Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.     

Signal transduction, chemotaxis, and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain

Dictyostelium discoideum cells have been generated that lack myosin heavy chain (MHC) due to antisense RNA inactivation of the endogenous mRNA or to insertional mutagenesis of the myosin gene. These cells retain chemotactic movement in gradients of the chemoattractant cAMP. Furthermore, cAMP does induce many biochemical and physiological responses in aggregative cells, including binding of cAMP to surface receptors, modification, and down-regulation of the receptor; activation of adenylate and guanylate cyclase, secretion of cAMP; and the association of actin to the Triton-insoluble cytoskeleton. Cells lacking MHC were found to have a requirement for bivalent cations in the medium for optimal chemotaxis and cell aggregation.     

Pregnenolone Sulfate and Cortisol Induce Secretion of Acyl-CoA-binding Protein and Its Conversion into Endozepines from Astrocytes

Acyl-CoA-binding protein (ACBP) functions both intracellularly as part of fatty acid metabolism and extracellularly as diazepam binding inhibitor, the precursor of endozepine peptides. Two of these peptides, ODN and TTN, bind to the GABA_A receptor and modulate its sensitivity to γ-aminobutyric acid (GABA). We have found that depolarization of mouse primary astrocytes induces the rapid release and processing of ACBP to the active peptides. We previously showed that ODN can trigger the rapid sporulation of the social amoeba Dictyostelium. Using this bioassay, we now show that astrocytes release the endozepine peptides within 10 min of exposure to the steroids cortisol, pregnenolone, pregnenolone sulfate, or progesterone. ACBP lacks a signal sequence for secretion through the endoplasmic reticulum/Golgi pathway and its secretion is not affected by addition of brefeldin A, a well known inhibitor of the classical secretion pathway, suggesting that it follows an unconventional pathway for secretion. Moreover, induction of autophagy by addition of rapamycin also resulted in rapid release of ACBP indicating that this protein uses components of the autophagy pathway for secretion. Following secretion, ACBP is proteolytically cleaved to the active neuropeptides by protease activity on the surface of astrocytes. Neurosteroids, such as pregnenolone sulfate, were previously shown to modulate the excitatory/inhibitory balance in brain through increased release of glutamate and decreased release of GABA. These effects of steroids in neurons will be reinforced by the release of endozepines from astrocytes shown here, and suggest an orchestrated astrocyte-neuron cross-talk that can affect a broad spectrum of behavioral functions.     

The phase behavior of hydrated cholesterol.

The thermotropic phase behavior of cholesterol monohydrate in water was investigated by differential scanning calorimetry, polarizing light microscopy, and x-ray diffraction. In contrast to anhydrous cholesterol which undergoes a polymorphic crystalline transition at 39 degrees C and a crystalline to liquid transition at 151 degrees C, the closed system of cholesterol monohydrate and water exhibited three reversible endothermic transitions at 86, 123, and 157 degrees C. At 86 degrees C, cholesterol monohydrate loses its water of hydration, forming the high temperature polymorph of anhydrous cholesterol. At least 24 hours were required for re-hydration of cholesterol and the rate of hydration was dependent on the polymorphic crystalline form of anhydrous cholesterol. At 123 degrees C, anhydrous crystalline cholesterol in the presence of excess water undergoes a sharp transition to a birefringent liquid crystalline phase of smectic texture. The x-ray diffraction pattern obtained from this phase contained two sharp low-angle reflections at 37.4 and 18.7 A and a diffuse wide-angle reflection centered at 5.7 A, indicating a layered smectic type of liquid crystalline structure with each layer being two cholesterol molecules thick. The liquid crystalline phase is stable over the temperature range of 123 to 157 degrees C before melting to a liquid dispersed in water. The observation of a smectic liquid crystalline phase for hydrated cholesterol correlates with its high surface activity and helps to explain its ability to exist in high concentrations in biological membranes.     

sn-1,2-Diacylglycerol kinase of Escherichia coli. Purification, reconstitution, and partial amino- and carboxyl-terminal analysis

