Purification and thermal dependence of glutathione reductase from two forage legume species

Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from `Cimarron' alfalfa and `PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45°C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25°C), and (c) the thermal kinetic windows (6-19°C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season.     

Regulation of cyclic AMP accumulation in lymphoid cells

We have examined several features of the regulation of cyclic AMP accumulation in lymphoid cells isolated from peripheral blood of human subjects and in the murine T-lymphoma cell line, S49, S49 cells are unique because of the availability of variant clones with lesions in the pathway of cyclic AMP generation and response. We found that human lymphoid cells prepared at 4 degrees C showed substantially greater cyclic AMP accumulation in response to histamine and the beta-adrenergic agonist isoproterenol than did cells prepared at ambient temperature. The muscarinic cholinergic agonist carbamylcholine and peptide hormone somatostatin failed to inhibit cyclic AMP accumulation in human lymphoid cells and treatment with pertussis toxin (which blocks function of Gi, the guanine nucleotide binding protein that mediates inhibition of adenylate cyclase) only minimally increased cyclic AMP levels in these cells. Thus the Gi component of adenylate cyclase appears to play only a small role in modulating cyclic AMP levels in this mixed population of lymphoid cells. Incubation of whole blood with isoproterenol desensitized human lymphocytes to subsequent stimulation with beta agonist. This desensitization was associated with a redistribution of beta-adrenergic receptors such that a substantial portion of the receptors in intact cells could no longer bind a hydrophilic antagonist. Wild-type S49 lymphoma cells showed a similar redistribution of beta-adrenergic receptors after a few minutes' incubation with agonist. Based on studies in S49 variants, this redistribution is independent of components distal to receptors in the adenylate cyclase/cyclic AMP pathway. By contrast, a more slowly developing, agonist-mediated down-regulation of beta-adrenergic receptors was blunted in variants with defective interaction between receptors and Gs, the guanine nucleotide binding protein that mediates stimulation of adenylate cyclase. Unlike results in human lymphoid cells, S49 cells show a prominent inhibition of cyclic AMP accumulation mediated by Gi; this inhibition is promoted by somatostatin and blocked by pertussis toxin. Inhibition by Gi is unable to account for the marked decrease in ability of the diterpene forskolin to maximally stimulate adenylate cyclase in S49 variants having defective Gs. These results emphasize that both Gs and Gi component are important in modulating cyclic AMP accumulation and receptors linked to adenylate cyclase in S49 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

A serum-free primary culture system for studying cell-substrate interactions during newt epidermal cell migration

Summary To study the interaction of migrating newt epidermal cells with purified extracellular matrix (ECM) molecules we have developed an in vitro migration assay using pieces of newt skin explanted onto culture dishes coated with various ECM molecules and cultured for 18 h in defined serum-free medium. Newt epidermal cells migrate out from explants placed on dishes coated with either collagen, vitronectin, fibronectin, or fibrinogen but not on albumin-coated or uncoated dishes. Explant outgrowth on collagen was best in CEM 2000 medium diluted to 60% of mammalian osmolarity. Other media such as RPMI 1640 or Ex-Cell 300, diluted similarly, may also be used although in our hands CEM 2000 always allowed more migration. We found no migration on collagen when skin explants were incubated in Holtfreter's solution (an amphibian saline solution that we have previously shown allows reepithelialization on amputated newt limbs). Supplementation of Holtfreter's solution with glucose did not improve its ability to support migration. By testing various supplement combinations in conjunction with CEM 2000 and RPMI 1640 we found that neither serum, insulin, selenium, transferrin, norl-glutamine is required for explant outgrowth. Of the additives tested, outgrowth was stimulated only by insulin. Epidermal cell outgrowth on collagen was inhibited by both puromycin and cycloheximide, indicating the necessity for protein synthesis in this system. Whether the effects of these protein synthesis inhibitors are specifically on migration-related events or on general metabolic requirements is not clear. Inasmuch as there was no correlation (r=−0.227) between DNA synthesis (measured by incorporation of tritiated thymidine) and the amount of outgrowth, we believe that our assay is a measure of cell migration alone rather than a combination of mitosis and migration. This explant outgrowth system represents a new and relatively simple assay that can be used in the study of cell-substrate interactions during newt epidermal cell migration over extracellular matrix molecules in a defined serum-free environment. This work was supported by NIH grant AR27940 awarded to D. J. D.     

