Regional variation in soil carbon and δ<Superscript>13</Superscript>C in forests and pastures of northeastern Costa Rica

Recent studies suggest that the direction and magnitude of changes in soil organic carbon (soil C) pools following forest-to-pasture conversion in the tropics are dependent upon initial soil conditions and local factors (e.g. pre-conversion soil C content, soil texture, vegetation productivity, and management practices). The goal of this study was to understand how landscape-scale variation in soil-forming factors influenced the response of soil C pools to forest clearing and pasture establishment in northeastern Costa Rica. We measured soil C and its stable isotopic composition in 24 paired pasture and reference forest sites distributed over large gradients of edaphic characteristics and slope throughout a 1400 km² region. We used the large difference in stable C isotopic signatures of C3 vegetation (rain forest) versus C4 vegetation (pasture grasses) as a tracer of soil C dynamics. Soil C pools to 30 cm depth ranged from 26% lower to 23% higher in pastures compared to paired forests. The presence of non-crystalline clays and percent slope explained between 27 and 37% of the variation in the direction and magnitude of the changes in soil C storage following pasture establishment. Stable carbon isotopes (¹³C) in the top soil (0–10 cm) showed a rapid incorporation of pasture-derived C following pasture establishment, but the vegetation in these pastures never became pure C4 communities. The amount of forest-derived soil C in pasture topsoils (0–10 cm) was negatively correlated to both pasture age and the concentrations of non-crystalline iron oxides. Together these results imply that site factors such as soil mineralogy are an important control over soil C storage and turnover in this region.     

Effect of ACTH and CRH on Plasma Levels of Cortisol and Prostaglandin F<Subscript>2α</Subscript> Metabolite in Cycling Gilts and Castrated Boars

The present study was designed to evaluate the effects of synthetic ACTH (1–24, tetracosactid) and porcine CRH on the plasma levels of cortisol and PGF_2α metabolite in cycling gilts (n = 3) and castrated boars (n = 3). The experiments were designed as crossover studies for each gender separately. Each animal received, during three consecutive days; 1) ACTH (Synacthen^® Depot) at a dose of 10 μg/kg body weight in 5 ml physiological saline, 2) porcine CRH at a dose 0.6 μg/kg body weight in 5 ml physiological saline or 3) physiological saline (5 ml). The test substances were administered via an indwelling jugular cannula in randomized order according to a Latin square. The administration of ACTH to cycling gilts resulted in concomitant elevations of cortisol and PGF_2α metabolite with peak levels reached at 70.0 ± 10.0 and 33.3 ± 6.7 min, respectively. Similarly, the administration of ACTH to castrated boars resulted in concomitant elevation of cortisol and PGF_2α metabolite with peak levels reached at 60.0 ± 0.0 and 20.0 ± 0.0 min, respectively. Cortisol peaked at 20 min after administration of CRH in both cycling gilts and castrated boars with maximum levels of 149.3 ± 16.5 nmol/1 and 138.3 ± 10.1 nmol/1, respectively. It can be concluded that administration of synthetic ACTH (tetracosactid) to pigs caused a concomitant elevation of cortisol and PGF_2α metabolite levels in both cycling gilts as well as castrated boars. The administration of CRH to pigs resulted in an elevation of cortisol levels in both cycling gilts and castrated boars. Conversely, PGF_2α metabolite levels were not influenced by the administration of CRH either in cycling gilts or in castrated boars.     

