Glycerotoxin stimulates neurotransmitter release from N-type Ca2+ channel expressing neurons

Glycerotoxin (GLTx) is capable of stimulating neurotransmitter release at the frog neuromuscular junction by directly interacting with N-type Ca2+ (Cav2.2) channels. Here we have utilized GLTx as a tool to investigate the functionality of Cav2.2 channels in various mammalian neuronal preparations. We first adapted a fluorescent-based high-throughput assay to monitor glutamate release from rat cortical synaptosomes. GLTx potently stimulates glutamate secretion and Ca2+ influx in synaptosomes with an EC50 of 50 pm. Both these effects were prevented using selective Cav2.2 channel blockers suggesting the functional involvement of Cav2.2 channels in mediating glutamate release in this system. We further show that both Cav2.1 (P/Q-type) and Cav2.2 channels contribute equally to depolarization-induced glutamate release. We then investigated the functionality of Cav2.2 channels at the neonatal rat neuromuscular junction. GLTx enhances both spontaneous and evoked neurotransmitter release causing a significant increase in the frequency of postsynaptic action potentials. These effects were blocked by specific Cav2.2 channel blockers demonstrating that either GLTx or its derivatives could be used to selectively enhance the neurotransmitter release from Cav2.2-expressing mammalian neurons.     

Understanding the Structure and Function of the Immunological Synapse

The immunological synapse has been an area of very active scientific interest over the last decade. Surprisingly, much about the synapse remains unknown or is controversial.  Here we review some of these current issues in the field:  how the synapse is defined, its potential role in T-cell function, and our current understanding about how the synapse is formed.T cells are activated when they recognize peptide-MHC complexes on the surface of antigen presenting cells (APC) (Babbitt et al. 1985). But the exact process regarding how antigenic pMHC complexes are recognized and transduced into signals is still incompletely understood. Naïve T cells enter secondary lymphoid organs such as the lymph node and scan dendritic cells for the presence of rare specific pMHC complexes (Miller et al. 2004). After recognizing less than 10 specific pMHC complexes, naïve T cells maintain long contacts (6–18 h) with dendritic cells before being committed to enter cell cycle and differentiate into effector T cells (Iezzi et al. 1998; Irvine et al. 2002; Mempel et al. 2004).The immunological synapse (IS) refers to the organization of membrane proteins that occurs at the interface between the T cell and the APC during these long contacts and also during the effector phase (Grakoui et al. 1999; Monks et al. 1998). Interest in studying the IS stems from ideas that the supramolecular structures that form at the IS underlies the high sensitivity of T cell recognition and that understanding these structures will lead to better insights into how antigen recognition leads to the decision of a T cell to proliferate, differentiate, and function.Springer first put forward the concept that receptors would segregate laterally during cell interactions (Springer 1990). Subsequently, Kupfer was the first to show that proteins in the contact area between a T cell and APC segregate laterally (Monks et al. 1998). Specifically, he noted that the integrin, LFA-1, became concentrated in an outer ring, known as the peripheral supramolecular activation complex (pSMAC) and the TCR became concentrated in the center, in a zone known as the central supramolecular activation complex (cSMAC) (Monks et al. 1998)(Fig. 1). We showed that CD2 could segregate from LFA-1 and concentrate in the center of a hybrid cell-planar bilayer junction and suggested that these patterns and those described by Monks et al. (1998) provided evidence for the previously hypothesized immunological synapse (Dustin et al. 1998; Norcross 1984). The function of this receptor segregation is still not completely understood but it was initially hypothesized that formation of this pattern might be related to T-cell activation and constitute a “molecular machine” that would be formed in response to the presence of antigenic ligand and that this “molecular machine” might function to sustain signaling for long periods of time and direct subsequent T-cell differentiation (Grakoui et al. 1999).Open in a separate windowFigure 1.Structure of the immunological synapse. The basic structure of the “organized” immunological synapse with SMACs is shown (left). In the center is the central supramolecular activation complex or cSMAC, which contains receptors like the TCR, CD28, CD4, CD8, and CD2. Newer studies suggest that the cSMAC may be divided into an outer area containing CD28 and an inner area containing the TCR (not shown). The ring that surrounds the cSMAC is called the peripheral supramolecular activation complex or pSMAC. This domain is mainly populated by the integrin molecule LFA-1. Outside of the pSMAC is another domain known as the distal supramolecular activation complex. Originally the dSMAC was thought not be important and contain all of the molecules that are not specifically recruited to the cSMAC or pSMAC but it is increasingly becoming appreciated that the dSMAC is an area of active membrane movement. This suggests that the pSMAC and dSMAC may be analogous to the actin structures known as the lamellae and lamellipodia, respectively (right).     

RNA-cleaving properties of human apurinic/apyrimidinic endonuclease 1 (APE1)

We have recently identified apurinic/apyrimidinic endonuclease 1 (APE1) as an endoribonuclease that cleaves c-myc mRNA in vitro and regulates c-myc mRNA levels and half-life in cells. This study was undertaken to further unravel the RNA-cleaving properties of APE1. Here, we show that APE1 cleaves RNA in the absence of divalent metal ions and, at 2 mM, Zn²⁺, Ni²⁺, Cu²⁺, or Co²⁺ inhibited the endoribonuclease activity of APE1. APE1 is able to cleave CD44 mRNA, microRNAs (miR-21, miR-10b), and three RNA components of SARS-corona virus (orf1b, orf3, spike) suggesting that, when challenged, it can cleave any RNAs in vitro. APE1 does not cleave strong doublestranded regions of RNA and it has a strong preference for 3’ of pyrimidine, especially towards UA, CA, and UG sites at single-stranded or weakly paired regions. It also cleaves RNA weakly at UC, CU, AC, and AU sites in single-stranded or weakly paired regions. Finally, we found that APE1 can reduce the ability of the Dicer enzyme to process premiRNAs in vitro. Overall, this study has revealed some previously unknown biochemical properties of APE1 which has implications for its role in vivo.     

