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.

INVESTIGATIONS ON THE INTRACELLULAR LOCALIZATION OF PHOSPHATIDYLSERINE SYNTHASE FROM ESCHERICHIA COLI

Phosphatidylserine synthase catalyzes the committed step in the synthesis of the major lipid of Escherichia coli, phosphatidylethanolamine, and may be involved in regulating the balance of the zwitterionic and anionic phospholipids in the membrane. Unlike the other enzymes involved in the biosynthesis of phospholipids in E. coli, phosphatidylserine synthase is not membrane associated but seems to have a high affinity for the ribosomal fraction of cells broken by various methods. Investigations on the enzyme in cell free extracts using glycerol gradient centrifugation revealed that the binding of the synthase to ribosomes may be prevented by the presence of highly basic compounds such as spermidine and by the presence of detergent-lipid substrate micelles under assay conditions. Thus phosphatidylserine synthase may not be ribosome associated under physiological conditions but associated with its membrane bound substrate (Louie and Dowhan (1980) J. Biol. Chem. 255, 1124).^ In addition homogeneous enzyme shows many of the properties of a membrane associated protein. It binds nonionic detergent such as Triton X-100, which is also required during purification of the enzyme. Optimal catalytic activity is also dependent on micelle or surface bound substrate. Phosphatidylserine synthase has been synthesized in vitro using a coupled transcription-translation system dependent on the presence of the cloned structural gene. The translation product was found to preferentially associate with the ribosomal fraction even in the presence of added E. coli membranes. Preferential membrane binding could be induced if the membranes were supplemented with the lipid substrate CDP-diacylglycerol. Similar effects were obtained with the acidic lipids phosphatidylglycerol and cardiolipin. On the other hand the zwitterionic lipid phosphatidylethanolamine and the lipid product phosphatidylserine did not cause any detectable membrane association. These results are consistent with the enzyme recognizing membrane bound substrate (Carman and Dowhan (1979) J. Biol. Chem. 254, 8391) and with the lipid charge influencing membrane interaction.^ Phosphatidylserine synthase is at a branch point in lipid metabolism, which may determine the distribution of the zwitterionic and anionic phospholipids in the membrane. The results obtained here indicate phosphatidylserine synthase may play a significant role in membrane lipid biosynthesis by maintaining charge balance of the E. coli membrane. In determining the localization of phosphatidylserine synthase in vitro one may have a better understanding of its function and control in vivo and may also have a better understanding of its role in membrane assembly.^

The strategies associated with the migration of networks to 4G

The networks need to provide higher speeds than those offered today. For it, considering that in the spectrum radio technologies is the scarcest resource in the development of these technologies and the new developments is essential to maximize the performance of bits per hertz transmitted. Long Term Evolution optimize spectral efficiency modulations with new air interface, and more advanced algorithms radius. These capabilities is the fact that LTE is an IPbased technology that enables end-to-end offer high transmission rates per user and very low latency, ie delay in the response times of the network around only 10 milliseconds, so you can offer any realtime application. LTE is the latest standard in mobile network technology and 3GPP ensure competitiveness in the future, may be considered a technology bridge between 3G networks - current 3.5G and future 4G networks, which are expected to reach speeds of up to 1G . LTE operators provide a simplified architecture but both robust, supporting services on IP technology. The objectives to be achieved through its implementation are ambitious, first users have a wide range of added services like capabilities that currently enjoys with residential broadband access at competitive prices, while the operator will have a network fully IP-based environment, reducing the complexity and cost of the same, which will give operators the opportunity to migrate to LTE directly. A major advantage of LTE is its ability to fuse with existing networks, ensuring interconnection with the same, increasing his current coverage and allowing a data connection established by a user in the environment continue when fade the coverage LTE. Moreover, the operator has the advantage of deploying network gradually, starting initially at areas of high demand for broadband services and expand progressively in line with this. RESUMEN. Las redes necesitan proporcionar velocidades mayores a las ofertadas a día de hoy. Para ello, teniendo en cuenta que en tecnologías radio el espectro es el recurso más escaso, en la evolución de estas tecnologías y en los nuevos desarrollos es esencial maximizar el rendimiento de bits por hercio transmitido. Long Term Evolution optimiza la eficiencia espectral con nuevas modulaciones en la interfaz aire, así como los algoritmos radio más avanzado. A estas capacidades se suma el hecho de que LTE es una tecnología basada en IP de extremo a extremo que permite ofrecer altas velocidades de transmisión por usuario y latencias muy bajas, es decir, retardos en los tiempos de respuesta de la red en torno a sólo 10 milisegundos, por lo que permite ofrecer cualquier tipo de aplicación en tiempo real. LTE es el último estándar en tecnología de redes móviles y asegurará la competitividad de 3GPP en el futuro, pudiendo ser considerada una tecnología puente entre las redes 3G – 3.5G actuales y las futuras redes 4G, de las que se esperan alcanzar velocidades de hasta 1G. LTE proporcionará a las operadoras una arquitectura simplificada pero robusta a la vez, soportando servicios sobre tecnología IP. Los objetivos que se persiguen con su implantación son ambiciosos, por una parte los usuarios dispondrá de una amplia oferta de servicios añadidos con capacidades similares a las que disfruta actualmente con accesos a banda ancha residencial y a precios competitivos, mientras que el operador dispondrá de una red basada en entorno totalmente IP, reduciendo la complejidad y el costo de la misma, lo que dará a las operadoras la oportunidad de migrar a LTE directamente. Una gran ventaja de LTE es su capacidad para fusionarse con las redes existentes, asegurando la interconexión con las mismas, aumentando su actual cobertura y permitiendo que una conexión de datos establecida por un usuario en el entorno LTE continúe cuando la cobertura LTE se desvanezca. Por otra parte el operador tiene la ventaja de desplegar la red LTE de forma gradual, comenzando inicialmente por las áreas de gran demanda de servicios de banda ancha y ampliarla progresivamente en función de ésta.