Birth and Death of Genes and Functions in the β-Esterase Cluster of <Emphasis Type="Italic">Drosophila</Emphasis>

Here we analyze the molecular evolution of the β-esterase gene cluster in the Drosophila genus using the recently released genome sequences of 12 Drosophila species. Molecular evolution in this small cluster is noteworthy because it contains contrasting examples of the types and stages of loss of gene function. Specifically, missing orthologs, pseudogenes, and null alleles are all inferred. Phylogenetic analyses also suggest a minimum of 9 gene gain–loss events; however, the exact number and age of these events is confounded by interparalog recombination. A previous enigma, in which allozyme loci were mapped to β-esterase genes that lacked catalytically essential amino acids, was resolved through the identification of neighbouring genes that contain the canonical catalytic residues and thus presumably encode the mapped allozymes. The originally identified genes are evolving with selective constraint, suggesting that they have a “noncatalytic” function. Curiously, 3 of the 4 paralogous β-esterase genes in the D. ananassae genome sequence have single inactivating (frame-shift or nonsense) mutations. To determine whether these putatively inactivating mutations were fixed, we sequenced other D. ananassae alleles of these four loci. We did not find any of the 3 inactivating mutations of the sequenced strain in 12 other strains; however, other inactivating mutations were observed in the same 3 genes. This is reminiscent of the high frequency of null alleles observed in one of the β-esterase genes (Est7/EstP) of D. melanogaster. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transmembrane transport of K<Superscript>+</Superscript> and Cl<Superscript>−</Superscript> during pollen grain activation in vivo and in vitro

We studied the possibility of K⁺ and Cl⁻ efflux from tobacco pollen grains during their activation in vitro or on the stigma of a pistil. For this purpose the X-ray microanalysis and spectrofluorometry were applied. We found that the relative content of potassium and chlorine in the microvolume of pollen grain decreases during its hydration and activation on stigma. Efflux of these ions was found both in vivo and in vitro. In model in vitro experiments anion channel inhibitor NPPB ((5-nitro-2-(3-phenylpropylamino) benzoic acid) in the concentration that was blocking pollen germination, reduced Cl⁻ efflux; potassium channel inhibitor TEA (tetraethylammonium chloride) partially reduced K⁺ efflux and lowered the percent of activated cells. Another blocker of potassium channels Ba²⁺ caused severe decrease in cell volume and blocked the activation. In general, the obtained data demonstrates that the initiation of pollen germination both in vivo and in vitro involves the activation of K⁺ and Cl⁻ release. An important role in these processes is played by NPPB-, TEA- and Ba²⁺-sensitive plasmalemma ion channels.     

The tumor-growth inhibitory activity of flavanone and 2′-OH flavanone <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> through induction of cell cycle arrest and suppression of cyclins and CDKs

Summary Natural products, including flavonoids, are suggested to be involved in the protective effects of fruits and vegetables against cancer. However, studies concerning the effect of flavonoids frequently lacked data regarding to flavanones. In this study, we investigated the inhibitory effect of flavanone compounds, including flavanone, 2′-OH flavanone, 4′-OH flavanone, 6-OH flavanone, naringin and naringenin, on cell growth of various cancer cells. We determined that flavanone and 2′-OH flavanone inhibited cell growth of A549, LLC, AGS, SK-Hepl and HA22T cancer cells, while other flavanones showed little or no inhibition. We evaluated growth-inhibitory activity of flavanone and 2′-OH flavanone against highly proliferative human lung cancer cells (A549) via anchorage-independent and -dependent colony formation assay, and further showed that treatment of flavanone resulted in a G1 cell cycle arrest with reduction of cyclin D, E and cyclin-dependent kinase (CDK) 2, while treatment of 2′-OH flavanone led to a G2/M phase accumulation with reduction of cyclin B, D and Cdc2. Moreover, we demonstrated the improvement effect of flavanone and 2′-OH flavanone with anti-cancer drug, doxorubicin, on A549 cells. Finally, flavanone and 2′-OH flavanone were evidenced by its inhibition on the growth of A549 and Lewis lung carcinoma cells in vivo. Yung-Chin Hsiao and Yih-Shou Hsieh are equally contributed to this work.     