The sn-1,2-diacylglycerol kinase structural gene from Escherichia coli was demonstrated to be the dgkA locus previously sequenced (Lightner, V. A., Bell, R. M., and Modrich, P. (1983) J. Biol. Chem. 258, 10856-10861). The dgkA gene product was shown by maxicell analysis to be an Mr = 14,000 membrane-bound protein. When dgkA was placed on a hybrid plasmid under control of the lambda pL promoter, a 100-fold overproduction of diacylglycerol kinase activity was obtained. Diacylglycerol kinase was solubilized from membranes with 2-propanol/heptane/trifluoroacetic acid and purified to near homogeneity by high performance liquid chromatography. Activity was reconstituted in a mixed micellar assay containing beta-octylglucoside, cardiolipin, and sn-1,2-dioleoylglycerol. Amino acid analysis, partial NH2-terminal analysis and COOH-terminal analysis permitted alignment of the polypeptide on the sequenced gene. The data establish that dgkA is the structural gene for the diacylglycerol kinase and establish the primary structure of the enzyme of 122 residues, 13,245 daltons. Secondary structure analysis predicted a protein conformation consisting of three transmembrane alpha-helical segments, an amphipathic helix, and an alpha-helix. Taken together, the predicted helical segments comprise more than 75% of the polypeptide.     

Impact of the invasive shrub Amur honeysuckle (Lonicera maackii) on shrub-layer insects in a deciduous forest in the eastern United States

Invasive Amur honeysuckle (Lonicera maackii) creates a dense shrub layer in deciduous forests in eastern North America that negatively impacts native herbs and tree seedlings. We predicted that higher vegetative cover caused by this shrub would increase abundance and diversity of insects and alter composition of insect assemblages. We used paired plots, one with and one without honeysuckle, in ten forest locations in southwestern Ohio, USA, to sample insects and measure diversity and vertical cover of vegetation in the shrub layer. Vertical cover of this vegetation was higher on honeysuckle-present plots, but diversity of shrub-layer vegetation did not differ between honeysuckle-present and honeysuckle-absent plots. Species diversity of Hexapoda, Coleoptera, and Psocoptera, richness of Hexapoda, Coleoptera, Diptera, Hymenoptera, and Psocoptera, and abundance of Hexapoda, Diptera, Hymenoptera, and Psocoptera were higher on honeysuckle-present than on honeysuckle-absent plots. Evenness did not differ between honeysuckle treatments. Nonmetric multidimensional scaling distinguished taxonomic composition in honeysuckle-present plots from that in honeysuckle-absent plots. Presence of vertical cover explained higher richness of Hexapoda and Coleoptera, and higher abundance of Hexapoda, Diptera, Hymenoptera, and Psocoptera. Attributes of honeysuckle, independent of its contribution to vertical cover, explained increases in richness of Hexapoda, Coleoptera, and Hymenoptera and abundance of Hexapoda, Hymenopera, and Psocoptera. These attributes of honeysuckle could include a more complex vegetative structure, a greater availability of resources such as food, detritus, or shelter, and/or a more favorable cooler and moister microenvironment. To more fully understand the mechanisms causing increased richness and abundance of insects in honeysuckle-present areas, detailed studies on these attributes of honeysuckle would be necessary.     

Ghrelin is dispensable for embryonic pancreatic islet development and differentiation

Ghrelin is a peptide hormone that has been implicated in the regulation of food intake and energy homeostasis. Ghrelin is predominantly produced in the stomach, but is also expressed in many other tissues where its functions are not well characterized. In the rodent and human pancreas, ghrelin levels peak at late gestation and gradually decline postnatally. Several studies have suggested that ghrelin regulates beta cell function during embryonic development and in the adult. In addition, in a number of mouse models, ghrelin cells appear to replace insulin- and glucagon-producing cells in the islet. In this analysis, we investigated whether the absence or overexpression of ghrelin influenced the development and differentiation of the pancreatic islet during embryonic development. These studies revealed that ghrelin is dispensable for normal pancreas development during gestation. Conversely, we demonstrated that elevated ghrelin in the Nkx2.2 null islets is not responsible for the absence of insulin- and glucagon-producing cells. Finally, we have also determined that in the absence of insulin, ghrelin cells form in their normal numbers and ghrelin is expressed at wild type levels.