A 13,600-year diatom oxygen isotope record from the South Carpathians (Romania): Reflection of winter conditions and possible links with North Atlantic circulation changes

This study provides a continuous lateglacial and Holocene record of diatom silica oxygen isotope changes (delta O-18(DIAT)) in a subalpine lake sediment sequence obtained from the Retezat Mts (Taul dintre Brazi, 1740 m a.s.l.). This through-flow, shallow, high-altitude lake with a surface area of only 0.4 ha has short water residence time and is predominantly fed by snowmelt and rainwater. Its delta O-18(DIAT) record principally reflects the oxygen isotope composition of the winter and spring precipitation, as diatom blooms occur mainly in the spring and early summer. Hence, changes in delta O-18(DIAT) are interpreted as seasonal scale changes: in the amount of winter precipitation. Low oxygen isotope values (27-28.5 parts per thousand) occurred during the lateglacial until 12,300 cal BP, followed by a sharp increase thereafter. In the Holocene delta O-18(DIAT) values ranged from 29 to 31 parts per thousand until 3200 cal BP, followed by generally lower values during the late Holocene (27-30 parts per thousand). Short-term decreases in the isotopic values were found between 10,140-9570, 9000-8500, 7800-7300, 6300-5800, 5500-5000 and at 8015, 4400, 4000 cal BP. After 3200 cal BP a decreasing trend was visible with the lowest values between 3100-2500 and after 2100 cal BP The general trend in the record suggests that contribution of winter precipitation was generally lower between 11,680 and 3200 cal BP, followed by increased contribution during the last millennia. The late Holocene decrease in delta O-18(DIAT) shows good agreement with the speleothem delta O-18, lake level and testate amoebae records from the Carpathian Mountains that also display gradual delta O-18 decrease and lake level/mire water table level rise after 3200 cal BR Strong positive correlation with North Atlantic circulation and solar activity proxies, such as the Austrian and Hungarian speleothem records, furthermore suggested that short-term increases in the isotopic ratios in the early and mid Holocene are likely connectable to high solar activity phases and high frequency of positive North Atlantic Oscillation indexes that may have resulted in decreased winter precipitation in this region.

Palaeoenvironmental changes in Central Europe (NE Poland) during the last 6200 years reconstructed from a high-resolution multi-proxy peat archive

Peat deposits from an ombrotrophic bog (north-eastern Poland) were analysed to reconstruct peatland development and environmental changes. This paper presents reconstructions of hydrological changes and plant succession over the last 6000 years. The methods included the high-resolution analysis of plant macrofossils, pollen and testate amoebae, supported by radiocarbon dating. Three main phases were identified in the history of the bog and surrounding woodland vegetation: 4000–400 BC, 400 BC–AD 1700 and AD 1700–2011. Except for terrestrialisation and the fen-to-bog transition phase, the development of bog vegetation was mainly dependent on the climate until approximately AD 1700. The dominant taxon in Gązwa bog was Sphagnum fuscum/rubellum. Woodland development was significantly affected by human activity at several time periods. The most visible human activity, manifested by the decline of deciduous species, occurred ca. 350 BC, ca. AD 250, ca. AD 1350 and after AD 1700. These events correspond to phases of human settlement in the area. During 400–300 BC, the decline of deciduous trees, primarily Carpinus, coincided with an increase in indicators of human activity and fire frequency. At ca. AD 200, Carpinus and Tilia abundance decreased, corresponding to an increased importance of cereals (Secale and Triticum). Since ca. AD 1350, the impact of Teutonic settlement is apparent, and after AD 1700, deciduous forests largely disappeared.

