Direct evidence for an intracellular role for IFN-gamma. Microinjection of human IFN-gamma induces Ia expression on murine macrophages

An intracellular action for IFN-gamma was detected by using microinjection technology. Human IFN-gamma (huIFN-gamma) does not ordinarily act on murine cells because it fails to bind to murine cell surface receptors. However, when huIFN-gamma was microinjected into murine macrophages, a time and dose-dependent induction of Ia was detected by autoradiography on the surface of injected and neighboring cells. These results imply a direct role for internalized IFN-gamma and show that huIFN-gamma, although it fails to be recognized by murine cell surface receptors, can act internally on murine cells. The effect on Ia gene expression induced by microinjected huIFN-gamma was in part indirect: granulocyte/macrophage-CSF (GM-CSF) was released by IFN-gamma-injected macrophages, and this secondary mediator appeared to induce Ia on neighboring cells, inasmuch as anti-GM-CSF blocked Ia induction. Anti-GM-CSF also partially blocked Ia induction by extracellular murine IFN-gamma on murine macrophages. Thus, at least some of the Ia induction attributed to IFN-gamma was mediated by GM-CSF.     

Transformation of chicken embryo fibroblasts by direct DNA transfection of single oncogenes: comparative analyses of src, erbB, myc, and ras.

Chicken embryo fibroblasts (CEF) have been used extensively to study the transformation parameters of a number of avian sarcoma-leukemia viruses. Previously, oncogene transformation of CEF has been conducted almost exclusively with replicating viruses, because of perceived difficulties with direct DNA transfection. Here, we show that CEF can be efficiently and stably transfected by selection for the neomycin resistance gene (neo). Cotransfection of neo with various oncogenes resulted in CEF transformation in vitro and, in several instances, sarcoma formation in vivo. Transfection of src, myc, erbB, and ras, either singly or in combination, resulted in soft-agar colonies with unique morphologies. Transfection of a family of v-src, c-src, and v/c-src chimeric constructs demonstrated the ability of the assay to discriminate between transforming and nontransforming genes. Transfection of a number of erbB variants showed that internal mutations, primarily in the kinase domain, contribute significantly to the ability to transform fibroblasts. The tumorigenic potential detected by transfection of oncogenes faithfully reproduced those previously reported by using viral infections. Our studies establish the utility of CEF transformation by direct DNA transfection. This method should prove useful in analyzing oncogenes, (e.g., myc) that do not readily transform rodent cell lines and in studying host-range mutants of oncogenes, such as those recently identified for src and erbB.     

Root distribution in relation to soil nitrogen availability in field-grown tomatoes

Tomato root growth and distribution were related to inorganic nitrogen (N) availability and turnover to determine 1) if roots were located in soil zones where N supply was highest, and 2) whether roots effectively depleted soil N so that losses of inorganic N were minimized. Tomatoes were direct-seeded in an unfertilized field in Central California. A trench profile/monolith sampling method was used. Concentrations of nitrate (NO₃ ^-) exceeded those of ammonium (NH₄ ⁺) several fold, and differences were greater at the soil surface (0–15 cm) than at lower depths (45–60 cm or 90–120 cm). Ammonium and NO₃ ^- levels peaked in April before planting, as did mineralizable N and nitrification potential. Soon afterwards, NO₃ ^- concentrations decreased, especially in the lower part of the profile, most likely as a result of leaching after application of irrigation water. Nitrogen pool sizes and rates of microbial processes declined gradually through the summer.Tomato plants utilized only a small percentage of the inorganic N available in the large volume of soil explored by their deep root systems; maximum daily uptake was approximately 3% of the soil pool. Root distribution, except for the zone around the taproot, was uniformly sparse (ca. 0.15 mg dry wt g^-1 soil or 0.5 cm g^-1 soil) throughout the soil profile regardless of depth, distance from the plant stem, or distance from the irrigation furrow. It bore no relation to N availability. Poor root development, especially in the N-rich top layer of soil, could explain low fertilizer N use by tomatoes.     

Synthesis and applications of an aldehyde-containing analogue of SCH-23390

SCH-23390 is a high-affinity antagonist selective for D1 dopamine receptors (Ki = 2.5 nM). It does not contain a functional group that can be conveniently coupled to commercially available resins for affinity chromatography or to prepare photolabels for photoaffinity labeling of receptors. To construct an affinity resin for purification of dopamine D1 receptors, an aldehyde analogue of SCH-23390, (+/-)-7-chloro-8-hydroxy-1-(4'-formylphenyl)-3-methyl-2,3,4,5-tetrahydro -1H- 3-benzazepine (ASCH), was synthesized. 8-Methoxy-1-(4'-bromophenyl)-SCH-23390 was lithiated, formylated, and O-demethylated to form the aldehyde. NMR and IR analyses were performed to characterize the product. Assays were performed with the radioligand [125I]SCH-23982 to define the biological activity of the aldehyde. ASCH displaced [125I]SCH-23982 binding from caudate membranes with a Ki value of 7.1 nM. ASCH has been coupled through the aldehyde group on the phenyl ring to diaminodipropylamine-agarose for affinity chromatography. After solubilization of caudate membranes in 1% digitonin, the affinity resin retained binding sites for [125I]SCH-23982 that were eluted with 10 mM SCH-23390. The aldehyde was also covalently coupled to biotin hydrazide for fluorescence labeling of dopamine D1 receptors. The biotin-conjugated aldehyde of SCH-23390 displaced [125I]SCH-23982 binding from caudate membranes with a Ki value of 9.3 nM.     

