Allergenicity of the ornamental urban flora: ecological and aerobiological analyses in C��rdoba (Spain) and Ascoli Piceno (Italy)

Comparative ecological and aerobiological analyses of ornamental urban flora in the cities of Córdoba (Spain) and Ascoli Piceno (Italy) identified a group of plants with allergenic pollen whose behaviour is influenced by a number of factors. The geographical position and the resulting climate of both cities favoured the presence of Mediterranean species. In Córdoba, strongly allergenic evergreens introduced during the Moorish period predominated (Cupressus sempervirens, among others), while in the urban area of Ascoli Piceno, Pinaceae were abundant. In both cities, many species of American origin have been introduced for aesthetic reasons, contributing to an increase in the overall allergenicity of urban greenery. The pollen spectrum differed between the two cities: airborne Oleaceae and Cupressaceae pollen abounded in Córdoba, whilst allergenic pollen from surrounding natural environments (Corylaceae) predominated in Ascoli Piceno. These results pointed to a large number of potentially allergenic species in cities, thus highlighting the importance of greater ecological and aerobiological knowledge of allergenic species of urban ornamental flora. Avoidance of more allergenic species when planning new urban green areas could ensure healthier environments for pollen-allergy sufferers.     

Cell Adhesion, the Backbone of the Synapse: ��Vertebrate�� and ��Invertebrate�� Perspectives

