SSU‐rRNA Gene Sequencing Survey of Benthic Microbial Eukaryotes from Guaymas Basin Hydrothermal Vent

Microbial eukaryotes have important roles in marine food webs, but their diversity and activities in hydrothermal vent ecosystems are poorly characterized. In this study, we analyzed microbial eukaryotic communities associated with bacterial (Beggiatoa) mats in the 2,000 m deep‐sea Guaymas Basin hydrothermal vent system using 18S rRNA gene high‐throughput sequencing of the V4 region. We detected 6,954 distinct Operational Taxonomic Units (OTUs) across various mat systems. Of the sequences that aligned with known protistan phylotypes, most were affiliated with alveolates (especially dinoflagellates and ciliates) and cercozoans. OTU richness and community structure differed among sediment habitats (e.g. different mat types and cold sediments away from mats). Additionally, full‐length 18S rRNA genes amplified and cloned from single cells revealed the identities of some of the most commonly encountered, active ciliates in this hydrothermal vent ecosystem. Observations and experiments were also conducted to demonstrate that ciliates were trophically active and ingesting fluorescent bacteria or Beggiatoa trichomes. Our work suggests that the active and diverse protistan community at the Guaymas Basin hydrothermal vent ecosystem likely consumes substantial amounts of bacterial biomass, and that the different habitats, often defined by distances of just a few 10s of cm, select for particular assemblages and levels of diversity.     

Demography of an endangered,long-lived fish: Informing management options in the face of cyclic and stochastic climate variation

June sucker (Chasmistes liorus) is a long-lived, endangered fish endemic to Utah Lake, Utah. For several decades June sucker have failed to recruit sufficient numbers to the adult size classes such that the current wild population consists of a small number of old adults and it continues to decline. Vital rates of June sucker are influenced by climate-driven variation in lake level and inflow from the Provo River. We used population projection matrix modeling to assess effects of cyclic and stochastic environmental variation on population growth trajectories of June sucker in Utah Lake. The stable stage distribution is dominated by stage 1 individuals (93% of the total population) in contrast to the current situation where old age classes are the most abundant. Total population size is highly influenced by the stochastic component of climate variation; whereas, the adult population of June sucker closely tracks the systematic drought cycle. If changes in survival of larvae and juveniles can be coordinated such that positive changes in both parameters can occur somewhat simultaneously, then each parameter would only have to be increased by a factor of about 8.8 to achieve sustainable population growth (compared to a 77-fold increase for each parameter separately). Stochastic climatic variation has relatively little long-term effect on population growth. However, the multidecadal cyclic pattern of lake level and river discharge imposes a similar pattern on population growth rates of the June sucker, such that during some periods, populations decline even when the long-term trend is positive.     

Empty class II major histocompatibility complex created by peptide photolysis establishes the role of DM in peptide association

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.     

Natural microbial control of crickets populations (Orthoptera: Gryllotalpidae: Scapteriscus borellii): regulation of populations aggregated in time and space

From 1983 through 1988, a total of 1,762 collections, containing 31,312 individuals of the mole cricket, Scapteriscus borellii, were made, principally in the State of S?o Paulo, Brazil. Collections were found to fit a negative binomial distribution both as whole and when divided into monthly collections. In these collections, an iridovirus, a entomogenous nematode, and the fungi Metarhizium anisopliae, Beauveria bassiana, Paecilomyces sp., and Entomophtora sp., were found to be agents of natural mortality, although usually as endozootics and relatively rarely as epizootics and panzootics. As a group, these diseases were also distributed in a binomial negative. These data suggest that the temporal and spatial aggregations of the mole crickets, produced by high rates of migration among suitable habitats, are adaptations to outbreaks of epidemics, which also serve as mole cricket population regulators. These ideas are develop and derived from simple mathematical models of population change.     

Beneficial effect of intracellular trehalose on the membrane integrity of dried mammalian cells

Recently, there has been much interest in using trehalose and other small carbohydrates to preserve mammalian cells in the dried state as an alternative to cryopreservation. Here, we report on the successful preservation of plasma membrane integrity after drying, as a first step toward full preservation of mammalian cells. Trehalose was introduced into cells using a genetically engineered version of alpha-hemolysin, a pore-forming protein; the cells were then dried and stored for weeks at different temperatures with approximately 90% recovery of the intact plasma membrane. We show that protection of the plasma membrane by internal trehalose is dose dependent and estimate the amount of internal trehalose required for adequate protection to be approximately 10(10) molecules/cell. In addition, a minimal amount of water (approximately 15 wt%) appears to be necessary. These results show that a key component of mammalian cells can be preserved in a dried state for weeks under mild conditions (-20 degrees C and 5% relative humidity) and thereby suggest new approaches to preserving mammalian cells.     