Influence of source and micronization of soya bean meal on growth performance, nutrient digestibility and ileal mucosal morphology of Iberian piglets

The effects of inclusion in the diet of different sources of soya bean meal (SBM) on growth performance, total tract apparent digestibility (TTAD) and apparent ileal digestibility (AID) of major dietary components and mucosal ileum morphology were studied in Iberian pigs weaned at 30 days of age. From 30 to 51 days of age (phase I), there was a control diet based on regular soya bean meal (R-SBM; 44% CP) of Argentina (ARG) origin and five extra diets in which a high-protein soya bean meal (HP-SBM; 49% CP) of the USA or ARG origin, either ground (990 μm) or micronized (60 μm), or a soya protein concentrate (SPC; 65% CP) substituted the R-SBM. From 51 to 61 days of age (phase II), all pigs were fed a common commercial diet in mash form. The following pre-planned orthogonal contrasts were conducted: (1) R-SBM v. all the other diets, (2) SPC v. all the HP-SBM diets, (3) micronized HP-SBM v. ground HP-SBM, (4) HP-SBM of ARG origin v. HP-SBM of US origin and (5) interaction between source and the degree of grinding of the HP-SBM. Dietary treatment did not affect growth performance of the pigs at any age but from 30 to 51 days of age, post weaning diarrhoea (PWD) was higher (P<0.001) and the TTAD and AID of all nutrients were lower for pigs fed the R-SBM diet than for pigs fed the HP-SBM or the SPC diets. However, no differences between the HP-SBM and the SPC containing diets were detected for any trait. The TTAD of organic matter (P=0.07) and gross energy (GE) (P=0.05) tended to be higher for the micronized HP-SBM than for the ground HP-SBM and that of GE was higher (P<0.05) for US meal than for the ARG meal. Pigs fed R-SBM had lower villus height (P<0.01) than pigs fed HP-SBM or SPC but no differences in ileal mucosal morphology were detected between SPC and HP-SBM containing diets. It is concluded that feeding the HP-SBM or SPC-reduced PWD and improved nutrient digestibility and ileal morphology as compared with feeding the R-SBM, but had no effect on pig performance. The inclusion in the diet of added value soya products (micronized SBM or SPC) in substitution of the R-SBM increased the TTAD of all nutrients and reduced PWD but had no advantage in terms of growth performance over the use of ground HP-SBM.

Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia

Primitive subsets of leukemic cells isolated by using fluorescence-activated cell sorting from patients with newly diagnosed Ph+/BCR–ABL+ chronic myeloid leukemia display an abnormal ability to proliferate in vitro in the absence of added growth factors. We now show from analyses of growth-factor gene expression, protein production, and antibody inhibition studies that this deregulated growth can be explained, at least in part, by a novel differentiation-controlled autocrine mechanism. This mechanism involves the consistent and selective activation of IL-3 and granulocyte colony-stimulating factor (G-CSF) production and a stimulation of STAT5 phosphorylation in CD34+ leukemic cells. When these cells differentiate into CD34− cells in vivo, IL-3 and G-CSF production declines, and the cells concomitantly lose their capacity for autonomous growth in vitro despite their continued expression of BCR–ABL. Based on previous studies of normal cells, excessive exposure of the most primitive chronic myeloid leukemia cells to IL-3 and G-CSF through an autocrine mechanism could explain their paradoxically decreased self-renewal in vitro and slow accumulation in vivo, in spite of an increased cycling activity and selective expansion of later compartments.