Immobilization of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> α-galactosidase in gelatin and its application in removal of flatulence-inducing sugars in soymilk

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean-derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. In this study, α-galactosidase from Aspergillus oryzae was entrapped in gelatin using formaldehyde as the hardener. The immobilization yield was 64.3% under the optimum conditions of immobilization. The immobilized α-galactosidase showed a shift in optimum pH from 4.8 to 5.4 in acetate buffer. The optimum temperature also shifted from 50°C to 57°C compared with soluble enzyme. Immobilized α-galactosidase was used in batch, repeated batch and continuous mode to degrade RO present in soymilk. In the repeated batch, 45% reduction of RO was obtained in the fourth cycle. The performance of immobilized α-galactosidase was tested in a fluidized bed reactor at different flow rates and 86% reduction of RO in soymilk was obtained at 25 ml h⁻¹ flow rate. The study revealed that immobilized α-galactosidase in continuous mode is efficient in reduction of RO present in soymilk.     

Cooperation of adenosine and prostaglandin E<Subscript>2</Subscript> (PGE<Subscript>2</Subscript>) in amplification of cAMP–PKA signaling and immunosuppression

INTRODUCTION: We hypothesize that adenosine and PGE(2) could have a complementary immunosuppressive effect that is mediated via common cAMP-PKA signaling. MATERIALS AND METHODS: To test this hypothesis, the effect of adenosine and PGE(2) on the cytotoxic activity and cytokine production of lymphokine activated killer (LAK) cells was investigated. RESULTS: PGE(2) and adenosine inhibited LAK cells cytotoxic activity and production of INF-gamma, GM-CSF and TNF-alpha. In combination they showed substantially higher inhibition than each modality used alone. Using agonists and antagonists specific for PGE(2) and adenosine receptors we found that cooperation of PGE(2) and adenosine in their inhibitory effects are mediated via EP(2) and A(2A) receptors, respectively. LAK cells have 35-fold higher expression of EP(2) than A(2A). Combined PGE(2) and adenosine treatment resulted in augmentation of cAMP production, PKA activity, CREB phosphorylation and inhibition of Akt phosphorylation. Wortmannin and LY294002 enhanced the suppressive effects of adenosine and PGE(2). In contrast, Rp-8-Br-cAMPS, an inhibitor of PKA type I blocked their immunosuppressive effects, suggesting that the inhibitory effects of PGE(2) and adenosine are mediated via common pathway with activation of cAMP-PKA and inhibition of Akt. CONCLUSION: In comparison to other immunosuppressive molecules (TGF-beta and IL-10), adenosine and PGE(2) are unique in their ability to inhibit the executive function of highly cytotoxic cells. High intratumor levels of adenosine and PGE(2) could protect tumor from immune-mediated destruction by inactivation of the tumor infiltrating functionally active immune cells.     

Acquisition and Evolution of Plant Pathogenesis–Associated Gene Clusters and Candidate Determinants of Tissue-Specificity in Xanthomonas

Background

Xanthomonas is a large genus of plant-associated and plant-pathogenic bacteria. Collectively, members cause diseases on over 392 plant species. Individually, they exhibit marked host- and tissue-specificity. The determinants of this specificity are unknown.

Methodology/Principal Findings

To assess potential contributions to host- and tissue-specificity, pathogenesis-associated gene clusters were compared across genomes of eight Xanthomonas strains representing vascular or non-vascular pathogens of rice, brassicas, pepper and tomato, and citrus. The gum cluster for extracellular polysaccharide is conserved except for gumN and sequences downstream. The xcs and xps clusters for type II secretion are conserved, except in the rice pathogens, in which xcs is missing. In the otherwise conserved hrp cluster, sequences flanking the core genes for type III secretion vary with respect to insertion sequence element and putative effector gene content. Variation at the rpf (regulation of pathogenicity factors) cluster is more pronounced, though genes with established functional relevance are conserved. A cluster for synthesis of lipopolysaccharide varies highly, suggesting multiple horizontal gene transfers and reassortments, but this variation does not correlate with host- or tissue-specificity. Phylogenetic trees based on amino acid alignments of gum, xps, xcs, hrp, and rpf cluster products generally reflect strain phylogeny. However, amino acid residues at four positions correlate with tissue specificity, revealing hpaA and xpsD as candidate determinants. Examination of genome sequences of xanthomonads Xylella fastidiosa and Stenotrophomonas maltophilia revealed that the hrp, gum, and xcs clusters are recent acquisitions in the Xanthomonas lineage.

