Fructan Accumulation and Sucrose Metabolism in Transgenic Maize Endosperm Expressing a Bacillus amyloliquefaciens SacB Gene

Over 40,000 species of plants accumulate fructan, [beta]-2-1- and [beta]-2-6-linked polymers of fructose as a storage reserve. Due to their high fructose content, several commercial applications for fructans have been proposed. However, plants that accumulate these polymers are not agronomically suited for large-scale cultivation or processing. This study describes the transformation of a Bacillus amyloliquefaciens SacB gene into maize (Zea mays L.) callus by particle bombardment. Tissue-specific expression and targeting of the SacB protein to endosperm vacuoles resulted in stable accumulation of high-molecular-weight fructan in mature seeds. Accumulation of fructan in the vacuole had no detectable effect on kernel development or germination. Fructan levels were found to be approximately 9-fold higher in sh2 mutants compared to wild-type maize kernels. In contrast to vacuole-targeted expression, starch synthesis and endosperm development in mature seeds containing a cytosolically expressed SacB gene were severely affected. The data demonstrate that hexose resulting from cytosolic SacB activity was not utilized for starch synthesis. Transgenic seeds containing a chimeric SacB gene provide further evidence that the dominant pathway for starch synthesis in maize endosperm is through uridine diphosphoglucose catalyzed by the enzyme sucrose synthase.     

Water in channel-like cavities: structure and dynamics.

Ion channels contain narrow columns of water molecules. It is of interest to compare the structure and dynamics of such intrapore water with those of the bulk solvent. Molecular dynamics simulations of modified TIP3P water molecules confined within channel-like cavities have been performed and the orientation and dynamics of the water molecules analyzed. Channels were modeled as cylindrical cavities with lengths ranging from 15 to 60 A and radii from 3 to 12 A. At the end of the molecular dynamics simulations water molecules were observed to be ordered into approximately concentric cylindrical shells. The waters of the outermost shell were oriented such that their dipoles were on average perpendicular to the normal of the wall of the cavity. Water dynamics were analyzed in terms of self-diffusion coefficients and rotational reorientation rates. For cavities of radii 3 and 6 A, water mobility was reduced relative to that of simulated bulk water. For 9- and 12-A radii confined water molecules exhibited mobilities comparable with that of the bulk solvent. If water molecules were confined within an hourglass-shaped cavity (with a central radius of 3 A increasing to 12 A at either end) a gradient of water mobility was observed along the cavity axis. Thus, water within simple models of transbilayer channels exhibits perturbations of structure and dynamics relative to bulk water. In particular the reduction of rotational reorientation rate is expected to alter the local dielectric constant within a transbilayer pore.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

Gonadal Steroid Hormone Receptors and Sex Differences in the Hypothalamo-Pituitary-Adrenal Axis

The rapid activation of stress-responsive neuroendocrine systems is a basic reaction of animals to perturbations in their environment. One well-established response is that of the hypothalamo-pituitary-adrenal (HPA) axis. In rats, corticosterone is the major adrenal steroid secreted and is released in direct response to adrenocorticotropin (ACTH) secreted from the anterior pituitary gland. ACTH in turn is regulated by the hypothalamic factor, corticotropin-releasing hormone. A sex difference exists in the response of the HPA axis to stress, with females reacting more robustly than males. It has been demonstrated that in both sexes, products of the HPA axis inhibit reproductive function. Conversely, the sex differences in HPA function are in part due to differences in the circulating gonadal steroid hormone milieu. It appears that testosterone can act to inhibit HPA function, whereas estrogen can enhance HPA function. One mechanism by which androgens and estrogens modulate stress responses is through the binding to their cognate receptors in the central nervous system. The distribution and regulation of androgen and estrogen receptors within the CNS suggest possible sites and mechanisms by which gonadal steroid hormones can influence stress responses. In the case of androgens, data suggest that the control of the hypothalamic paraventricular nucleus is mediated trans-synaptically. For estrogen, modulation of the HPA axis may be due to changes in glucocorticoid receptor-mediated negative feedback mechanisms. The results of a variety of studies suggest that gonadal steroid hormones, particularly testosterone, modulate HPA activity in an attempt to prevent the deleterious effects of HPA activation on reproductive function.     

Components Acting in Localization of Bicoid mRNA Are Conserved among Drosophila Species

Substantial insights into basic strategies for embryonic body patterning have been obtained from genetic analyses of Drosophila melanogaster. This knowledge has been used in evolutionary comparisons to ask if genes and functions are conserved. To begin to ask how highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning, we have focused on the localization of bcd mRNA to the anterior pole of the embryo. Here we consider two components involved in that process: the exuperantia (exu) gene, required for an early step in localization; and the cis-acting signal that directs bcd mRNA localization. First, we use the cloned D. melanogaster exu gene to identify the exu genes from Drosophila virilis and Drosophila pseudoobscura and to isolate them for comparisons at the structural and functional levels. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of bcd mRNA, demonstrating that function is conserved. Second, we reevaluate the ability of the D. pseudoobscura bcd mRNA localization signal to function in D. melanogaster. In contrast to a previous report, we find that function is retained. Thus, among these Drosophila species there is substantial conservation of components acting in mRNA localization, and presumably the mechanisms underlying this process.     

