Neutral red as a probe for confocal laser scanning microscopy studies of plant roots

BACKGROUND AND AIMS: Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology. METHODS: NR was used as a fluorochrome for living roots of Phaseolus vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM. The effect of NR on the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye. KEY RESULTS: The main advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylem elements, Casparian bands in the endodermis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular-arbuscular mycorrhizas. CONCLUSIONS: Ease, low cost and absence of tissue processing make NR a useful probe for structural, developmental and vacuole-biogenetic studies of plant roots with CLSM.     

Effects of Cold Shock on Microtubule Organization and Cell Cycle in Gynogenetically Activated Eggs of Olive Flounder (Paralichthys olivaceus)

Cytological changes and subsequent mitotic processes were studied in gynogenetically activated eggs of olive flounder subjected to cold-shock treatment using indirect immunofluorescence staining of isolated blastodisks. Obvious differences between controls and treated eggs were detected during early cell division. The developmental process of haploid control was similar to that of the diploid control except several minutes delayed. Spindles disassembled by the cold-shock treatment regenerated soon after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole was easily depolymerized by cold-shock treatment, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle, resulting in chromosome set doubling. Some two-cell stage eggs had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These eggs had a high potency developing into haploid-diploid mosaics. To the best of our knowledge, this study is the first to clarify the mechanism of chromosome set doubling in marine fishes and provides a preliminary cytological basis for developing a reliable and efficient protocol for mitotic gynogenesis induction by cold-shock treatment in olive flounder.     

EFL GTPase in cryptomonads and the distribution of EFL and EF-1alpha in chromalveolates

EFL (EF-like protein) is a member of the GTPase superfamily that includes several translation factors. Because it has only been found in a few eukaryotic lineages and its presence correlates with the absence of the related core translation factor EF-1alpha, its distribution is hypothesized to be the result of lateral gene transfer and replacement of EF-1alpha. In one supergroup of eukaryotes, the chromalveolates, two major lineages were found to contain EFL (dinoflagellates and haptophytes), while the others encode EF-1alpha (apicomplexans, ciliates, heterokonts and cryptomonads). For each of these groups, this distribution was deduced from whole genome sequence or expressed sequence tag (EST) data from several species, with the exception of cryptomonads from which only a single EF-1alpha PCR product from one species was known. By sequencing ESTs from two cryptomonads, Guillardia theta and Rhodomonas salina, and searching for all GTPase translation factors, we revealed that EFL is present in both species, but, contrary to expectations, we found EF-1alpha in neither. On balance, we suggest the previously reported EF-1alpha from Rhodomonas salina is likely an artefact of contamination. We also identified EFL in EST data from two members of the dinoflagellate lineage, Karlodinium micrum and Oxyrrhis marina, and from an ongoing genomic sequence project from a third, Perkinsus marinus. Karlodinium micrum is a symbiotic pairing of two lineages that would have both had EFL (a dinoflagellate and a haptophyte), but only the dinoflagellate gene remains. Oxyrrhis marina and Perkinsus marinus are early diverging sister-groups to dinoflagellates, and together show that EFL originated early in this lineage. Phylogenetic analysis confirmed that these genes are all EFL homologues, and showed that cryptomonad genes are not detectably related to EFL from other chromalveolates, which collectively form several distinct groups. The known distribution of EFL now includes a third group of chromalveolates, cryptomonads. Of the six major subgroups of chromalveolates, EFL is found in half and EF-1alpha in the other half, and none as yet unambiguously possess both genes. Phylogenetic analysis indicates EFL likely arose early within each subgroup where it is found, but suggests it may have originated multiple times within chromalveolates as a whole.     

Mathematical modelling of the aerobic degradation of two-phase olive mill effluents in a batch reactor