Preassembly of interleukin 2 (IL-2) receptor subunits on resting Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: A fluorescence resonance energy transfer study

Assembly and mutual proximities of α, β, and γc subunits of the interleukin 2 receptors (IL-2R) in plasma membranes of Kit 225 K6 T lymphoma cells were investigated by fluorescence resonance energy transfer (FRET) using fluorescein isothiocyanate- and Cy3-conjugated monoclonal antibodies (mAbs) that were directed against the IL-2Rα, IL-2Rβ, and γc subunits of IL-2R. The cell-surface distribution of subunits was analyzed at the nanometer scale (2–10 nm) by FRET on a cell-by-cell basis. The cells were probed in resting phase and after coculture with saturating concentrations of IL-2, IL-7, and IL-15. FRET data from donor- and acceptor-labeled IL-2Rβ-α, γ-α, and γ-β pairs demonstrated close proximity of all subunits to each other in the plasma membrane of resting T cells. These mutual proximities do not appear to represent mAb-induced microaggregation, because FRET measurements with Fab fragments of the mAbs gave similar results. The relative proximities were meaningfully modulated by binding of IL-2, IL-7, and IL-15. Based on FRET analysis the topology of the three subunits at the surface of resting cells can be best described by a “triangular model” in the absence of added interleukins. IL-2 strengthens the bridges between the subunits, making the triangle more compact. IL-7 and IL-15 act in the opposite direction by opening the triangle possibly because they associate their private specific α receptors with the β and/or γc subunits of the IL-2R complex. These data suggest that IL-2R subunits are already colocalized in resting T cells and do not require cytokine-induced redistribution. This colocalization is significantly modulated by binding of relevant interleukins in a cytokine-specific manner.

Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol—disulfide status

The Escherichia coli transcription factor OxyR is activated by the formation of an intramolecular disulfide bond and subsequently is deactivated by enzymatic reduction of the disulfide bond. Here we show that OxyR can be activated by two possible pathways. In mutants defective in the cellular disulfide-reducing systems, OxyR is constitutively activated by a change in the thiol—disulfide redox status in the absence of added oxidants. In wild-type cells, OxyR is activated by hydrogen peroxide. By monitoring the presence of the OxyR disulfide bond after exposure to hydrogen peroxide in vivo and in vitro, we also show that the kinetics of OxyR oxidation by low concentrations of hydrogen peroxide is significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall reducing environment. We propose that the activity of OxyR in vivo is determined by the balance between hydrogen peroxide levels and the cellular redox environment.

Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis

RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse–chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A1 and A2 are inhibited and delayed at site A0. Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.

In Vivo Binding and Hierarchy of Assembly of the Yeast RNA Polymerase I Transcription Factors

Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a series of transcription factors that have been genetically and biochemically identified. In particular, the core factor (CF) and the upstream activation factor (UAF) have been shown in vitro to bind the core element and the upstream promoter element, respectively. We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking one specific transcription factor at the time. In this way we were able to unambiguously attribute the protections by the CF and the UAF to their respective putative binding sites. In addition, we have found that in vivo a binding hierarchy exists, the UAF being necessary for CF binding. Because the CF footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affects binding of the CF, stabilizing its contact with DNA. Thus, in vivo, the CF is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.

Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR

We have used 19F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of 19F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg2+. The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg2+ with an association constant in the range of 100 to 500 M−1, depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range.

Three Mechanisms for the Calcium Alleviation of Mineral Toxicities

Ca2+ in rooting medium is essential for root elongation, even in the absence of added toxicants. In the presence of rhizotoxic levels of Al3+, H+, or Na+ (or other cationic toxicants), supplementation of the medium with higher levels of Ca2+ alleviates growth inhibition. Experiments to determine the mechanisms of alleviation entailed measurements of root elongation in wheat (Triticum aestivum L. cv Scout 66) seedlings in controlled medium. A Gouy-Chapman-Stern model was used to compute the electrical potentials and the activities of ions at the root-cell plasma membrane surfaces. Analysis of root elongation relative to the computed surface activities of ions revealed three separate mechanisms of Ca2+ alleviation. Mechanism I is the displacement of cell-surface toxicant by the Ca2+-induced reduction in cell-surface negativity. Mechanism II is the restoration of Ca2+ at the cell surface if the surface Ca2+ has been reduced by the toxicant to growth-limiting levels. Mechanism III is the collective ameliorative effect of Ca2+ beyond mechanisms I and II, and may involve Ca2+-toxicant interactions at the cell surface other than the displacement interactions of mechanisms I and II. Mechanism I operated in the alleviation of all of the tested toxicities; mechanism II was generally a minor component of alleviation; and mechanism III was toxicant specific and operated strongly in the alleviation of Na+ toxicity, moderately in the alleviation of H+ toxicity, and not at all in the alleviation of Al3+ toxicity.