Fibronectin binding protein A of Staphylococcus aureus can mediate human T lymphocyte adhesion and coactivation

The extracellular matrix protein fibronectin (FN) mediates the adhesion of bacteria as well as T lymphocytes. Mammalian cells express integrins alpha(4)beta(1) and alpha(5)beta(1) as the major FN-binding cell surface receptors. Bacteria such as Staphylococcus aureus, also express FN-binding receptors that are important for adherence to host tissue and initiation of infection. The S. aureus FN-binding protein, FnbpA, has been previously identified, and recombinant proteins that correspond to distinct functional regions of this protein have been made. Three recombinant truncated forms of FnbpA, rFnbpA(37-881), rFnbpA(37-605), and rFnbpA(620-881), were examined for effects on in vitro adhesion and coactivation of human T lymphocytes. These proteins, when coimmobilized with anti-CD3 mAb, activated T lymphocyte proliferation. The coactivation signal generated by the rFnbpA proteins required medium containing serum with FN. Furthermore, the costimulatory signal could be restored in FN-depleted serum when the rFnbpAs were preloaded with soluble FN. Monoclonal Ab blocking studies revealed that integrin alpha(5)beta(1) is the major receptor responsible for the rFnbpA costimulatory signal. Shear flow cell detachment assays confirmed that lymphocytes can bind to FN captured by the rFnbpA proteins. These results suggest that the S. aureus rFnbpA can interact with integrin alpha(5)beta(1) via an FN bridge to mediate adhesion and costimulatory signals to T lymphocytes.     

The folding nucleus of a fibronectin type III domain is composed of core residues of the immunoglobulin-like fold

To identify the contacts that stabilise the rate-limiting transition state for folding of FNfn10 (the tenth fnIII domain of human fibronectin), 42 mutants have been analysed at 29 positions across this domain. An anomalous response to mutation means that structure formation in the A, B and G strands cannot be evaluated by this method. In all the residues analysed, phi-values are fractional and no completely structured region is observed. The analysis reveals that hydrophobic residues from the central strands of the beta-sandwich form a large core of interactions in the transition state. Br?nsted analysis shows that the stabilisation energy from the amino acid side-chains in the transition state is approximately 40 % of that in the native state. The protein folds by a nucleation-condensation mechanism, and tertiary interactions within the core make up the folding nucleus. Local interactions, in turns and loops, are apparently much less significant. Comparison with an homologous domain from human tenascin (TNfn3), shows that FNfn10 has a more extended, structured transition state spanning three different "layers" of the beta-sandwich. The results support the hypothesis that interactions in the common structural core guide the folding of these domains.     

Effects of neuropathic and non-neuropathic isomers of tricresyl phosphate and their microsomal activation on the production of axon-like processes by differentiating mouse N2a neuroblastoma cells

The aim of this work was to investigate the sublethal neuropathic effects of tricresyl phosphate (TCP: mixed isomers), triorthocresyl phosphate (TO:CP) and triparacresyl phosphate (TP:CP) on differentiating mouse N2a neuroblastoma cells. This was achieved by a combination of measurements of cell viability, axon outgrowth and the levels of cytoskeletal proteins detectable on western blots of extracts from cells induced to differentiate in the presence and absence of the compounds. In a time-course experiment TCP inhibited the outgrowth of axon-like processes following exposure times of 24 h or longer. Dose-response experiments indicated that TCP and TO:CP exhibited similar sustained levels of toxicity following both 24 and 48 h exposure, with no significant difference between their respective IC(50) values. By contrast, TP:CP demonstrated a transient effect on the outgrowth of axon-like processes, which was detectable after 24 but not 48 h of exposure. Isomer-specific patterns of toxicity were also evident at earlier time-points, with only the ortho isomer showing significant levels of inhibition of axon outgrowth following 4-8 h exposure. Probing of western blots with antibodies against cytoskeletal proteins indicated that the inhibition of axon outgrowth by these compounds was associated with a sustained reduction in the levels of phosphorylated neurofilament heavy chain. The inhibitory effect on axon outgrowth of TO:CP but not TP:CP was enhanced in the presence of a microsomal activation system. Since TO:CP is the most neuropathic of the isomers of TCP in vivo, differentiating N2a cells provide a useful cellular system for mechanistic studies of the neurodegenerative effects of this organophosphate.     

The aspartic proteinase from the rodent parasite Plasmodium berghei as a potential model for plasmepsins from the human malaria parasite, Plasmodium falciparum

The gene encoding an aspartic proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin inhibitors is discussed.