A laboratory-scale study was conducted on the aerobic degradation of two-phase olive mill effluents (TPOME) made up of the mixture of the washwaters derived from the initial cleansing of the olives and those obtained in the washing and purification of virgin olive oil. The process was carried out in a 1-l working volume stirred tank reactor operating in batch mode at room temperature (25 °C). The reactor was operated at influent substrate concentrations of 2.80 g COD/l (TPOME 25%), 5.45 g COD/l (TPOME 50%), 8.18 g COD/l (TPOME 75%) and 10.90 g COD/l (TPOME 100%). After five days of operation time, total and soluble COD removal efficiencies of 64.3% and 66.6% were achieved respectively for the most concentrated influent used (TPOME 100%). A simplified kinetic model for studying the hydrolysis of insoluble organic matter, oxidation of soluble substrate and biomass production was proposed on the basis of the experimental results obtained. The following kinetic constants with their standard deviations were obtained for the above stages in the case of the most concentrated influent used (TPOME 100%): k₁ (kinetic constant for hydrolysis of suspended organic matter): 0.11 ± 0.01 l/(g VSS day); k₂ (kinetic constant for total consumption of soluble substrate): 0.30 ± 0.02 l/(g VSS day); k₃ (endogenous metabolism constant): 0.07 ± 0.01 per day). Finally, the biomass yield coefficient was found to be 0.30 g VSS/g COD_removed. The values of non-biodegradable total and soluble CODs obtained from the model were found to be 3 and 2 g/l, respectively. The kinetic constants obtained and the proposed equations were used to simulate the aerobic degradation process of TPOME and to obtain the theoretical values of non-soluble and soluble CODs and biomass concentration. The small deviations obtained (equal or lower than 10%) between the theoretical and experimental values suggest that the parameters obtained represent and predict the activity of the microorganisms involved in the overall aerobic degradation process of this wastewater.     

Efforts to Explain and Control the Prolonged Thermophilic Period in Two-phase Olive Oil Mill Sludge Composting

The aim of this paper was to evaluate the use of different bulking agents in different ratios as a means to control, optimise and eventually reduce the duration of the thermophilic period in two-phase olive oil mill sludge (OOMS) composting. The bulking agents used were: (i) olive tree leaves (OTL), (ii) olive tree shredded branches (OTB) and (iii) woodchips (WDC). The selection of these materials was based on their abundance and availability on the island of Crete, the southernmost point of Greece. The ratios studied were: Pile 1, OOMS:OTL in 1:1 v/v; Pile 2, OOMS:WDC in 1:1.5 v/v; Pile 3, OOMS:OTL in 1:2 v/v; Pile 4, OOMS:OTL:OTB in 1:1:1 v/v; and Pile 5, OOMS:OTL:OTB in 1:1:2 v/v. The composting system used was that of windrows with the volume of each pile approximately 20–25 m³. The experiments took place over two consecutive years. A composting turner was used and turnings were performed at one and two week intervals. In each pile a variety of physiochemical parameters were monitored. Temperature remained high in all five trials. Piles 1, 2, 3, 4 and 5 temperatures recorded values of above 50 °C for 106, 158, 160, 175 and 183 days, respectively. Volumes were reduced by approximately 67%, 62%, 63%, 80% and 84%, respectively. Temperature remained high, mainly due to the presence in large amounts of oily substances which during their complete oxidation release important amounts of energy and aid the cometabolism of more stable molecules such as lignin. This process is better described as the slow “burning” of a “fuel” mixture in an “engine” than composting. This approach is based on the extensive similarities of this process to that of crude oil sludge or similar waste composting.     

A multivariate population density model of the dLGN/PGN relay

Using a population density approach we study the dynamics of two interacting collections of integrate-and-fire-or-burst (IFB) neurons representing thalamocortical (TC) cells from the dorsal lateral geniculate nucleus (dLGN) and thalamic reticular (RE) cells from the perigeniculate nucleus (PGN). Each population of neurons is described by a multivariate probability density function that satisfies a conservation equation with appropriately defined probability fluxes and boundary conditions. The state variables of each neuron are the membrane potential and the inactivation gating variable of the low-threshold Ca²⁺ current I_T. The synaptic coupling of the populations and external excitatory drive are modeled by instantaneous jumps in the membrane potential of postsynaptic neurons. The population density model is validated by comparing its response to time-varying retinal input to Monte Carlo simulations of the corresponding IFB network composed of 100 to 1000 cells per population. In the absence of retinal input, the population density model exhibits rhythmic bursting similar to the 7 to 14 Hz oscillations associated with slow wave sleep that require feedback inhibition from RE to TC cells. When the TC and RE cell potassium leakage conductances are adjusted to represent cholingergic neuromodulation and arousal of the network, rhythmic bursting of the probability density model may either persists or be eliminated depending on the number of excitatory (TC to RE) or inhibitory (RE to TC) connections made by each presynaptic cell. When the probability density model is stimulated with constant retinal input (10–100 spikes/sec), a wide range of responses are observed depending on cellular parameters and network connectivity. These include asynchronous burst and tonic spikes, sleep spindle-like rhythmic bursting, and oscillations in population firing rate that are distinguishable from sleep spindles due to their amplitude, frequency, or the presence of tonic spikes. In this context of dLGN/PGN network modeling, we find the population density approach using 2,500 mesh points and resolving membrane voltage to 0.7 mV is over 30 times more efficient than 1000-cell Monte Carlo simulations. Action Editor: David Golomb     