Conclusions/Significance

Our results provide insight into the ancestral Xanthomonas genome and indicate that differentiation with respect to host- and tissue-specificity involved not major modifications or wholesale exchange of clusters, but subtle changes in a small number of genes or in non-coding sequences, and/or differences outside the clusters, potentially among regulatory targets or secretory substrates.     

Variability and directionality of temporal changes in δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N of aquatic invertebrate primary consumers

Seasonal oscillations in the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope signatures of aquatic algae can cause seasonal enrichment–depletion cycles in the isotopic composition of planktonic invertebrates (e.g., copepods). Yet, there is growing evidence that seasonal enrichment–depletion cycles also occur in the isotope signatures of larger invertebrate consumers, taxa used to define reference points in isotope-based trophic models (e.g., trophic baselines). To evaluate the general assumption of temporal stability in non-zooplankton aquatic invertebrates, δ¹³C and δ¹⁵N time series data from the literature were analyzed for seasonality and the influence of biotic (feeding group) and abiotic (trophic state, climate regime) factors on isotope temporal patterns. The amplitude of δ¹³C and δ¹⁵N enrichment–depletion cycles was negatively related to body size, although all size-classes of invertebrates displayed a winter-to-summer enrichment in δ¹³C and depletion in δ¹⁵N. Among feeding groups, periphytic grazers were more variable and displayed larger temporal changes in δ¹³C than detritivores. For nitrogen, temporal variability and magnitude of directional change of δ¹⁵N was most strongly related to ecosystem trophic state (eutrophic > mesotrophic, oligotrophic). This study provides evidence of seasonality in the isotopic composition of aquatic invertebrates across very broad geographical and ecological gradients as well as identifying factors that are likely to modulate the strength and variability of seasonality. These results emphasize the need for researchers to recognize the likelihood of temporal changes in non-zooplankton aquatic invertebrate consumers at time scales relevant to seasonal studies and, if present, to account for temporal dynamics in isotope trophic models.     

Effective and ecological half-lives of <Superscript>137</Superscript>Cs in cow’s milk in alpine agriculture

In the mountainous “Hohe Tauern” region of Salzburg (Austria), milk samples have been collected in a long-term montitoring programme since 1988, at eight alpine sites used for extensive, seasonal stock farming. For this alpine environment with its acidic soils developed on silicate bedrock, high soil-to-plant transfer factors and long-lasting ¹³⁷Cs contamination levels in milk—the main product of seasonal agriculture at elevated altitudes—are characteristic features. The decrease in ¹³⁷Cs concentration in milk measured since 1988 turned out to be best described by one or two effective half-lives. For the period from 1993 to 2007, which can be modelled with one effective half-life for all sites, effective half-lives between 3.7 and 15.0 years (ecological half-lives: 4.3–29.9 years) were obtained. The effective half-life increases with mean altitude of the investigated graze pastures, probably due to reduced migration velocities of ¹³⁷Cs and low ¹³⁷Cs half-value depths of a few centimetres in the soil.     