Cryptophytes: An emerging algal group in the rapidly changing Antarctic Peninsula marine environments

The western Antarctic Peninsula (WAP) is a climatically sensitive region where foundational changes at the basis of the food web have been recorded; cryptophytes are gradually outgrowing diatoms together with a decreased size spectrum of the phytoplankton community. Based on a 11-year (2008–2018) in-situ dataset, we demonstrate a strong coupling between biomass accumulation of cryptophytes, summer upper ocean stability, and the mixed layer depth. Our results shed light on the environmental conditions favoring the cryptophyte success in coastal regions of the WAP, especially during situations of shallower mixed layers associated with lower diatom biomass, which evidences a clear competition or niche segregation between diatoms and cryptophytes. We also unravel the cryptophyte photo-physiological niche by exploring its capacity to thrive under high light stress normally found in confined stratified upper layers. Such conditions are becoming more frequent in the Antarctic coastal waters and will likely have significant future implications at various levels of the marine food web. The competitive advantage of cryptophytes in environments with significant light level fluctuations was supported by laboratory experiments that revealed a high flexibility of cryptophytes to grow in different light conditions driven by a fast photo-regulating response. All tested physiological parameters support the hypothesis that cryptophytes are highly flexible regarding their growing light conditions and extremely efficient in rapidly photo-regulating changes to environmental light levels. This plasticity would give them a competitive advantage in exploiting an ecological niche where light levels fluctuate quickly. These findings provide new insights on niche separation between diatoms and cryptophytes, which is vital for a thorough understanding of the WAP marine ecosystem.     

Post-exercise metabolic rate in Atlantic cod and its dependence upon the method of exhaustion

This study tests whether or not post-exercise oxygen consumption rates ( M o₂) in fish are dependent upon how exhaustion is induced. A group of eight Atlantic cod ( Gadus morhua ) were each exercised using (1) a critical swimming speed ( U _crit) protocol, (2) an exercise protocol designed to measure anaerobic capacity of fish ( U _burst), and (3) a protocol in which the fish were chased to exhaustion manually. M o₂ was measured for a 2-h period following exhaustion induced by all three exercise regimes ( U _crit, U _burst and chase). Post-exercise M o₂ following exhaustion from the U _burst and chase protocols were significantly higher than post-exercise M o₂ following the U _crit protocol. Each fish during the U _crit protocol exhibited maximal M o₂ during exercise rather than during recovery, yet 75% of the fish during U _brust recovery and 100% during chase recovery exhibited M o₂ higher than that measured during U _crit exercise. These results, as well as the large interindividual variations in M o₂ among the eight fish, show that post-exhaustion M o₂ is specific to the exercise regime employed, thus, investigators must exercise caution when combining results from different exercise protocols and/or individuals.     

Packing interactions of aib-containing helices: Molecular modeling of parallel dimers of simple hydrophobic helices and of alamethicin

α-Aminoisobutyric acid (Aib) is a helicogenic α,α-dimethyl amino acid found in channel-forming peptaibols such as alamethicin. Possible effects of Aib on helix–helix packing are analyzed. Simulated annealing via restrained molecular dynamics is used to generate ensembles of approximately parallel helix dimers. Analysis of variations in geometrical and energetic parameters within ensembles defines how tightly a pair of helices interact. Simple hydrophobic helix dimers are compared: Ala₂₀, Leu₂₀, Aib₂₀, and P20, the latter a simple channel-forming peptide [G. Menestrina, K. P. Voges, G, Jung, and G. Boheim (1986) Journal of Membrane Biology, Vol. 93, pp. 111–132]. Ala₂₀ and Leu₂₀ dimers exhibit well-defined ridges-in-grooves packing with helix crossing angles (Ω) of the order of +20°. Aib₂₀ α-helix dimers are much more loosely packed, as evidenced by a wide range of Ω values and small helix-helix interaction energies. However, when in a 3₁₀ conformation Aib₂₀ helices pack in three well-defined parallel modes, with Ω ca. ?15°, +5°, and 10°. Comparison of helix–helix interaction energies suggests that dimerization may favor the 3₁₀ conformation. P20, with 8 Aib residues, also shows looser packing of α-helices. The results of these studies of hydrophobic helix dimers are analyzed in the context of the ridges-in-grooves packing model. Simulations are extended to dimers of alamethicin, and of an alamethicin derivative in which all Aib residues are replaced by Leu. This substitution has little effect on helix–helix packing. Rather, such interactions appear to be sensitive to interactions between polar side chains. Overall, the results suggest that Aib may modulate the packing of simple hydrophobic helices, in favor of looser interactions. For more complex amphipathic helices, interactions between polar side chains may be more critical. © 1995 John Wiley & Sons, Inc.