Synapses are asymmetric intercellular junctions that mediate neuronal communication. The number, type, and connectivity patterns of synapses determine the formation, maintenance, and function of neural circuitries. The complexity and specificity of synaptogenesis relies upon modulation of adhesive properties, which regulate contact initiation, synapse formation, maturation, and functional plasticity. Disruption of adhesion may result in structural and functional imbalance that may lead to neurodevelopmental diseases, such as autism, or neurodegeneration, such as Alzheimer''s disease. Therefore, understanding the roles of different adhesion protein families in synapse formation is crucial for unraveling the biology of neuronal circuit formation, as well as the pathogenesis of some brain disorders. The present review summarizes some of the knowledge that has been acquired in vertebrate and invertebrate genetic model organisms.Synapses are asymmetric, intercellular junctions that are the basic structural units of neuronal transmission. The correct development of synaptic specializations and the establishment of appropriate connectivity patterns are crucial for the assembly of functional neuronal circuits. Improper synapse formation and function may cause neurodevelopmental disorders, such as mental retardation (MsR) and autism spectrum disorders (ASD) (McAllister 2007; Sudhof 2008), and likely play a role in neurodegenerative disorders, such as Alzheimer''s disease (AD) (Haass and Selkoe 2007).At chemical synapses (reviewed in Sudhof 2004; Zhai and Bellen 2004; Waites et al. 2005; McAllister 2007; Jin and Garner 2008), the presynaptic compartment contains synaptic vesicles (SV), organized in functionally distinct subcellular pools. A subset of SVs docks to the presynaptic membrane around protein-dense release sites, named active zones (AZ). Upon the arrival of an action potential at the terminal, the docked and “primed” SVs fuse with the plasma membrane and release neurotransmitter molecules into the synaptic cleft. Depending on the type of synapse (i.e., excitatory vs. inhibitory synapses), neurotransmitters ultimately activate an appropriate set of postsynaptic receptors that are accurately apposed to the AZ.Synapse formation occurs in several steps (Fig. 1) (reviewed in Eaton and Davis 2003; Goda and Davis 2003; Waites et al. 2005; Garner et al. 2006; Gerrow and El-Husseini 2006; McAllister 2007). Spatiotemporal signals guide axons through heterogeneous cellular environments to contact appropriate postsynaptic targets. At their destination, axonal growth cones initiate synaptogenesis through adhesive interactions with target cells. In the mammalian central nervous system (CNS), immature postsynaptic dendritic spines initially protrude as thin, actin-rich filopodia on the surface of dendrites. Similarly, at the Drosophila neuromuscular junction (NMJ), myopodia develop from the muscles (Ritzenthaler et al. 2000). The stabilization of intercellular contacts and their elaboration into mature, functional synapses involves cytoskeletal arrangements and recruitment of pre- and postsynaptic components to contact sites in spines and boutons. Conversely, retraction of contacts results in synaptic elimination. Both stabilization and retraction sculpt a functional neuronal circuitry.Open in a separate windowFigure 1.(A–C) Different stages of synapse formation. (A) Target selection, (B) Synapse assembly, (C) Synapse maturation and stabilization. (D–F) The role of cell adhesion molecules in synapse formation is exemplified by the paradigm of N-cadherin and catenins in regulation of the morphology and strength of dendritic spine heads. (D) At an early stage the dendritic spines are elongated from motile structures “seeking” their synaptic partners. (E) The contacts between the presynaptic and postsynaptic compartments are stabilized by recruitment of additional cell adhesion molecules. Adhesional interactions activate downstream pathways that remodel the cytoskeleton and organize pre- and postsynaptic apparatuses. (F) Cell adhesion complexes, stabilized by increased synaptic activity, promote the expansion of the dendritic spine head and the maturation/ stabilization of the synapse. Retraction and expansion is dependent on synaptic plasticity.In addition to the plastic nature of synapse formation, the vast heterogeneity of synapses (in terms of target selection, morphology, and type of neurotransmitter released) greatly enhances the complexity of synaptogenesis (reviewed in Craig and Boudin 2001; Craig et al. 2006; Gerrow and El-Husseini 2006). The complexity and specificity of synaptogenesis relies upon the modulation of adhesion between the pre- and postsynaptic components (reviewed in Craig et al. 2006; Gerrow and El-Husseini 2006; Piechotta et al. 2006; Dalva et al. 2007; Shapiro et al. 2007; Yamada and Nelson 2007; Gottmann 2008). Cell adhesive interactions enable cell–cell recognition via extracellular domains and also mediate intracellular signaling cascades that affect synapse morphology and organize scaffolding complexes. Thus, cell adhesion molecules (CAMs) coordinate multiple synaptogenic steps.However, in vitro and in vivo studies of vertebrate CAMs are often at odds with each other. Indeed, there are no examples of mutants for synaptic CAMs that exhibit prominent defects in synapse formation. This apparent “resilience” of synapses is probably caused by functional redundancy or compensatory effects among different CAMs (Piechotta et al. 2006). Hence, studies using simpler organisms less riddled by redundancy, such as Caenorhabditis elegans and Drosophila, have aided in our understanding of the role that these molecules play in organizing synapses.In this survey, we discuss the roles of the best characterized CAM families of proteins involved in synaptogenesis. Our focus is to highlight the complex principles that govern the molecular basis of synapse formation and function from a comparative perspective. We will present results from cell culture studies as well as in vivo analyses in vertebrate systems and refer to invertebrate studies, mainly performed in Drosophila and C. elegans, when they have provided important insights into the role of particular CAM protein families. However, we do not discuss secreted factors, for which we refer the reader to numerous excellent reviews (as for example Washbourne et al. 2004; Salinas 2005; Piechotta et al. 2006; Shapiro et al. 2006; Dalva 2007; Yamada and Nelson 2007; Biederer and Stagi 2008; Salinas and Zou 2008).     

Increased ��-synuclein phosphorylation and nitration in the aging primate substantia nigra

Post-translational modifications of α-synuclein occur in the brain of patients affected by Parkinson''s disease and other α-synucleinopathies, as indicated by the accumulation of Lewy inclusions containing phosphorylated (at serine 129) and nitrated α-synuclein. Here we found that phospho-Ser 129 and nitrated α-synuclein are also formed within dopaminergic neurons of the monkey substantia nigra as a result of normal aging. Dopaminergic cell bodies immunoreactive for phospho-Ser 129 and nitrated α-synuclein were rarely seen in adult mature animals but became significantly more frequent in the substantia nigra of old primates. Dual labeling with antibodies against phospho-Ser 129 and nitrated α-synuclein revealed only limited colocalization and mostly stained distinct sub-populations of dopaminergic neurons. Age-related elevations of modified protein paralleled an increase in the number of neurons immunoreactive for unmodified α-synuclein, supporting a relationship between higher levels of normal protein and enhanced phosphorylation/nitration. Other mechanisms were also identified that likely contribute to α-synuclein modifications. In particular, increased expression of Polo-like kinase 2 within neurons of older animals could contribute to phospho-Ser 129 α-synuclein production. Data also indicate that a pro-oxidant environment characterizes older neurons and favors α-synuclein nitration. Aging is an unequivocal risk factor for human α-synucleinopathies. These findings are consistent with a mechanistic link between aging, α-synuclein abnormalities and enhanced vulnerability to neurodegenerative processes.     