Dichotomous versus palm-type mechanisms of lateral assembly of amyloid fibrils

Despite possessing a common cross-beta core, amyloid fibrils are known to exhibit great variations in their morphologies. To date, the mechanism responsible for the polymorphism in amyloid fibrils is poorly understood. Here we report that two variants of mammalian full-length prion protein (PrP), hamster (Ha) and mouse (Mo) PrPs, produced morphologically distinguishable subsets of mature fibrils under identical solvent conditions. To gain insight into the origin of this morphological diversity we analyzed the early stages of polymerization. Unexpectedly, we found that despite a highly conserved amyloidogenic region (94% identity within the residues 90-230), Ha and Mo PrPs followed two distinct pathways for lateral assembly of protofibrils into mature, higher order fibrils. The protofibrils of Ha PrP first formed irregular bundles characterized by a peculiar palm-type shape, which ultimately condensed into mature fibrils. The protofibrils of Mo PrP, on the other hand, associated in pairs in a pattern resembling dichotomous coalescence. These pathways are referred to here as the palm-type and dichotomous mechanisms. Two distinct mechanisms for lateral assembly explain striking differences in morphology of mature fibrils produced from closely related Mo and Ha PrPs. Remarkable similarities between subtypes of amyloid fibrils generated from different proteins and peptides suggest that the two mechanisms of lateral assembly may not be limited to prion proteins but may be a common characteristic of polymerization of amyloidogenic proteins and peptides in general.     

Phenolic removal in olive oil mill wastewater using loofah-immobilized Phanerochaete chrysosporium

Summary Olive oil mill wastewater (OMW) has a high organic load, and this is a serious concern of the olive industry. Conventional biological wastewater treatments, despite their simplicity and suitable performance are ineffective for OMW treatment since phenolics possess antimicrobial activity. In order to carry out a proper treatment of OMW, use of a microorganism able to degrade the phenolics is thus necessary. In this study the ability of Phanerochaete chrysosporium to degrade the phenolic compounds of OMW and to decrease the chemical oxygen demand (COD) using cells immobilized on loofah was examined. The basal mineral salt solution along with glucose, ammonium sulfate and yeast extract was used to dilute the OMW appropriately. The fungus did not grow on the concentrated OMW. The extent of removal in this bio-treatment, of total phenols (TP) and the COD were 90 and 50%, respectively, while the color and aromaticity decreased by 60 and 95%, respectively. The kinetic behavior of the loofah-immobilized fungus was found to follow the Monod equation. The maximum growth rate μ_max was 0.045 h⁻¹ while the Monod constant based on the consumed TP and COD were (mg/l) 370 and 6900, respectively.     

Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu-Zn, Zn-Zn and as-isolated wild-type enzymes

Human Cu-Zn superoxide dismutase (SOD1) protects cells from the effects of oxidative stress. Mutations in SOD1 are linked to the familial form of amyotrophic lateral sclerosis. Several hypotheses for their toxicity involve the mis-metallation of the enzyme. We present atomic-resolution crystal structures and biophysical data for human SOD1 in three metallation states: Zn-Zn, Cu-Zn and as-isolated. These data represent the first atomic-resolution structures for human SOD1, the first structure of a reduced SOD1, and the first structure of a fully Zn-substituted SOD1 enzyme. Recombinantly expressed as-isolated SOD1 contains a mixture of Zn and Cu at the Cu-binding site. The Zn-Zn structure appears to be at least as stable as the correctly (Cu-Zn) metallated enzyme. These data raise the possibility that in a cellular environment with low availability of free copper, Zn-Zn may be the preferred metallation state of SOD1 prior to its interaction with the copper chaperone.     

Loss of metabotropic glutamate receptor-mediated regulation of glutamate transport in chemically activated astrocytes in a rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.