Recognition of Carbohydrate Epitopes Specific for Electron-Dense Granule Antigens from Entamoeba histolytica by Monoclonal Antibodies in the Cecal Content of Infected Hamsters

Saccharomyces cerevisiae CGR1 encodes a 120-amino acid protein with a predominant nucleolar localization. In this study we report the identification and cloning of the ortholog, cgrA, from Aspergillus nidulans. The cgrA gene is comprised of three exons on A. nidulans Chromosome 7. The cDNA contains a single open reading frame (ORF) that would encode a protein of 114 amino acids with 44% sequence identity to yeast Cgr1p. A plasmid expressing cgrA complemented the impaired growth phenotype of a yeast strain that can be inducibly depleted of CGR1, and a green fluorescent protein (GFP)-tagged CgrA protein had the same nucleolar localization as the corresponding yeast protein. These results identify cgrA as the A. nidulans ortholog of yeast CGR1 and suggest evolutionary conservation of nucleolar localization mechanisms used by these proteins. Received: 14 September 2000 / Accepted: 13 November 2000     

Occurrence and Diversity of <Emphasis Type="Italic">Legionellaceae</Emphasis> in Polar Lakes of the Antarctic Peninsula

Legionellaceae is a family of Gram-negative, mesophilic, and facultative intracellular parasitic bacteria that inhabits freshwater environments. In this article, the Legionella population of water samples from the North and South Lake, located close to the Brazilian Scientific Station on King George Island, Keller Peninsula, Antarctica has been characterized. Culture onto selective medium and a independent-culture method were applied to the samples. In our attempt to isolate Legionella species from Antarctic lakes, we were able to obtain one L. pneumophila colony by an amoebic coculture procedure followed by plate culture onto a selective medium. In addition, results obtained from phylogenetic inference showed the presence of noncharacterized specimens of Legionella spp. These findings indicated the presence of legionellae in Antarctica and suggest that these bacteria can adapt to extreme conditions and open new possibilities for understanding the survival strategies of mesophilic Legionellaceae living in Antarctic environments. Furthermore, the isolation of these symbiotic bacteria in Antarctic lakes will allow future studies on cold-resistant mechanisms of legionellae in polar environments.     

Effectiveness of different species of entomopathogenic nematodes for biocontrol of the Mediterranean flatheaded rootborer, Capnodis tenebrionis (Linné) (Coleoptera: Buprestidae) in potted peach tree

The susceptibility of larvae of the Mediterranean flatheaded rootborer (Capnodis tenebrionis) to 13 isolates of entomopathogenic nematodes was examined using GF-677 potted trees (peachxalmond hybrid) as the host plant. The nematode strains tested included nine Steinernema feltiae, one S. affine, one S. carpocapsae and two Heterorhabditis bacteriophora. Nematodes showed the ability to locate and kill larvae of C. tenebrionis just after they enter into the roots of the tree. S. feltiae strains provided an efficacy ranging from 79.68% to 88.24%. H. bacteriophora strains resulted in control of 71.66-76.47%. S. carpocapsae (B14) and S. affine (Gspe3) caused lower control of C. tenebrionis larvae (62.03% and 34.76%, respectively). The influence of foraging strategy and the use of autochthonous nematodes to control C. tenebrionis larvae inside the roots is discussed.     

β-Alanine transport into plasma membrane vesicles derived from rat brain synaptosomes

Transport of -alanine has been demonstrated in membrane vesicles isolated from rat brain, using artificially imposed ion gradients as the sole energy source. The uptake of -alanine is strictly dependent on the presence of Na⁺ and Cl^– in the medium, and the process can be driven either by an Na⁺ gradient (out > in) or by a Cl^– gradient (out > in) when the other essential ion is present. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of ionophore valinomycin and anions with different permeabilities. -Alanine uptake is inhibited by the presence of GABA.     

Spatial but not temporal co-divergence of a virus and its mammalian host

Co-divergence between host and parasites suggests that evolutionary processes act across similar spatial and temporal scales. Although there has been considerable work on the extent and correlates of co-divergence of RNA viruses and their mammalian hosts, relatively little is known about the extent to which virus evolution is determined by the phylogeographic history of host species. To test hypotheses related to co-divergence across a variety of spatial and temporal scales, we explored phylogenetic signatures in Andes virus (ANDV) sampled from Chile and its host rodent, Oligoryzomys longicaudatus. ANDV showed strong spatial subdivision, a phylogeographic pattern also recovered in the host using both spatial and genealogical approaches, and despite incomplete lineage sorting. Lineage structure in the virus seemed to be a response to current population dynamics in the host at the spatial scale of ecoregions. However, finer scale analyses revealed contrasting patterns of genetic structure across a latitudinal gradient. As predicted by their higher substitution rates, ANDV showed greater genealogical resolution than the rodent, with topological congruence influenced by the degree of lineage sorting within the host. However, despite these major differences in evolutionary dynamics, the geographic structure of host and virus converged across large spatial scales.     

Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells

The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl^-release in intestinal cells, including the human colonic carcinoma T₈₄ cell line, involving increased cGMP and membrane alkalization due to reduced Na⁺/H⁺ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pH_i), and NHE1, NHE2, and NHE4 are expressed in T₈₄ cells, we characterized the STa role as modulator of these exchangers. pH_i was assayed by the NH₄Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pH_i recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H⁺ efflux (J _H ⁺) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J _H ⁺ (~63%), without altering basal pH_i (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pH_i units. The dpHi/dt and J _H ⁺ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa–decreased dpHi/dt and J _H ⁺ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T₈₄ cells with STa results in reduced NHE4 activity leading to a lower capacity of pH_i recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.     

Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

Background

Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood.

Methodology/Principal findings

Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone.We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus.

Conclusions/significance

The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials.     

Plant life on gypsum: a review of its multiple facets

The adaptation of plants to particular soil types has long intrigued biologists. Gypsum soils occupy large areas in many regions of the world and host a striking biological diversity, but their vegetation has been much less studied than that developing over serpentine or saline soils. Herein, we review all aspects of plant life on gypsum ecosystems, discuss the main processes driving their structure and functioning, and highlight the main conservation threats that they face. Plant communities in gypsum habitats typically show distinctive bands at very small spatial scales, which are mainly determined by topography. Plants living on gypsum soils can be classified into three categories: (i) wide gypsophiles are specialists that can penetrate the physical soil crust during early life stages and have physiological adjustments to cope with the chemical limitations imposed by gypsum soils; (ii) narrow gypsophiles are refugee plants which successfully deal with the physical soil crust and can tolerate these chemical limitations but do not show specific adaptations for this type of soils; and (iii) gypsovags are non‐specialist gypsum plants that can only thrive in gypsum soils when the physical crust is absent or reduced. Their ability to survive in gypsum soils may also be mediated by below‐ground interactions with soil microorganisms. Gypsophiles and gypsovags show efficient germination at low temperatures, seed and fruit heteromorphism within and among populations, and variation in seed dormancy among plants and populations. In gypsum ecosystems, spatio‐temporal changes in the composition and structure of above‐ground vegetation are closely related to those of the soil seed bank. Biological soil crusts (BSCs) dominated by cyanobacteria, lichens and mosses are conspicuous in gypsum environments worldwide, and are important drivers of ecosystem processes such as carbon and nitrogen cycling, water infiltration and run‐off and soil stability. These organisms are also important determinants of the structure of annual plant communities living on gypsum soils. The short‐distance seed dispersal of gypsophiles is responsible for the high number of very narrow endemisms typically found in gypsum outcrops, and suggests that these species are evolutionarily old taxa due to the time they need to colonize isolated gypsum outcrops by chance. Climate change and habitat fragmentation negatively affect both plants and BSCs in gypsum habitats, and are among the major threats to these ecosystems. Gypsum habitats and specialists offer the chance to advance our knowledge on restrictive soils, and are ideal models not only to test important evolutionary questions such as tolerance to low Ca/Mg proportions in soils, but also to improve the theoretical framework of community ecology and ecosystem functioning.     

The partial inhibition of soil urease by naturally occurring polyphenols

Summary Urea applied to soils is converted to ammonium carbonate by the action of the urease enzyme system. The nitrogen can then be lost by volatilisation as ammonia. Data is presented in this paper to show that naturally occuring polyphenolic compounds can be used to effectively inhibit urease activity at soil surfaces, thereby preventing the loss of nitrogen.     

Live cell imaging of the assembly, disassembly, and actin cable-dependent movement of endosomes and actin patches in the budding yeast, Saccharomyces cerevisiae

Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64–labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement. Rather, linear actin patch movement occurs at the same velocity and direction as the movement of actin cables. Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement. Our studies support a mechanism whereby actin cables serve as “conveyor belts” for retrograde movement and delivery of actin patches/endosomes to FM4-64–labeled internal compartments.