Uniform cAMP stimulation of Dictyostelium cells induces localized patches of signal transduction and pseudopodia

The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1microM cAMP we observed two distinct phases of PHCrac-GFP translocation. The first translocation is transient and occurs to nearly the entire boundary of the cell; the response is maximal at 6-8 s after stimulation and disappears after approximately 20 s. A second translocation of PHCrac-GFP starts after approximately 30 s and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of approximately 4-5 microm, each covering approximately 10% of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at approximately 10 s from the area with a PHCrac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP.     

A new concept for DNA-arrays

We will insert a cleavage site in an oligodeoxynucleotide, which can be used for a selective and quantitative cleavage. For that reason we synthesized the four 5'-S-(4,4'-dimethoxytrityl)-mercapto-2'-deoxynucleotide-3'-O-(2-cyanoethoxydiisopropylamino)-phosphites (5a-d). The cleavage of P-S and C-S bonds is described (Mag, M.; Lücking, S.; Engels, J.W. Synthesis and selective cleavage of an oligodeoxy-nucleotide containing a bridged internucleotide 5'-phosphorthioate linkage. Nucleic Acids Res. 1991, 19 (7), 1437-1441; Marriott, J.H.; Mottahedeh, M.; Reese, C.B. 9-(4-methoxyphenyl)xanthen-9-thiol: A useful reagent for the preparation of thiols. Tetrahedron Lett. 1990, 31 (51), 7485-7488; Divakar, K.J.; Mottoh, A.; Reese, C.B.; Shanghvi, Y.S. Approaches to the synthesis of 2' thio analogues of pyrimidine ribosides. J. Chem. Sc., Perkin Trans. 1 1990, 969-974). The oligodeoxynucleotides with an achiral bridged 5'-phosphorothioate linkage 5'-O-P-S-3' are synthesized by the phosphoramidite procedure.     

Interactions of trimeric purine nucleoside phosphorylases with ground state analogues--calorimetric and fluorimetric studies

Binding enthalpies, dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen and Cellulomonas sp. purine nucleoside phosphorylases with their ground state analogues (substrates and inhibitors) were studied by calorimetric and spectrofluorimetric methods. Data for all ligands, with possible exception of hypoxanthine, are consistent with three identical non-interacting binding sites.     

Characterization of a Dopamine D1 Receptor from Apis mellifera: Cloning, Functional Expression, Pharmacology, and mRNA Localization in the Brain

Abstract: The neurotransmitter dopamine is an important regulator of physiological and behavioral functions in both vertebrates and invertebrates. We have isolated a homologue of the vertebrate dopamine D1 receptor subfamily from the honeybee Apis mellifera . [³H]Lysergic acid diethylamide specifically binds to the heterologously expressed receptor with K _D∼5 n M . Dopaminergic receptor ligands compete for this high-affinity binding, with the following order of potency: R (+)-lisuride > chlorpromazine = cis ( Z )-flupentixol > dopamine > S (+)-butaclamol > R (+)-SCH 23390 > haloperidol. Activation of the heterologously expressed receptor of Apis mellifera leads to cyclic AMP production. Receptor mRNA is expressed in perikarya of different brain neuropils, including those of mushroom body intrinsic neurons. These results suggest that this dopamine receptor is involved in signal processing of visual and olfactory information in the honeybee.     

Bronchial airway deposition and retention of particles in inhaled boluses: effect of anatomic dead space