Epitheliocystis and the importance of Chlamydia-related bacteria on fish health

This book offers unique coverage of all presently known amoeba-resistant microorganisms and their significance in the study of infectious diseases. It highlights the role of free-living amoebae as a widespread evolutionary crib for the development of virulence traits in resistant microbes, including the ability of intracellular bacteria to survive to other phagocytic cells such as human macrophages. The emphasis is on public health risks associated with the presence in drinking water of intra-amoebal bacteria as well as the ecology and pathogenic role of amoebae-resisting bacteria as new emerging human pathogens

Long-term hydrological dynamics and fire history over the last 2000 years in CE Europe reconstructed from a high-resolution peat archive

Sphagnum peatlands in the oceanic-continental transition zone of Poland are currently influenced by climatic and anthropogenic factors that lead to peat desiccation and susceptibility to fire. Little is known about the response of Sphagnum peatland testate amoebae (TA) to the combined effects of drought and fire. To understand the relationships between hydrology and fire dynamics, we used high-resolution multi-proxy palaeoecological data to reconstruct 2000 years of mire history in northern Poland. We employed a new approach for Polish peatlands – joint TA-based water table depth and charcoal-inferred fire activity reconstructions. In addition, the response of most abundant TA hydrological indicators to charcoal-inferred fire activity was assessed. The results show four hydrological stages of peatland development: moderately wet (from ∼35 BC to 800 AD), wet (from ∼800 to 1390 AD), dry (from ∼1390 to 1700 AD) and with an instable water table (from ∼1700 to 2012 AD). Fire activity has increased in the last millennium after constant human presence in the mire surroundings. Higher fire activity caused a rise in the water table, but later an abrupt drought appeared at the onset of the Little Ice Age. This dry phase is characterized by high ash contents and high charcoal-inferred fire activity. Fires preceded hydrological change and the response of TA to fire was indirect. Peatland drying and hydrological instability was connected with TA community changes from wet (dominance of Archerella flavum, Hyalosphenia papilio, Amphitrema wrightianum) to dry (dominance of Cryptodifflugia oviformis, Euglypha rotunda); however, no clear fire indicator species was found. Anthropogenic activities can increase peat fires and cause substantial hydrology changes. Our data suggest that increased human fire activity was one of the main factors that influenced peatland hydrology, though the mire response through hydrological changes towards drier conditions was delayed in relation to the surrounding vegetation changes.

Human and climatic impact on mires: a case study of Les Amburnex mire, Swiss Jura Mountains

Modern period long-term human and climatic impacts on a small mire in the Jura Mountains were assessed using testate amoebae, macrofossils and pollen. This multiproxy data analysis permitted detailed interpretations of local and regional environmental change and thus a partial disentanglement of the different variables that influence long-term mire development. From the Middle Ages until a.d. 1700 the mire vegetation was characterised by ferns, Caltha and Vaccinium, but then abruptly changed into the modern vegetation characterised by Cyperaceae, Potentilla and Sphagnum. The cause for this change was most probably deforestation, possibly enhanced by climatic cooling. A decrease in trampling intensity by domestic animals from a.d. 1950 onwards allowed Sphagnum growth and climatic warming in the a.d. 1980s and 1990s may have been responsible for considerable changes in the species composition. The mire investigated is an example of the rapid changes in mire vegetation and peat development that occurred throughout the central European mountain region during the past centuries as a result of changing climate and land-use practice. These processes are still active today and will determine the future development of high-altitude mires.

Investigating the Affects of Cucurbitacin-I on Cellular Motility

Cellular migration is an integral component of many biological processes including immune function, wound healing and cancer cell metastasis. A complete model illustrating the mechanism by which cells accomplish movement is still lacking. Exploring the affects of various drugs on cell motility may be instrumental in discovering new proteins which mediate cell movement. This project aims ultimately to characterize the molecular target of the drug Cucurbitacin-I, a natural plant product. This drug has been shown to inhibit migration of epithelial sheets and may have anti-tumor activity. In this paper, we show that Cucurbitacin-I inhibits the migration of MDCK and B16F1 cells. The drug also affects the integrity of the actin cytoskeleton of these cells by indirectly stabilizing filamentous actin. Cucurbitacin-I does not, however, have an effect on the motility or cytoskeletal morphology of the soil amoeba, Dictyostelium discoidium.