Overexpression of Nitrate Reductase in Tobacco Delays Drought-Induced Decreases in Nitrate Reductase Activity and mRNA1

Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3− decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit.

Characterization of a Gene for Spinach CAP160 and Expression of Two Spinach Cold-Acclimation Proteins in Tobacco1

The cDNA sequence for CAP160, an acidic protein previously linked with cold acclimation in spinach (Spinacia oleracea L.), was characterized and found to encode a novel acidic protein of 780 amino acids having very limited homology to a pair of Arabidopsis thaliana stress-regulated proteins, rd29A and rd29B. The lack of similarity in the structural organization of the spinach and Arabidopsis genes highlights the absence of a high degree of conservation of this cold-stress gene across taxonomic boundaries. The protein has several unique motifs that may relate to its function during cold stress. Expression of the CAP160 mRNA was increased by low-temperature exposure and water stress in a manner consistent with a probable function during stresses that involve dehydration. The coding sequences for CAP160 and CAP85, another spinach cold-stress protein, were introduced into tobacco (Nicotiana tabacum) under the control of the 35S promoter using Agrobacterium tumefaciens-based transformation. Tobacco plants expressing the proteins individually or coexpressing both proteins were evaluated for relative freezing-stress tolerance. The killing temperature for 50% of the cells of the transgenic plants was not different from that of the wild-type plants. As determined by a more sensitive time/temperature kinetic study, plants expressing the spinach proteins had slightly lower levels of electrolyte leakage than wild-type plants, indicative of a small reduction of freezing-stress injury. Clearly, the heterologous expression of two cold-stress proteins had no profound influence on stress tolerance, a result that is consistent with the quantitative nature of cold-stress-tolerance traits.

Ligand induction of a transcriptionally active thyroid hormone receptor coactivator complex.

Transcriptional regulation by nuclear hormone receptors is thought to involve interactions with putative cofactors that may potentiate receptor function. Here we show that human thyroid hormone receptor alpha purified from HeLa cells grown in the presence of thyroid hormone (T3) is associated with a group of distinct nuclear proteins termed thyroid hormone receptor-associated proteins (TRAPs). In an in vitro system reconstituted with general initiation factors and cofactors (and in the absence of added T3), the "liganded" thyroid hormone receptor (TR)/TRAP complex markedly activates transcription from a promoter template containing T3-response elements. Moreover, whereas the retinoid X receptor is not detected in the TR/TRAP complex, its presence is required for the function of the complex. In contrast, human thyroid hormone receptor alpha purified from cells grown in the absence of T3 lacks the TRAPs and effects only a low level of activation that is dependent on added ligand. These findings demonstrate the ligand-dependent in vivo formation of a transcriptionally active TR-multisubunit protein complex and suggest a role for TRAPs as positive coactivators for gene-specific transcriptional activation.

The transcription factors c-myb and GATA-2 act independently in the regulation of normal hematopoiesis.

The transcription factors c-myb and GATA-2 are both required for blood cell development in vivo and in vitro. However, very little is known on their mechanism(s) of action and whether they impact on complementary or overlapping pathways of hematopoietic proliferation and differentiation. We report here that embryonic stem (ES) cells transfected with c-myb or GATA-2 cDNAs, individually or in combination, underwent hematopoietic commitment and differentiation in the absence of added hematopoietic growth factors but that stimulation with c-kit and flt-3 ligands enhanced colony formation only in the c-myb transfectants. This enhancement correlated with c-kit and flt-3 surface receptor up-regulation in c-myb-(but not GATA-2-) transfected ES cells. Transfection of ES cells with either a c-myb or a GATA-2 antisense construct abrogated erythromyeloid colony-forming ability in methyl cellulose; however, introduction of a full-length GATA-2 or c-myb cDNA, respectively, rescued the hematopoiesis-deficient phenotype, although only c-myb-rescued ES cells expressed c-kit and flt-3 surface receptors and formed increased numbers of hematopoietic colonies upon stimulation with the cognate ligands. These results are in agreement with previous studies indicating a fundamental role of c-myb and GATA-2 in hematopoiesis. Of greater importance, our studies suggest that GATA-2 and c-myb exert their roles in hematopoietic gene regulation through distinct mechanisms of action in nonoverlapping pathways.