The fractionaldeposition of particles in boluses delivered to shallow lung depths andtheir subsequent retention in the airways may depend on the relativevolume and size of an individual's airways. To evaluate the effect ofvariable anatomic dead space (ADS) on aerosol bolus delivery we hadhealthy subjects inhale radiolabeled, monodisperse aerosol(^99mTc-iron oxide, 3.5 µm meanmondispersed aerosol diameter) boluses (40 ml) to a volumetric frontdepth of 70 ml into the lung at a lung volume of 70% total lungcapacity end inhalation. By using filter techniques, aerosolphotometry, and gamma camera analysis, we estimated the fraction of theinhaled boluses deposited in intrathoracic airways (IDF). ADS bysingle-breath N₂ washout was alsomeasured from 70% total lung capacity. Results showed that among allsubjects IDF was variable (range = 0.04-0.43, coefficient ofvariation = 0.54) and increased with decreasing ADS(r = 0.76, P = 0.001, n = 16). We found significantlygreater deposition in the left (L) vs. right (R) lungs; mean L/R (ratioof deposition in L lung to R lung, normalized to ratio of L-to-R lungvolume) was 1.58 ± 0.42 (SD; P < 0.001 for comparison with 1.0). Retention of deposited particles at 2 hwas independent of ADS or IDF. There was significant retention ofparticles at 24 h postdeposition (0.27 ± 0.05) andslow clearance of these particles continued through 48 hpostdeposition. Finally, analysis of central-to-peripheral ratios ofinitial deposition and 24-h-retention gamma-camera images suggestsignificant retention of insoluble particles in large bronchial airwaysat 24 h postdeposition (i.e., 24 h central-to-peripheral ratio = 1.40 ± 0.44 and 1.82 ± 0.54 in the R and L lung, respectively; P < 0.02 for comparison with 1.0).These data may prove useful for 1)designing aerosol delivery techniques to target bronchial airways and2) understanding airway retention ofinhaled particles.

     

Regulation of Ncx1 expression. Identification of regulatory elements mediating cardiac-specific expression and up-regulation

The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.     

Beak length analysis of the Southern Ocean squid Psychroteuthis glacialis (Cephalopoda: Psychroteuthidae) and its use for size and biomass estimation

A detailed analysis of beak length to body size and mass measurements was carried out for the glacial squid Psychroteuthis glacialis, which is an endemic cephalopod species in the Southern Ocean. Beak lengths (lower rostral length) were measured from 211 specimens which had been sampled in the Atlantic sector of the Southern Ocean. The basic idea was to find some calibration model in order to inter- or extrapolate missing mantle length and/or wet body mass data by means of beak lengths. The relationships between beak length and mantle length/wet body mass bear essential information for future use in biomass estimates in Southern Ocean top predators, since beaks of P. glacialis occur frequently in the stomach contents of Antarctic seabirds, seals and toothed whales. Therefore, lower rostral lengths were plotted against both mantle length and wet body mass to determine the relationship between these variables. The relationships had limited scatter and very high coefficients of determination, showing that lower rostral length is a good predictor of the squid's mantle length and wet mass. A non-linear 3rd order polynomial regression of lower rostral length against mantle length was identified as the best fitted calibration model, explaining 93% (R ²) of the associated variance. The relationship between lower rostral length and wet body mass was empirically well fitted through regressing ln-transformed values of lower rostral length against wet body mass, explaining 95% (R ²) of the associated variance. The present investigation provides measurements for a wide size range of P. glacialis individuals compared to earlier studies, which were limited on very small data sets. Accepted: 23 August 1999     

Characterisation of mouse monoclonal antibodies against rhesus macaque killer immunoglobulin-like receptors KIR3D

Killer immunoglobulin-like receptors (KIRs) represent a highly polymorphic and diverse gene family in rhesus macaques. Analyses of the respective gene products have been hampered until now due to non-availability of specific monoclonal antibodies and failure of cross-reactivity of anti-human KIR antibodies. We utilised one activating (KIR3DSW08) and two inhibitory (KIR3DLW03 and KIR3DL05) rhesus macaque KIR-Fc fusion proteins for generation of monoclonal antibodies in mice. Besides broadly reacting ones, we obtained anti-rhesus macaque KIR antibodies with intermediate and with single specificity. These monoclonal antibodies were tested for binding to a panel of rhesus macaque KIR proteins after heterologous expression on transiently transfected cells. Epitope mapping identified two polymorphic regions that are located next to each other in the mature KIR proteins. The availability of monoclonal antibodies against rhesus macaque KIR proteins will enable future studies on KIR at the protein level in rhesus macaques as important animal models of human infectious diseases.     

Purification of the fengycin synthetase multienzyme system from Bacillus subtilis b213

The purification of the multienzyme system producing the lipodecapeptide fengycin in Bacillus subtilis b213 was investigated. By gel filtration of a cell free extract of this organism three enzyme fractions were obtained from which five multifunctional components of fengycin synthetase were separated by high resolution anion-exchange FPLC procedures. These proteins were characterized by their thioester formation activities with ¹⁴C-labeled substrate amino acids and by N-terminal sequencing. Correlation of these data with the DNA sequences of the pps (fen) operons in three B. subtilis strains provided detailed knowledge on the structural and functional organization of fengycin synthetase.