The role of Cl<Superscript>−</Superscript> in pollen germination and tube growth

The involvement of Cl^? in cytoplasm polarization in the pollen tube and membrane potential control during pollen germination in vitro was studied by fluorescence techniques in Nicotiana tabacum. Cl^? release from cells was blocked by the anion channel inhibitor nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or by the addition of Cl^? to the incubation medium. The concentrations of the inhibitor (40 μM) and extracellular Cl^? completely inhibiting pollen germination (200 mM) and pollen tube growth (100 mM) were used. The release of anions from the pollen grain has been revealed in the first minutes of hydration also in the presence of 200 mM Cl^?. The inhibitor blocked this process completely, which points to the significance of the NPPB-sensitive anion channels in the transmembrane Cl^? transport at the early activation stage. The pollen tube membrane was hyperpolarized in the presence of 100 mM Cl^?; however, exogenous Cl^? had no effect on the compartmentalization and organelle movement in the tube. The inhibitor depolarized the plasma membrane in the pollen grain and tube and affected the polar organization of the cytoplasm and organelle movement. Thus, activity of NPPB-sensitive chloride channels was required to regulate the potential on the plasma membrane and to maintain the functional compartmentalization of the cytoplasm, which provides for the polar growth.     

Effect of ammonium ions and cytokinins on hyperhydricity and multiplication rate of in vitro regenerated shoots of <Emphasis Type="Italic">Aloe polyphylla</Emphasis>

In search for the optimal culture conditions resulting in a high production of healthy plants and low occurrence of hyperhydricity in tissue cultured regenerants of Aloe polyphylla, we investigated the relationship between ammonium ions in the medium, applied cytokinins (CKs) and CK concentrations in the induction of hyperhydricity. Shoots were grown on media with different NH₄ ⁺ concentrations (10.3, 20.6 and 61.8 mM) and supplemented with N⁶-benzyladenine (BA), zeatin or thidiazuron (TDZ) at 0, 5 or 15 μM. Elevating the levels of NH₄ ⁺, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH₄ ⁺ (10.3 mM). However, in the presence of higher NH₄ ⁺ concentrations, CKs increased hyperhydricity in a concentration-dependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH₄ ⁺ and low BA or zeatin (5 μM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. Identifying the factors responsible for hyperhydricity is an important step in the successful use of the micropropagation technique for the conservation of this species.     

Paradigm shifts in pathophysiology and management of atrial fibrillation—a tale of the RACE trials in the Netherlands

In the past 20 years the Netherlands-based RACE trials have investigated important concepts in clinical atrial fibrillation (AF). Their scope ranged from rhythm versus rate control to early or delayed cardioversion and also included early comprehensive management of AF in two trials, one focusing on early ‘upstream therapy’ and risk factor management and the other on integrated chronic nurse-led care. Studies were mostly triggered by simple clinical observations including futility of electrical cardioversion in persistent AF; many patients with permanent AF tolerating day-after-day ‘uncontrolled’ resting heart rates of up till 110 beats/min; patients being threatened more by vascular risks than AF itself; and insufficient guideline-based treatments for AF. Also the observation that recent-onset atrial fibrillation generally converts spontaneously, obviating cardioversion, triggered one of the studies. The RACE trials shifted a number of paradigms and by that could change the AF guidelines. The initial ‘shock-and-forget’ attitude made place for increased attention for anticoagulation, and in turn, broader vascular risks were recognised. In a nutshell, the adage eventually became: ‘look beyond the ECG, treat the patient’.

     

Interplay of the magnitude and time-course of postsynaptic Ca<Superscript>2 + </Superscript> concentration in producing spike timing-dependent plasticity

Synaptic strength can be modified by the relative timing of pre- and postsynaptic activity, a phenomenon termed spike timing-dependent plasticity (STDP). Studies of neurons in the hippocampus and in other regions have found that when presynaptic activity occurs within a narrow time window, typically 10 or 20 ms, before postsynaptic activity, long-term potentiation (LTP) is induced, while if presynaptic activity occurs within a similar time window after postsynaptic activity, long-term depression (LTD) results. The mechanisms underlying these modifications are not completely understood, although there is strong evidence that the postsynaptic Ca ^2 + concentration plays a central role. Some previous modeling of STDP has focused on the dynamics of the postsynaptic Ca ^2 + concentration, while other work has studied biophysical mechanisms of how a synapse can exist in, and switch between, different states corresponding to LTP and LTD. Building on previous work in these two areas we have developed the first low level STDP model of a tristable biochemical system that incorporates induction and maintenance of both LTP and LTD. Our model is able to explain the STDP observed in hippocampal neurons in response to pre- and postsynaptic pulse pairs, using only parameters derived from previous work and without the need for parameter fine-tuning. Our results also give insight into how and why the time course of the postsynaptic Ca ^2 + concentration can lead to either LTP or LTD, and suggest that voltage dependent calcium channels play a key role.     