A graphical method for detecting recombination in phylogenetic data sets

Current phylogenetic tree reconstruction methods assume that there is a single underlying tree topology for all sites along the sequence. The presence of mosaic sequences due to recombination violates this assumption and will cause phylogenetic methods to give misleading results due to the imposition of a single tree topology on all sites. The detection of mosaic sequences caused by recombination is therefore an important first step in phylogenetic analysis. A graphical method for the detection of recombination, based on the least squares method of phylogenetic estimation, is presented here. This method locates putative recombination breakpoints by moving a window along the sequence. The performance of the method is assessed by simulation and by its application to a real data set.      

Temporal fluctuation in shrub species preferences of two native rodents: The effect of infection status on habitat use

Small mammals use plant species for gathering food resources and for shelter. Preferences for certain plant species are related to nutritional restrictions and behavioural patterns, which could be altered in the presence of an infectious disease. Several native small mammals are part of the wild cycle of the protozoan Trypanosoma cruzi, responsible for Chagas disease in humans. This is a vector‐borne disease transmitted by insects of the subfamily Triatominae. We examined the effect of T. cruzi infection status on the use and preference patterns of shrub species by two native rodent species: Octodon degus and Phyllotis darwini. This study was conducted during four sampling years (2010–2013) in a hyper‐endemic zone of Chagas disease located in a semiarid Mediterranean ecosystem. We captured individuals of 599 O. degus and 575 P. darwini (89% of the total captures), which were related to nine shrub species and examined for T. cruzi infection. In a community‐level analysis, infected and non‐infected O. degus used individual shrub species within the shrub community significantly non‐randomly relative to their availability; the same pattern was detected for non‐infected P. darwini individuals, whereas infected individuals used the shrub community according to the abundance of each shrub species. Examining individual preferences, both rodents showed a strong preference for Flourensia thurifera and Colliguaja odorifera regardless of their infection status. Preferences for specific shrub species were variable among years, showing a ‘core’ of preferred shrub species and variable levels of use of the remaining ones. Our results show that T. cruzi infection in wild small mammals can modify habitat use patterns and preferences for certain shrub species, probably affecting processes acting at community level.     

Inability of newt epidermal cells to migrate over concanavalin A-coated substrates

Pieces of coverslip glass coated with various proteins were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Despite the fact that concanavalin A (Con A) receptors could be demonstrated on newt epidermal cells with fluorescein isothiocyanate (FITC)-conjugated lectin, Con A-coated implants supported practically no migration, an even poorer response than the modest amount of migration that occurred on uncoated glass. Coomassie blue staining verified that the lectin formed a complete film over the glass, and peroxidase binding assays showed that even after several hours in the wound, the Con A binding sites for mannose were still available. Migration on fibrinogen-coated glass (a good migration substrate) was not affected by placing the implants next to Con A-coated implants. Thus, the failure to migrate on Con A cannot be explained by soluble Con A effects from lectin leaching off the implants. These data suggest that linkages between cell surface mannose and the substrate are not part of the strategy by which newt epidermal cells migrate.     

Isolation, characterization, and developmental expression of pig intestinal fatty acid-binding proteins

The goal of this study was to characterize and quantify intestinal fatty acid-binding proteins of the pig. Small intestinal mucosa from 13-19 kg pigs was homogenized and centrifuged to obtain cytosol. Isolation of fatty acid-binding proteins from delipidated cytosol was achieved using molecular sieve, oleic acid affinity, and ion exchange chromatography. Fatty acid-binding protein isolation was monitored using a fatty-acid binding assay in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Antisera to rat liver-fatty acid-binding protein cross reacted with an isolated intestinal fatty acid-binding protein of Mr = 13,000, whereas antisera to rat intestine-fatty acid-binding protein was not cross reactive with isolated pig intestinal proteins. These experiments identify a pig intestinal fatty acid-binding protein that exhibits strong immunochemical similarity to rat liver-fatty acid-binding protein. Cytosol prepared from intestinal mucosa of pigs at -4, 2, 4, 7, 15, 22, 28, and 35 d of age was assayed for fatty acid-binding protein activity. Preweaning fatty acid-binding protein activity in cytosol was maximal at 7 days of age when expressed as total jejunal fatty acid binding per kilogram bodyweight, intestinal or mucosal weight or milligram total protein. After weaning (21 d), fatty acid-binding protein activities declined to 28 days, but increased again by 35 days. Total soluble fatty acid-binding protein activity in pig intestine is regulated during postnatal development and this may account in part for the altered intestinal absorption of lipids observed in young pigs at weaning.