Lipolytic and metabolic response to glucagon in fasting king penguins: phase II vs. phase III

This study aims to determine how glucagon intervenes in the regulation of fuel metabolism, especially lipolysis, at two stages of a spontaneous long-term fast characterized by marked differences in lipid and protein availability and/or utilization (phases II and III). Changes in the plasma concentration of various metabolites and hormones, and in lipolytic fluxes as determined by continuous infusion of [2-3H]glycerol and [1-14C]palmitate, were examined in vivo in a subantarctic bird (king penguin) before, during, and after a 2-h glucagon infusion. In the two fasting phases, glucagon infusion at a rate of 0.025 microg. kg(-1). min(-1) induced a three- to fourfold increase in the plasma concentration and in the rate of appearance (Ra) of glycerol and nonesterified fatty acids, the percentage of primary reesterification remaining unchanged. Infusion of glucagon also resulted in a progressive elevation of the plasma concentration of glucose and beta-hydroxybutyrate and in a twofold higher insulinemia. These changes were not significantly different between the two phases. The plasma concentrations of triacylglycerols and uric acid were unaffected by glucagon infusion, except for a 40% increase in plasma uric acid in phase II birds. Altogether, these results indicate that glucagon in a long-term fasting bird is highly lipolytic, hyperglycemic, ketogenic, and insulinogenic, these effects, however, being similar in phases II and III. The maintenance of the sensitivity of adipose tissue lipolysis to glucagon could suggest that the major role of the increase in basal glucagonemia observed in phase III is to stimulate gluconeogenesis rather than fatty acid delivery.     

Novel system for the simultaneous analysis of geminivirus DNA replication and plant interactions in Nicotiana benthamiana

The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based on Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replication-associated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R(2), R(5), R(7) and K(11) or H(56), L(57) and H(58) prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.     

Thermodynamic and kinetic analysis of a peptide-class I MHC interaction highlights the noncovalent nature and conformational dynamics of the class I heterotrimer

The class I major histocompatibility (MHC) molecule is a heterotrimer composed of a heavy chain, the small subunit beta(2)-microglobulin (beta(2)m), and a peptide. Fluorescence anisotropy has been used to assay the interaction of a labeled peptide with a recombinant, soluble form of the class I MHC HLA-A2. Consistent with earlier work, peptide binding is shown to be a two-step process limited by a conformational rearrangement in the heavy chain/beta(2)m heterodimer. However, we identify two pathways for peptide dissociation from the heterotrimer: (1) initial peptide dissociation leaving a heavy chain/beta(2)m heterodimer and (2) initial dissociation of beta(2)m, followed by peptide dissociation from the heavy chain. Eyring analyses of rate constants measured as a function of temperature permit for the first time a complete thermodynamic characterization of peptide binding. We find that in this case peptide binding is mostly entropically driven, likely reflecting the hydrophobic character of the peptide binding groove and the peptide anchor residues. Thermodynamic and kinetic analyses of peptide-MHC interactions as performed here may be of practical use in the engineering of peptides with desired binding properties and will aid in the interpretation of the effects of MHC and peptide substitutions on peptide binding and T cell reactivity. Finally, our data suggest a role for beta(2)m in dampening conformational dynamics in the heavy chain. Remaining conformational variability in the heavy chain once beta(2)m has bound may be a mechanism to promote promiscuity in peptide binding.     

Development of In Vivo Sponge Cultures: Particle Feeding by the Tropical Sponge Pseudosuberites aff. andrewsi

The rate of food particle uptake of the tropical sponge Pseudosuberites aff. andrewsi was studied in relation to particle concentrations and particle size. A range of different concentrations of either the marine microalga Dunaliella tertiolecta (∼5–8 μm) or the marine cyanobacterium Synechococcus sp. (∼1 μm) was supplied to the sponges. D. tertiolecta had a pronounced effect on the filtration activity of the sponges: at concentrations higher than approximately 4 × 10⁵ cells/cm³, the filtration rates dropped dramatically. Such a clear effect was not found for Synechococcus sp. The results further showed that the maximal amount of food (when expressed in organic carbon) that can be taken up per cubic centimeter of sponge volume per unit of time should in principle be sufficient to enable growth (irrespective of the food particle type). At the maximal food particle concentration that did not affect the filtration rates, the uptake of organic carbon is already highly in excess of the amount of organic carbon that the sponges need to cope with their respiratory demand. Based on these findings, a series of growth experiments was carried out in which the sponges were subjected to a constant concentration of different types of food particles (Synechococcus sp. and the microalgae Chlorella sorokiniana and Nannochloropsis sp). Although initial growth was sometimes observed, continuous growth at a constant rate could not be obtained. It is concluded that qualitative aspects of feeding rather than quantitative aspects are the key to successful in vivo sponge culture. Received December 20, 2000; accepted March 26, 2001     