Aquaporin-2 in the ��-omics�� Era

Vasopressin controls renal water excretion largely through actions to regulate the water channel aquaporin-2 in collecting duct principal cells. Our knowledge of the mechanisms involved has increased markedly in recent years with the advent of methods for large-scale systems-level profiling such as protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible aspects of everyday physiology. An outdoor tennis game on a hot summer day can result in substantial water losses by sweating, and the kidneys respond by reducing water excretion. In contrast, excessive intake of water, a frequent occurrence in everyday life, results in excretion of copious amounts of clear urine. These responses serve to exact tight control on the tonicity of body fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg of H₂O through the regulated return of water from the pro-urine in the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails. For example, polyuria (rapid uncontrolled excretion of water) is a sometimes devastating consequence of lithium therapy for bipolar disorder. On the other side of the coin are water balance disorders that result from excessive renal water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia due to excessive water retention can be seen with severe congestive heart failure, hepatic cirrhosis, and the syndrome of inappropriate antidiuresis.The chief regulator of water excretion is the peptide hormone AVP,² whereas the chief molecular target for regulation is the water channel AQP2. In this minireview, we describe new progress in the understanding of the molecular mechanisms involved in regulation of AQP2 by AVP in collecting duct cells, with emphasis on new information derived from “systems-level” approaches involving large-scale profiling and screening techniques such as oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid analysis. Most of the progress with these techniques is in the identification of individual molecules involved in AVP signaling and binding interactions with AQP2. Additional related issues are addressed in several recent reviews (1–4).     

Acidification of lakes in Šumava (Bohemia) and in the High Tatra Mountains (Slovakia)

Acidification of lakes takes place when pH of rainwater is less than 4.5 and the catchments lie on sensitive geology. Both conditions are met for most lakes in Bohemia and Slovakia. Since 1978 we have studied mountain lakes in the Sumava and in the High Tatra Mountains. In Šumava the three lakes under study are of glacial origin. The catchments are small, with steep sides covered by spruce. The bedrocks are biotite-rich paragneiss, together with gneiss, quartzite and granite. In summer 1936 surface pH was 5.7–6.9 in the Lake Čertovo and 6.9–7.0 in the Lake Černé. Now the pH values are 4.3–4.8 in the two lakes and in the Lake Prášilské as well. Old reports on zooplankton are from the years 1871, 1892–96, 1935–37, 1947 and 1960. Since 1979 we have not found any planktonic Crustacea in the lakes Černé and Čertovo. Lake Prášilské is inhabited by Daphnia longispina and Cyclops abyssorum. In July 1989 the pH values were 4.4, 4.7 and 4.7, concentrations of labile monomeric Al were 0.83, 0.68 and 0.24 mg l^-1 in the lakes Čertovo, Černé and Prašilské, respectively. High levels of toxic Al compounds might be responsible for the extinction of planktonic Crustacea in the lakes Čertovo and Černé. All the three lakes are void of fish at present. In the High Tatra Mts. we examined more than 40 lakes above timberline in altitudes 1612–2145 m. They are all clearwater, naturally fishless lakes. The bedrock is granite. Owing to different levels of calcium the lakes are now in different stages of acidification. According to recent changes in the zooplankton they can be divided into three groups: (1) Species composition of planktonic Crustacea has not changed. (2) Planktonic Crustacea were present until 1973 but are absent now. (3) From the original species of Crustacea only Chydorus sphaericus is present. The three groups are well separated along the gradients of calcium, ANC and pH. They can be identified with the Henriksen's bicarbonate (our group 1), intermediate (our group 2) and acid (our group 3) lakes. We suppose that in the process of acidification the lakes of the group (2) have been shifted from oligotrophy to ultraoligotrophy.     

