microscopy of freeze-fracture replicas from the sarcolemmas of fast-twitch muscle fibers reveals orthogonal arrays of particles. The biochemical nature of macromolecules associated with the sarcolemmal orthogonal array was investigated using muscle fragments and isolated sarcolemmal vesicles. Muscle fragments incubated in vitro with the lectin concanavalin A exhibited a clustering of orthogonal arrays into local patches. Treatment with other lectins did not result in the clustering of arrays. Clustering was inhibited by the addition of alpha-methyl-D-mannoside, a ligand which also binds concanavalin A. These results suggest that the orthogonal arrays (or associated components) specifically bind concanavalin A. Sarcolemmal vesicles from rabbit sacrospinalis (SAC) and rat extensor digitorum longus (EDL) (both primarily fast-twitch) and rat soleus (SOL) (primarily slow-twitch) were obtained by a combination of low-salt fractionation and sucrose density gradient centrifugation. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins and glycoproteins solubilized from these vesicles revealed several bands. Four of these bands were present in gels from both the rabbit and rat fast-twitch muscle sarcolemmal preparations (that contained arrays), yet were absent in gels from rat slow-twitch muscle sarcolemmal preparations (not bearing arrays). An enrichment in vesicles containing arrays was achieved by binding SAC sarcolemmal vesicles to Con A-Sepharose 4B beads. SDS-PAGE analysis of array-enriched vesicles from the concanavalin A beads revealed enrichment of three major bands at Mr 93,000, 54,000 and 49,000. These enriched bands correlate with three of the four bands common to fast-twitch EDL and SAC, yet absent in slow-twitch SOL sarcolemmal preparations. We conclude that at least one macromolecular component associated with the sarcolemmal orthogonal array is a concanavalin A binding glycoprotein. We further conclude that three candidates for this component co-purify with the morphological array, and have approximate molecular weights of 93,000, 54,000 and 49,000.     

Separation and concentration of amino acids using liquid emulsion membranes

The separation and concentration of amino acids using liquid emulsion membranes (LEMs) are discussed. Using L- phenylalanines as a model solute, it is experimentally shown using a facilitated transport system that separation and concentration can be simultaneously achieved. The rate of separation, final product concentration, and membrane swell are shown to increase with increasing chloride driving forces in the membrane, These effects are shown to be insensitive to the particular salt used as the driving force. Changes in the carrier concentration are shown to result in higher initial fluxes and higher swell rates. Hydrodynamically induced membrane breakage is minimal for the system under consideration. Experiments indicate that osmotically induced water transport ("swelling") in the LEM system is mediated by both the carrier and the emulsion-stabilizing surfactant. The data suggest that this swell is a diffusion-limited process. The specificity of the carrier is examined and is found to be directly related to the hydrophobicity of the solute. Strategies for optimizing LEM formulations are discussed. Emphasis is placed on the hydration characteristics of the surfactant and the specificity of the carrier.