Identification and characterization of distinct classes of cAMP-pulse-induced genes in Dictyostelium discoideum development

The unicellular amoeba Dictyostelium discoideum embarks on a developmental program upon starvation. During development, extracellular oscillatory cAMP signaling orchestrates the chemotaxis-mediated aggregation of ∼105 amoebae and is required for optimal induction of so-called pulse-induced genes. This requirement for pulsatile CAMP reflects adaptation of the cAMP-receptor-mediated pathways that regulate these genes. Through examination of a collection of pulse-induced genes, we defined two distinct gene classes based on their induction kinetics and the impact of mutations that impair PKA signaling. The first class (represented by D2 and prtA) is highly dependent on PKA signaling, whereas the second class (represented by carA, gpaB, and acaA) is not. Analysis of expression kinetics revealed that these classes are sequentially expressed with the PKA-independent genes peaking in expression before the PKA-dependent class. Experiments with cycloheximide, an inhibitor of translation, demonstrated that the pulse induction of both classes depends on new protein synthesis early in development. carA and gpaB also exhibit pulse-independent, starvation-induced expression which, unlike their pulse induction, was found to be insensitive to cycloheximide added at the outset of starvation. This result indicates that the mechanism of starvation induction pre-exists in growing cells and is distinct from the pulse induction mechanism for these genes. In order to identify cis-acting elements that are critical for induction of carA, we constructed a GFP reporter controlled by a 914-base-pair portion of its promoter and verified that its expression was PKA-independent, pulse-inducible, and developmentally regulated like the endogenous carA gene. By a combination of truncation, internal deletion, and site-directed mutation, we defined several distinct functional elements within the carA promoter, including a 39-bp region required for pulse induction between base pairs -321 and -282 (relative to the transcription start site), a 131-bp region proximal to the start site that is sufficient for starvation induction, and two separate enhancer domains. Identification of factors that interact with these promoter elements and genetic approaches exploiting the GFP reporter described here should help complete our understanding of the mechanisms regulating these genes, including adaptation mechanisms that likely also govern chemotaxis of Dictyostelium and mammalian cells. ^

Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

Constitutively active cAMP receptor mutants dominantly inhibit receptor-mediated signaling pathways in Dictyostelium discoideum

Dictyostelium, a soil amoeba, is able to develop from free-living cells to multicellular fruiting bodies upon starvation using extracellular cAMP to mediate cell-cell communication, chemotaxis and developmental gene expression. The seven transmembrane G protein-coupled cAMP receptor-1 (cAR1) mediated responses, such as the activation of adenylyl cyclase and guanylyl cyclase, are transient, due to the existence of poorly understood adaptation mechanisms. For this dissertation, the powerful genetics of the Dictyostelium system was employed to study the adaptation mechanism of cAR1-mediated cAMP signaling as well as mechanisms intrinsic to cAR1 that regulate its activation. ^ We proposed that constitutively active cAR1 would cause constant adaptation, thus inhibiting downstream pathways that are essential for aggregation and development. Therefore, a screen for dominant negative cAR1 mutants was undertaken to identify constitutively active receptor mutants. Three dominant negative cAR1 mutants were identified. All appear to be constitutively active receptor mutants because they are constitutively phosphorylated and possess high affinity for cAMP. Biochemical studies showed that these mutant receptors prevented the activation of downstream effectors, including adenylyl and guanylyl cyclases. In addition, these cells also were defective in cAMP chemotaxis and cAR1-mediated gene expression. These findings suggest that the mutant receptors block development by constantly activating multiple adaptation pathways. ^ Sequence analysis revealed that these mutations (I104N, L100H) are clustered in a conserved region of the third transmembrane helix (TM3) of cAR1. To investigate the role of this region in receptor activation, one of these residues, I104, was mutated to all the other 19 possible amino acids. We found that all but the most conservative substitutions increase the receptor's affinity about 20- to 70-fold. However, only highly polar substitutions of I104, particularly basic residues, resulted in receptors that are constitutively phosphorylated and dominantly inhibit development, suggesting that highly polar substitutions not only disrupt an interaction constraining the receptor in its low-affinity, inactive state but also promote an additional conformational change that resembles the ligand-bound conformation. Our findings suggest that I104 plays a specific role in constraining the receptor in its inactive state and that substituting it with highly polar residues results in constitutive activation. ^