cAMP controls cytosolic Ca<Superscript>2+</Superscript> levels in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) that is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca²⁺]_i.     

Ca<Superscript>2+</Superscript> reduces the effect of hypoxia in mosses <Emphasis Type="Italic">Mnium undulatum</Emphasis> and <Emphasis Type="Italic">Polytrichum commune</Emphasis>

Gametophores of mosses Mnium undulatum and Polytrichum commune were submerged in distilled water or in calcium chloride solution (0.9 mM Ca²⁺) to induce hypoxia. The net photosynthetic (P_N) and dark respiration rate (R_D) were measured in the air containing 300–400 μmol(CO₂)·mol⁻¹(air) and 0.21 mol(O₂)·mol⁻¹(air). P_N of M. undulatum gametophores decreased to 58 % of the control after 1-h submersion in water, whereas to 80 % of the control in P. commune gametophores. A smaller decrease in P_N was observed when the gametophores were immersed in CaCl₂ solution. In hypoxia, R_D in the tested mosses species was a little higher than in the control.     

Ca<Superscript>2+</Superscript> regulation in the absence of the <Emphasis Type="Italic">iplA</Emphasis> gene product in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). The [Ca²⁺]_i-change is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-entry. The significance of the [Ca²⁺]_i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca²⁺-buffers in the cytosol indicates that a [Ca²⁺]_i-increase is required for chemotaxis. Yet, the iplA ^- mutant disrupted in a gene bearing similarity to IP₃-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca²⁺]_i-transient which favours the view that [Ca²⁺]_i-changes are insignificant for chemotaxis.     

Ca<Superscript>2+ </Superscript>and Cu<Superscript>2+ </Superscript>supplementation increases mannitol production by <Emphasis Type="Italic">Candida magnoliae</Emphasis>

Supplementation with CaCl₂·2H₂O (50 mg l⁻¹) or CuSO₄·5H₂O (10 mg l⁻¹) improved mannitol production by Candida magnoliae by 14.5 and 18.6% (25 and 32 g/L), respectively. When used in combination, they acted synergistically: Ca²⁺ decreased the intracellular concentration of mannitol 30%, whereas Cu²⁺ increased the intracellular activity of mannitol dehydrogenase 1.6-times more than control. Ca²⁺ probably works by altering the permeability of cells to mannitol, whereas, Cu²⁺ increases the activity of an enzyme responsible for mannitol biosynthesis.     

Membrane Stretching Triggers Mechanosensitive Ca<Superscript>2+</Superscript> Channel Activation in <Emphasis Type="Italic">Chara</Emphasis>

In order to confirm that mechanosensitive Ca²⁺ channels are activated by membrane stretching, we stretched or compressed the plasma membrane of Chara by applying osmotic shrinkage or swelling of the cell by varying the osmotic potential of the bathing medium. Aequorin studies revealed that treatments causing membrane stretching induced a transient but large increase in cytoplasmic concentration of Ca²⁺ (Δ[Ca²⁺]_c). However, the observed Δ[Ca²⁺]_c decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (ΔE _m) and stretching or compression of the plasma membrane. Significant ΔE _m values, often accompanied by an action potential, were observed during the initial exchange of the bathing medium from a hypotonic medium to a hypertonic one (plasmolysis). ΔE _m appears to be triggered by a partial stretching of the membrane as it was peeled from the cell wall. After plasmolysis, other exchanges from hypertonic to hypotonic media, with their accompanying membrane stretching, always induced large ΔE _m values and were often accompanied by an action potential. By contrast, action potentials were scarcely observed during other exchanges from hypotonic to hypertonic solutions (=membrane compression). Thus, we concluded that activation of the mechanosensitive channels is triggered by membrane stretching in Chara.     

Enhanced intracellular Ca<Superscript>2+</Superscript> concentrations in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Bacillus subtilis</Emphasis> after addition of oligosaccharide elicitors

Elicitation can lead to overproduction of secondary metabolites in plants and microbes. Potential changes in cytosolic Ca²⁺ levels in bacteria were studied in response to elicitation. We report, for the first time, the effect of oligosaccharide elicitors on intracellular Ca²⁺ levels. The apoaequorin gene was cloned into Escherichia coli DH5α and Bacillus subtilis 1604 cultures. Addition of elicitors, oligoguluronate and mannan oligosaccharides, to the cultures caused up to 11-fold increase in cytosolic Ca²⁺ in E. coli and tenfold increase in B. subtilis. These increases in Ca²⁺ levels could therefore contribute to the enhancement of secondary metabolite levels.     