A Genetic Engineering Solution to the ��Arginine Conversion Problem�� in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the “arginine conversion problem” may be more broadly applicable to organisms amenable to genetic manipulation.Stable isotope labeling by amino acids in cell culture (SILAC)¹ (1) is one of the key methods for large scale quantitative proteomics (2, 3). In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing either normal (“light”) or heavy isotope-labeled amino acids, typically lysine and arginine. Peptides derived from the light and heavy cells are thus distinguishable by mass spectrometry and can be mixed for accurate quantitation. SILAC is also possible at the whole-organism level (4).An inherent problem in SILAC is the metabolic conversion of labeled arginine to other amino acids, as this complicates quantitative analysis of peptides containing these amino acids. Arginine conversion to proline is well described in mammalian cells, although the extent of conversion varies among cell types (5). When conversion is observed, typically 10–25% of the total proline pool is found to contain label (6–11). Arginine conversion has also been reported in SILAC experiments with budding yeast Saccharomyces cerevisiae (3, 12, 13).Because more than 50% of tryptic peptides in large data sets contain proline (7), it is not practical simply to disregard proline-containing peptides during quantitation. Several methods have been proposed to either reduce arginine conversion or correct for its effects on quantitation. In some cell types, arginine conversion can be prevented by lowering the concentration of exogenous arginine (6, 14–16) or by adding exogenous proline (9). However, these methods can involve significant changes to growth media and may need to be tested for each experimental condition used. Given the importance of arginine in many metabolic pathways, careful empirical titration of exogenous arginine concentration is required to minimize negative effects on cell growth (14). In addition, low arginine medium can lead to incomplete arginine labeling, although the reasons for this are not entirely clear (7). An alternative strategy is to omit labeled arginine altogether (3, 13, 17), but this reduces the number of quantifiable peptides. Correction methods include using two different forms of labeled arginine (7) or computationally compensating for proline-containing peptides (11, 12, 18). Ultimately, none of these methods address the problem at its root, the utilization of arginine in cellular metabolism.To develop a differential proteomics work flow for the fission yeast Schizosaccharomyces pombe, we sought to adapt SILAC for use in this organism, a widely used model eukaryote with excellent classical and reverse genetics. Here we describe extremely high conversion of labeled arginine to other amino acids in fission yeast as well as a novel general solution to the problem that should be applicable to other organisms. As proof of principle, we quantitated changes in protein levels before and after cell division on a proteome-wide scale. We identified both up- and down-regulated proteins, including a novel protein involved in septation.     

��The Climax of Reconciliation��: Transgression, Apology, Forgiveness and the Body in Conflict Resolution

According to Charles Hauss, “[i]n the last few years, reconciliation has become one of the ‘hottest’ topics in the increasingly ‘hot’ field of conflict resolution” (2003, ?1). However, despite the apparent interest in this “hot” academic topic (which is becoming increasingly warm in Canada as our own Truth and Reconciliation Commission commences), reconciliation studies have been dominated by Truth-based approaches. The restrictions of these approaches, which emphasize objectivity and rationality, often elide the body and the primacy of emotions in the reparative process. This essay begins a conversation on the role of the body and emotion in the study of reconciliation by engaging the work being done in the social sciences with contemporary trends in critical theory and literature. I argue that by looking at the fundamental role the body plays on the “road to reconciliation” we can devise a more vital approach to conflict resolution and the various processes that make it up.     

Phosphorylation of Thr-516 and Ser-520 in the Kinase Activation Loop of MEKK3 Is Required for Lysophosphatidic Acid-mediated Optimal I��B Kinase �� (IKK��)/Nuclear Factor-��B (NF-��B) Activation

MEKK3 serves as a critical intermediate signaling molecule in lysophosphatidic acid-mediated nuclear factor-κB (NF-κB) activation. However, the precise regulation for MEKK3 activation at the molecular level is still not fully understood. Here we report the identification of two regulatory phosphorylation sites at Thr-516 and Ser-520 within the kinase activation loop that is essential for MEKK3-mediated IκB kinase β (IKKβ)/NF-κB activation. Substitution of these two residues with alanine abolished the ability of MEKK3 to activate IKKβ/NF-κB, whereas replacement with acidic residues rendered MEKK3 constitutively active. Furthermore, substitution of these two residues with alanine abolished the ability of MEKK3 to mediate lysophosphatidic acid-induced optimal IKKβ/NF-κB activation.