Do low oxygen environments facilitate marine invasions? Relative tolerance of native and invasive species to low oxygen conditions

Biological invasions are one of the biggest threats to global biodiversity. Marine artificial structures are proliferating worldwide and provide a haven for marine invasive species. Such structures disrupt local hydrodynamics, which can lead to the formation of oxygen‐depleted microsites. The extent to which native fauna can cope with such low oxygen conditions, and whether invasive species, long associated with artificial structures in flow‐restricted habitats, have adapted to these conditions remains unclear. We measured water flow and oxygen availability in marinas and piers at the scales relevant to sessile marine invertebrates (mm). We then measured the capacity of invasive and native marine invertebrates to maintain metabolic rates under decreasing levels of oxygen using standard laboratory assays. We found that marinas reduce water flow relative to piers, and that local oxygen levels can be zero in low flow conditions. We also found that for species with erect growth forms, invasive species can tolerate much lower levels of oxygen relative to native species. Integrating the field and laboratory data showed that up to 30% of available microhabitats within low flow environments are physiologically stressful for native species, while only 18% of the same habitat is physiologically stressful for invasive species. These results suggest that invasive species have adapted to low oxygen habitats associated with manmade habitats, and artificial structures may be creating niche opportunities for invasive species.     

A century of landscape disturbance and urbanization of the San Francisco Bay region affects the present-day genetic diversity of the California Ridgway’s rail (<Emphasis Type="Italic">Rallus obsoletus obsoletus</Emphasis>)

Fragmentation and loss of natural habitat have important consequences for wild populations and can negatively affect long-term viability and resilience to environmental change. Salt marsh obligate species, such as those that occupy the San Francisco Bay Estuary in western North America, occupy already impaired habitats as result of human development and modifications and are highly susceptible to increased habitat loss and fragmentation due to global climate change. We examined the genetic variation of the California Ridgway’s rail (Rallus obsoletus obsoletus), a state and federally endangered species that occurs within the fragmented salt marsh of the San Francisco Bay Estuary. We genotyped 107 rails across 11 microsatellite loci and a single mitochondrial gene to estimate genetic diversity and population structure among seven salt marsh fragments and assessed demographic connectivity by inferring patterns of gene flow and migration rates. We found pronounced genetic structuring among four geographically separate genetic clusters across the San Francisco Bay. Gene flow analyses supported a stepping stone model of gene flow from south-to-north. However, contemporary gene flow among the regional embayments was low. Genetic diversity among occupied salt marshes and genetic clusters were not significantly different. We detected low effective population sizes and significantly high relatedness among individuals within salt marshes. Preserving genetic diversity and connectivity throughout the San Francisco Bay may require attention to salt marsh restoration in the Central Bay where habitat is both most limited and most fragmented. Incorporating periodic genetic sampling into the management regime may help evaluate population trends and guide long-term management priorities.     

Are numbers enough? Colonizer phenotype and abundance interact to affect population dynamics

1. Ecologists have long recognized that the number of colonizers entering a population can be a major driver of population dynamics, but still struggle to explain why the importance of colonizer supply varies so dramatically. While there are indications that differences in the phenotype among dispersing individuals could also be important to populations, the role of phenotypic variation relative to the number of individuals, and the extent to which they interact, remains unknown. 2. We simultaneously manipulated the phenotype (dispersal duration) and abundance of settlers of a marine bryozoan and measured subsequent population structure in the field. 3. Increases in the number of colonizing individuals increased the subsequent recruitment and biomass of populations, regardless of colonizer phenotype. However, the relationship between colonizer abundance and the subsequent reproductive yield of the population was strongly reduced in populations containing individuals that had long dispersal durations. 4. The interactive effects of colonizer phenotype and abundance on the reproductive yield of populations occurred because longer dispersal durations decreased the proportion of individuals that reproduced. In fact, populations established from a few individuals with short dispersal durations had similar reproductive yield to populations c. 30 times larger established from individuals with long dispersal durations. 5. Interactions between colonizer phenotype and abundance have important implications for predicting population dynamics beyond those previously provided by numerical abundance or recruit phenotype alone.     

Fitness consequences of larval traits persist across the metamorphic boundary

Metamorphosis is thought to provide an adaptive decoupling between traits specialized for each life-history stage in species with complex life cycles. However, an increasing number of studies are finding that larval traits can carry-over to influence postmetamorphic performance, suggesting that these life-history stages may not be free to evolve independently of each other. We used a phenotypic selection framework to compare the relative and interactive effects of larval size, time to hatching, and time to settlement on postmetamorphic survival and growth in a marine invertebrate, Styela plicata. Time to hatching was the only larval trait found to be under directional selection, individuals that took more time to hatch into larvae survived better after metamorphosis but grew more slowly. Nonlinear selection was found to act on multivariate trait combinations, once again acting in opposite directions for selection acting via survival and growth. Individuals with above average values of larval traits were most likely to survive, but surviving individuals with intermediate larval traits grew to the largest size. These results demonstrate that larval traits can have multiple, complex fitness consequences that persist across the metamorphic boundary; and thus postmetamorphic selection pressures may constrain the evolution of larval traits.