Antagonistic effect of divalent cations Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> on the morphological development of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> var. <Emphasis Type="Italic">geldanus</Emphasis>

The automated docking program DOCK 5.3.0 was applied to screening for quorum sensing inhibitors (QSIs) of Peudomonus aeruginosa from a database containing 51 active components of Traditional Chinese Medicines with antibacterial activity. Five potential QSIs were revealed by the computer-based virtual screening. The compounds 3, 4, 5, 6, 7 inhibit biofilm formation of P. aeruginosa at a concentration of 200 μM. Compound 4 (baicalein) does not inhibit the growth of P. aeruginosa; however, it significantly inhibits biofilm formation of the bacteria at a lower concentration of 20 μM and promoted proteolysis of the signal receptor TraR protein in Escherichia coli at 4–40 mM. Baicalein and ampicillin showed synergistic activity against P. aeruginosa. These results suggested that baicalein can interfere with quorum sensing system of P. aeruginosa and will be developed as antibacterial agent with novel target.     

Crystal structure of wild-type centrin 1 from <Emphasis Type="Italic">Mus musculus</Emphasis> occupied by Ca<Superscript>2+</Superscript>

Mus musculus centrin 1 (MmCen1) is located at the cilium of photoreceptor cells connecting the outer segment through signal transduction components to the metabolically active inner segment. In the cilium, MmCen1 is involved in the translocation of transducin between compartments as a result of photoreceptor activation. In this study, we report the crystal structure of wild-type MmCen1 and its Ca²⁺-binding properties using structure-based mutagenesis. The crystal structure exhibits three structural features, i.e. four Ca²⁺ equally occupied at each EF-hand motif, structural changes accompanying helix motion at the N- and C-lobes, and adoption of N–C type dimerization when Ca²⁺ binds to EF-hand I and II in the N-lobe. The presence of MmCen1 dimers was confirmed in solution by native PAGE. Isothermal titration calorimetry data showed sequential binding of Ca²⁺ at four independent sites. Mutations S45A and D49A in EF-hand I alone disrupted the Ca²⁺-binding property of the wild-type protein. Based on the crystal structure of MmCen1, we suggest that a dimerization mode between the N- and C-lobes may be required by Ca²⁺ binding at the N-lobe.     

Characterisation of electrogenic nutrient absorption in the <Emphasis Type="Italic">Cftr</Emphasis><Superscript><Emphasis Type="Italic">TgH(neoim)Hgu</Emphasis></Superscript> mouse model

Most cystic fibrosis (CF) patients show an exocrine pancreatic insufficiency that results in lower enzyme and bicarbonate secretion. To test whether an altered function of nutrient transporters might additionally attribute to the lower bodyweight of CF patients we investigated electrogenic absorption of alanine, glycyl-glutamine, glucose and the effect of pH on nutrient absorption by Ussing chambers in a CF mouse model carrying the Cftr TgH(neoim)Hgu mutation. The electrogenic transport of all three nutrients was similar between the D2.129P2(CF/3)-Cftr TgH(neoim)Hgu congenic strain and DBA/2J mice as well as between the B6.129P2(CF/3)-Cftr TgH(neoim)Hgu congenic strain and C57BL/6J mice. This indicates that the Cftr TgH(neoim)Hgu mutation does not affect the electrogenic absorption of alanine, glycyl-glutamine and glucose. In contrast, electrogenic nutrient absorption was reduced in the CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu inbred strains compared to the HsdOla:MF1, D2.129P2(CF/3)-Cftr TgH(neoim)Hgu and B6.129P2(CF/3)-Cftr TgH(neoim)Hgu strains, whereas no difference was found among the wild-type strains. This indicates that not the Cftr TgH(neoim)Hgu mutation but differences in the genetic background of the CF/1-Cftr TgH(neoim)Hgu and CF/3-Cftr TgH(neoim)Hgu strains compared to HsdOla:MF1, D2.129P2(CF/3)-Cftr TgH(neoim)Hgu and B6.129P2(CF/3)-Cftr TgH(neoim)Hgu strains are associated with the differences in electrogenic nutrient absorption. The electrogenic absorption of alanine, glycyl-glutamine and glucose was not influenced by an acidic pH (5.4) compared to absorption at pH 7.4.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Cdkn1c</Emphasis> (<Emphasis Type="Italic">p57</Emphasis><Superscript><Emphasis Type="Italic">Kip2</Emphasis></Superscript>) is the major regulator of embryonic growth within its imprinted domain on mouse distal chromosome 7

Background  

Cdkn1c encodes an embryonic cyclin-dependant kinase inhibitor that acts to negatively regulate cell proliferation and, in some tissues, to actively direct differentiation. This gene, which is an imprinted gene expressed only from the maternal allele, lies within a complex region on mouse distal chromosome 7, called the IC2 domain, which contains several other imprinted genes. Studies on mouse embryos suggest a key role for genomic imprinting in regulating embryonic growth and this has led to the proposal that imprinting evolved as a consequence of the mismatched contribution of parental resources in mammals.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

The synthesis of <Emphasis Type="Italic">N</Emphasis><Superscript>2</Superscript>,<Emphasis Type="Italic">N</Emphasis><Superscript>6</Superscript>-substituted diaminopurine ribosides

A series of new 2,6-substituted diaminopurine riboside derivatives were synthesized by activation of protected xantosine with sulfonyl chlorides followed by treatment with various amines. The relationship between the reactivity of intermediates and the nature of the activating agents was studied.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.