Influence of galls of Phanacis taraxaci on carbon partitioning within common dandelion, Taraxacum officinale

We examined how leaf galls, induced by the cynipid wasp Phanacis taraxaci, influence the partitioning of photoassimilates within the host, the common dandelion, Taraxacum officinale. Galled and ungalled plants were exposed to ¹⁴CO₂ and the labelled photoassimilates accumulating within galls and other parts of the host were measured. During the growth phase of the gall they were physiological sinks for photoassimilates, accumulating 9% to 70% of total carbon produced by the host, depending upon the number of galls per plant. High levels of ¹⁴C assimilation in the leaves of galled plants compared to controls, suggest that galls actively redirect carbon resources from unattacked leaves of their host plant. This represents a significant drain on the carbon resources of the host, which increases with the number and size of galls per plant. Active assimilation of ¹⁴C by the gall is greatest in the growth phase and is several orders of magnitude lower in the maturation phase. This finding is consistent with physiological and anatomical changes that occur during the two phases of gall development and represents a key developmental strategy by cynipids to ensure adequate food resources before larval growth begins.     

Mapping of the variegate porphyria (VP) gene: Contradictory evidence for linkage between VP and microsatellite markers at chromosome 14q32

The gene for variegate porphyria (VP), an autosomal dominant disease with a high prevalence in South Africa, evidently due to a founder effect, was previously mapped to chromosome 14q32. In the current study this localization was evaluated by linkage and haplotype analyses using microsatellite markers spanning a region of more than 20 cM on chromosome 14q32. In many recent studies linkage disequilibrium between disease and marker loci has been utilized to map genes in founder populations, but we could not find any association between VP and the markers used in this study. Our data suggest that the allocation of VP to chromosome 14q32 may be incorrect. Received: 1 September 1995 / Revised: 1 November 1995     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

The MAN antigens are non-lamin constituents of the nuclear lamina in vertebrate cells

The characterization of the human antiserum designated MAN has led to the identification of a subset of non-lamin proteins that are exclusively located at the nuclear periphery in all vertebrate cell types examined, from human to fish. Immunoreactive protein species were whown to comprise three major polypeptides of M _r 78000, 58000 and 40000. These antigens co-partitioned with the nuclear lamina during in situ isolation of nuclear matrices from lamin A/C-positive and-negative mammlian cells. Using double immunofluorescence, the spatial relationship of MAN antigens to type-A and type-B lamins was further examined throughout the cell cycle of lamin A/C-positive mammalian cells. In interphase HeLa and 3t3 cells, MAN antigens colocalized with both types of lamins at the periphery of the nucleus, but were absent from intranuclear foci of lamin B. As HeLa cells proceeded into mitosis, MAN antigens were seen to segregate from lamins A/C and coredistribute with lamin B. Lamins A/C disassembled during late prophase/early prometaphase and reassociated with chromatin in telophase/cytokinesis. In contrast, MAN antigens and lamin B dispersed late during prometaphase and reassembled on chromosomes in anaphase. Altogether, our data suggest that MAN antigens may play key functions in the maintenance of the structural integrity of the nuclear compartment in vertebrate cells.     

Reactions of zoo visitors to free-ranging monkeys

This study assessed the potential of two types of primate exhibit both for enhancing zoo visitors' interest, knowledge, and enjoyment and for promoting conservation education. Visitors' reactions to a free-ranging group of cotton-top tamarins were compared with their responses to caged tamarins in three ways: 1) timing how long visitors spent at each exhibit, 2) recording the comments made by visitors about the exhibits, and 3) asking visitors to respond to questionnaires about the two exhibits. The results showed that the free-ranging group of tamarins provoked wider comment from members of the public than caged groups and that visitors were willing to spend much more time looking for and watching monkeys in trees than monkeys in cages. Visitors felt that improvements in the animals' welfare were obtained from allowing them to live free in the trees, thought that they could learn more from such groups than from caged animals, and enjoyed seeing the free-ranging tamarins more than the monkeys in cages. These results suggest that developing more naturalistic zoo exhibits can have considerable benefits not only for the animals involved, but also for public education in conservation issues. © 1994 Wiley-Liss, Inc.