Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.

Membrane Lipid Integrity Relies on a Threshold of ATP Production Rate in Potato Cell Cultures Submitted to Anoxia

In this paper we report on our study of the changes in biomass, lipid composition, and fermentation end products, as well as in the ATP level and synthesis rate in cultivated potato (Solanum tuberosum) cells submitted to anoxia stress. During the first phase of about 12 h, cells coped with the reduced energy supply brought about by fermentation and their membrane lipids remained intact. The second phase (12–24 h), during which the energy supply dropped down to 1% to 2% of its maximal theoretical normoxic value, was characterized by an extensive hydrolysis of membrane lipids to free fatty acids. This autolytic process was ascribed to the activation of a lipolytic acyl hydrolase. Cells were also treated under normoxia with inhibitors known to interfere with energy metabolism. Carbonyl-cyanide-4-trifluoromethoxyphenylhydrazone did not induce lipid hydrolysis, which was also the case when sodium azide or salicylhydroxamic acid were fed separately. However, the simultaneous use of sodium azide plus salicylhydroxamic acid or 2-deoxy-D-glucose plus iodoacetate with normoxic cells promoted a lipid hydrolysis pattern similar to that seen in anoxic cells. Therefore, a threshold exists in the rate of ATP synthesis (approximately 10 μmol g−1 fresh weight h−1), below which the integrity of the membranes in anoxic potato cells cannot be preserved.

Geological, geochemical and biogeochemical data on boundary zones of the Atlantic Ocean

Results of studies in two biogeochemically active zones of the Atlantic Ocean (the Benguela upwelling waters and the region influenced by the Congo River run-off) are reported in the book. A multidisciplinary approach included studies of the major elements of the ocean ecosystem: sea water, plankton, suspended matter, bottom sediments, interstitial waters, aerosols, as well as a wide complex of oceanographic studies carried out under a common program. Such an approach, as well as a use of new methodical solutions led to obtaining principally new information on different aspects of oceanology.

Liquid-Crystalline Dendrimers Designed by Click Chemistry

Liquid-crystalline dendrimers have been prepared from second-generation Percec-type poly(benzyl ether) dendrons or second-generation poly(aryl ester) dendrons carrying cyanobiphenyl mesogens. The Janus dendrimer, which combines the two types of dendromesogens, has also been synthesized. Those compounds have been prepared under copper-catalyzed azide–alkyne cycloaddition conditions. The mesomorphic properties have been studied by thermal analysis (POM, DSC) and small-angle X-ray scattering. Smectic A, nematic, and columnar phases have been observed depending on the dendritic building blocks. The click reaction has proven to be a powerful and elegant synthetic tool for the design of complex dendritic liquid-crystalline architectures.

Real time monitoring of click chemistry self-healing in polymer composites

A photo-healable rubber composite based on effective and fast thiol-alkyne click chemistry as a selfhealing agent prestored in glass capillaries is reported. The click reaction and its effect on the mechanical properties of the composite are monitored in real time by dynamic mechanical analysis, showing that the successful bleeding of healing agents to the crack areas and the effective photoinitiated click reaction result in a 30% storage modulus increase after only 5 min of UV light exposure. X-ray tomography confirms capillary-driven bleeding of reactants to the damaged areas. The effect of storing the click chemistry reactants in separate capillaries is also studied, and results show the importance of stoichiometry in achieving a significant level of repair of the composite. No reactant degradation or premature chemical reaction is observed over time in samples stored in the absence of UV radiation; they are able to undergo the self-healing reaction even one month after preparation.

Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide⋅cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

Interaction of the 82-kDa subunit of the vaccinia virus early transcription factor heterodimer with the promoter core sequence directs downstream DNA binding of the 70-kDa subunit.

The vaccinia virus early transcription factor (VETF), a heterodimeric protein composed of 82- and 70-kDa subunits, interacts with viral early promoters at both a sequence-specific core region upstream and a sequence-independent region downstream of the RNA start site. To determine the VETF subunit-promoter interactions, 32P-labeled DNA targets were chemically synthesized with uniquely positioned phosphorothioates to which azidophenacyl bromide moieties were coupled. After incubating the derivatized promoter with VETF and exposing the complex to 302-nm light, the protein was denatured and the individual subunits with or without covalently bound DNA were isolated with specific antiserum and analyzed by SDS/polyacrylamide gel electrophoresis. Using a set of 26 duplex probes, with uniquely positioned aryl azide moieties on the coding or template strands, we found that the 82-kDa subunit interacted primarily with the core region of the promoter, whereas the 70-kDa subunit interacted with the downstream region. Nucleotide substitutions in the core region that downregulate transcription affected the binding of both subunits: the 82-kDa subunit no longer exhibited specificity for upstream regions of the promoter but also bound to downstream regions, whereas the binding of the 70-kDa subunit was abolished even though the mutations were far upstream of its binding site. These results suggested mechanisms by which the interaction of the 82-kDa subunit with the core sequence directs binding of the 70-kDa subunit to DNA downstream.

Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell.

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).