Nitric oxide-endothelin-1 interactions after acute ductal constriction in fetal lambs

Acute partial compression of the fetal ductus arteriosus (DA) results in an initial increase in pulmonary blood flow (PBF) that is followed by acute vasoconstriction. The objective of the present study was to determine the role of nitric oxide (NO)-endothelin-1 (ET-1) interactions in the acute changes in pulmonary vascular tone after in utero partial constriction of the DA. Twelve late-gestation fetal lambs (132-140 days) were instrumented to measure vascular pressures and left PBF. After a 24-h recovery period, acute constriction of the DA was performed by partially inflating a vascular occluder, and the hemodynamic variables were observed for 4 h. In control lambs (n = 7), acute ductal constriction initially increased PBF by 627% (P < 0.05). However, this was followed by active vasoconstriction, such that PBF was restored to preconstriction values by 4 h. This was associated with a 43% decrease in total NO synthase (NOS) activity (P < 0.05) and a 106% increase in plasma ET-1 levels (P < 0.05). Western blot analysis demonstrated no changes in lung tissue endothelial NOS, preproET-1, endothelin-converting enzyme-1, or ET(B) receptor protein levels. The infusion of PD-156707 (an ET(A) receptor antagonist, n = 5) completely blocked the vasoconstriction and preserved NOS activity. These data suggest that the fetal pulmonary vasoconstriction after acute constriction of the DA is mediated by NO-ET-1 interactions. These include an increase in ET(A) receptor-mediated vasoconstriction and an ET(A) receptor-mediated decrease in NOS activity. The mechanisms of these NO-ET-1 interactions, and their role in mediating acute changes in PBF, warrant further studies.     

Morphological variation in populations of Bletia purpurea (Orchidaceae) and description of the new species B. riparia

Bletia purpurea is the most widespread species in its genus. Morphological variation has been recognized throughout the range of its distribution. In this paper, the morphological variation from 63 populations (583 individuals) ofBletia purpurea is assessed to determine whether more than one species were present. Forty-four quantitative and qualitative characters were examined by univariate analyses and exploratory multivariate analyses. Univariate analyses indicate that quantitative characters such as lateral sepal width, petal. width, lip length, and lip width are significantly different for populations from Acazónica, Mexico. Floral parts in the populations from Acazónica are the smallest among all populations. Qualitative characters such as petals covering the lip midlobe and horizontal lip position are found exclusively in the same populations. We concluded that these populations should be described as a new species,B. riparia. Multivariate analyses indicated that morphological variation among the other populations cannot be ascribed to geographic distribution or ecological factors.     

The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast two-hybrid library using the large cytoplasmic domain of the alpha4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The alpha4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant alpha4beta2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native alpha4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant alpha4beta2 AChRs up-regulated their surface expression levels approximately 2-fold and increased their agonist sensitivity to acetylcholine approximately 3-fold. The modulation of the recombinant alpha4beta2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of alpha4beta2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.     

The Endoplasmic reticulum-associated maize GL8 protein is a component of the acyl-coenzyme A elongase ivolved in the production of cuticular waxes

The gl8 gene is required for the normal accumulation of cuticular waxes on maize (Zea mays) seedling leaves. The predicted GL8 protein exhibits significant sequence similarity to a class of enzymes that catalyze the reduction of a ketone group to a hydroxyl group. Polyclonal antibodies raised against the recombinant Escherichia coli-expressed GL8 protein were used to investigate the function of this protein in planta. Subcellular fractionation experiments indicate that the GL8 protein is associated with the endoplasmic reticulum membranes. Furthermore, polyclonal antibodies raised against the partially purified leek (Allium porrum) microsomal acyl-coenzyme A (CoA) elongase can react with the E. coli-expressed GL8 protein. In addition, anti-GL8 immunoglobulin G inhibited the in vitro elongation of stearoyl-CoA by leek and maize microsomal acyl-CoA elongase. In combination, these findings indicate that the GL8 protein is a component of the acyl-CoA elongase. In addition, the finding that anti-GL8 immunoglobulin G did not significantly inhibit the 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA dehydrase, and (E) 2,3-enoyl-CoA reductase partial reactions of leek or maize acyl-CoA elongase lends further support to our previous hypothesis that the GL8 protein functions as a beta-ketoacyl reductase during the elongation of very long-chain fatty acids required for the production of cuticular waxes.