Species diversity of edaphic mites (Acari: Oribatida) and effects of topography,soil properties and litter gradients on their qualitative and quantitative composition in 64 km<Superscript>2</Superscript> of forest in Amazonia

Small-scale spatial distribution of oribatid mites has been investigated in Amazonia. In addition, medium- and large-scale studies are needed to establish the utility of these mites in detecting natural environmental variability, and to distinguish this variability from anthropogenic impacts. We are expanding the knowledge about oribatid mites in a wet upland forest reserve, and investigate whether a standardized and integrated protocol is an efficient way to assess the effects of environmental variables on their qualitative and quantitative composition on a large spatial scale inside an ecological reserve in Central Amazonia, Brazil. Samples for Berlese-Tullgren extraction were taken in 72 plots of 250 × 6 m distributed over 64 km². In total 3,182 adult individuals, from 82 species and 79 morphospecies were recorded, expanding the number of species known in the reserve from 149 to 254. Galumna, Rostrozetes and Scheloribates were the most speciose genera, and 57 species were rare. Rostrozetes ovulum, Pergalumna passimpuctata and Archegozetes longisetosus were the most abundant species, and the first two were the most frequent. Species number and abundance were not correlated with clay content, slope, pH and litter quantity. However, Principal Coordinate Analysis indicated that as the percentage of clay content, litter quantity and pH changed, the oribatid mite qualitative and quantitative composition also changed. The standardized protocol effectively captured the diversity, as we collected one of the largest registers of oribatid mites’ species for Amazonia. Moreover, biological and ecological data were integrated to capture the effects of environmental variables accounting for their diversity and abundance.     

Conservation <Emphasis Type="BoldItalic">in vitro</Emphasis> of rare and threatened ferns—case studies of biodiversity hotspot and island species

The importance of in vitro tools to complement other ex situ methods for saving plants from extinction is more relevant than ever before. More than 50% of the world’s plant species are endemic to the 34 global biodiversity hotspots (GBHs), each holding at least 1,500 endemic plant species. In addition, a large number of small islands hold a number of endemic species on the brink of extinction. Conservation support concentrating more on these hotspots and small islands would significantly reduce the loss of species that is currently occurring. In the majority of these cases, the resources are either locally scarce or difficult to access for in vitro conservation to support other ex situ measures. Most island countries are small, and their geographical position is a stumbling block to initiate active partnerships with other countries when they need to use in vitro tools to rescue plants that produce recalcitrant seeds/spores or propagate only by vegetative means. However, many biodiversity hotspot countries have facilities and expertise, and they concentrate on their own flora for in vitro conservation programmes. For decades, because of the grave threat these plants face, the Conservation Biotechnology Unit, previously known as the Micropropagation Unit, at Royal Botanic Gardens Kew (RBG Kew) has been at the forefront of assisting countries to save their valuable biodiversity through both in situ and ex situ methods. Approaches mentioned here highlight work on recalcitrant ferns from GBHs and small islands. Source materials from recalcitrant species, either spore or seed and in some cases vegetative material, need to be used immediately after collection for tangible results in vitro. This becomes more difficult when only a few plants or small populations are left in the wild. The task becomes harder when available material is small in quantity, and there is greater restriction on the use of available genetic diversity in the wild. This paper highlights the importance of proper collection measures, in vitro culture procedures and cryopreservation and methods for the integrated conservation of threatened ferns from both GBHs and small islands. The importance of international networking to achieve these conservation goals also will be discussed.