The Unique Chemistry of Eastern Mediterranean Water Masses Selects for Distinct Microbial Communities by Depth

The waters of the Eastern Mediterranean are characterized by unique physical and chemical properties within separate water masses occupying different depths. Distinct water masses are present throughout the oceans, which drive thermohaline circulation. These water masses may contain specific microbial assemblages. The goal of this study was to examine the effect of physical and geological phenomena on the microbial community of the Eastern Mediterranean water column. Chemical measurements were combined with phospholipid fatty acid (PLFA) analysis and high-throughput 16S rRNA sequencing to characterize the microbial community in the water column at five sites. We demonstrate that the chemistry and microbial community of the water column were stratified into three distinct water masses. The salinity and nutrient concentrations vary between these water masses. Nutrient concentrations increased with depth, and salinity was highest in the intermediate water mass. Our PLFA analysis indicated different lipid classes were abundant in each water mass, suggesting that distinct groups of microbes inhabit these water masses. 16S rRNA gene sequencing confirmed the presence of distinct microbial communities in each water mass. Taxa involved in autotrophic nitrogen cycling were enriched in the intermediate water mass suggesting that microbes in this water mass may be important to the nitrogen cycle of the Eastern Mediterranean. The Eastern Mediterranean also contains numerous active hydrocarbon seeps. We sampled above the North Alex Mud Volcano, in order to test the effect of these geological features on the microbial community in the adjacent water column. The community in the waters overlaying the mud volcano was distinct from other communities collected at similar depths and was enriched in known hydrocarbon degrading taxa. Our results demonstrate that physical phenomena such stratification as well as geological phenomena such as mud volcanoes strongly affect microbial community structure in the Eastern Mediterranean water column.     

Multiphasic On/Off Pheromone Signalling in Moths as Neural Correlates of a Search Strategy

Insects and robots searching for odour sources in turbulent plumes face the same problem: the random nature of mixing causes fluctuations and intermittency in perception. Pheromone-tracking male moths appear to deal with discontinuous flows of information by surging upwind, upon sensing a pheromone patch, and casting crosswind, upon losing the plume. Using a combination of neurophysiological recordings, computational modelling and experiments with a cyborg, we propose a neuronal mechanism that promotes a behavioural switch between surge and casting. We show how multiphasic On/Off pheromone-sensitive neurons may guide action selection based on signalling presence or loss of the pheromone. A Hodgkin-Huxley-type neuron model with a small-conductance calcium-activated potassium (SK) channel reproduces physiological On/Off responses. Using this model as a command neuron and the antennae of tethered moths as pheromone sensors, we demonstrate the efficiency of multiphasic patterning in driving a robotic searcher toward the source. Taken together, our results suggest that multiphasic On/Off responses may mediate olfactory navigation and that SK channels may account for these responses.     

Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion.     

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.     

Butyrate-induced cell differentiation of cell-cycle mutants and ''wild-type'' mastocytoma cells: Histamine, 5-hydroxytryptamine and metachromatic granules as independently regulated differentiation markers

In cultures of heat-sensitive (hs; arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (cs; arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants that had been isolated from the same subclone (K21) of the murine P-815-X2 mastocytoma line, the degree of cell differentiation was assessed by determining the cellular histamine and 5-hydroxytryptamine (5-HT) content as well as the number of metachromatic granules per cell. The findings were compared with those obtained for 'wild-type' K21 and P-815-X2 cells. The addition of butyrate to 'wild-type' cells or to mutant cells maintained at the respective permissive temperature resulted in a relative increase in the level of all three differentiation markers. In cs mutant cells, essentially the same pronounced increase in granule numbers was observed during butyrate treatment at 39.5 degrees C and during incubation at 33 degrees C without butyrate, thereby suggesting that butyrate induces morphological cell differentiation in cs mutants via the same mechanisms as exposure to the nonpermissive temperature. In contrast, the histamine and 5-HT levels reached in hs and cs mutant cells in the presence of butyrate were higher than those observed during incubation at the nonpermissive temperature. Large quantitative differences were detected with respect to the potential of individual cell lines to express the three differentiation parameters. High levels of histamine were characteristic of 'wild-type' P-815-X2 cells treated at 33 degrees C with butyrate, while low amine levels and small numbers of granules were observed in K21 cells (i.e., the parent line of hs and cs mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of a pituitary corticotropin-releasing factor binding protein by chemical cross-linking

A corticotropin-releasing factor (CRF) binding protein has been identified based on the chemical cross-linking of ovine [Nle21,m-125I-Tyr32]CRF (125I-oCRF) to bovine anterior pituitary membranes using disuccinimidyl suberate (DSS). The apparent molecular weight of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 75,000 and was slightly decreased in its nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the molecular weight of 125I-oCRF, the binding protein appeared to have a molecular weight of approximately 70,000. The cross-linking was specific since an excess (1 microM) of an unrelated peptide (insulin) did not affect the appearance of the Mr 75,000 band. The concentration of CRF required to inhibit cross-linking by 50% was found to be similar to that determined for bovine pituitary CRF receptors by radioreceptor assay. The nonhydrolyzable GTP analogue 5'-guanylylimidodiphosphate dose dependently inhibited the cross-linking of 125I-oCRF to the Mr 70,000 protein. 50 nM of the inactive CRF analogue, [Ala14]oCRF, had no effect on the cross-linking, an observation which is consistent with this compound's low potencies in bioassays and radioreceptor assays. These results strongly suggest that this Mr 70,000 protein is the biological bovine anterior pituitary CRF receptor.     

Directional fixation of mutations in vertebrate evolution

Summary We have made pairwise comparisons between the coding sequences of 21 genes from coldblooded vertebrates and 41 homologous sequences from warm-blooded vertebrates. In the case of 12 genes, GC levels were higher, especially in third codon positions, in warm-blooded vertebrates compared to cold-blooded vertebrates. Six genes showed no remarkable difference in GC level and three showed a lower level. In the first case, higher GC levels appear to be due to a directional fixation of mutations, presumably under the influence of body temperature (see Bernardi and Bernardi 1986b). These GC-richer genes of warm-blooded vertebrates were located, in all cases studied, in isochores higher in GC than those comprising the homologous genes of cold-blooded vertebrates. In the third case, increases appear to be due to a limited formation of GC-rich isochores which took place in some cold-blooded vertebrates after the divergence of warm-blooded vertebrates. The directional changes in the GC content of coding sequences and the evolutionary conservation of both increased and unchanged GC levels are in keeping with the existence of compositional constraints on the genome.     

Species specificity of bacterial palindromic units

We described previously a family of dispersed palindromic sequences highly repeated in Escherichia coli and Salmonella typhimurium genomes. These sequences, called PU (palindromic units), are located outside structural genes. We report here observations suggesting that PU may have a role in bacterial speciation.     

Nerve Growth Factor Effect on Adenosine Transport in Cultured Chromaffin Cells

Chromaffin cells both recently isolated or in culture present a high-affinity adenosine transporter with a Km value of 1 microM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3 microM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (approximately 200-260%) in NGF-cultured cells throughout the period studied.     

CpG and TpA frequencies in the plant system.

Higher plant nuclear sequences reveal avoidance of CpG and TpA doublets. Chloroplast sequences avoid the TpA doublet in all codon positions. The chloroplast genome is not methylated but codon positions II-III and untranslated regions avoid CpG. The mitochondrial genome, also unmethylated, avoids CpG in all codon positions. We therefore deduce that methylation is not sufficient to explain CpG avoidance in the higher plant systems. Other factors must be taken into account such as amino acid composition, codon choices and perhaps stability of the DNA helix.