Morphogenetic movements driving neural tube closure in Xenopus require myosin IIB

Vertebrate neural tube formation involves two distinct morphogenetic events — convergent extension (CE) driven by mediolateral cell intercalation, and bending of the neural plate driven largely by cellular apical constriction. However, the cellular and molecular biomechanics of these processes are not understood. Here, using tissue-targeting techniques, we show that the myosin IIB motor protein complex is essential for both these processes, as well as for conferring resistance to deformation to the neural plate tissue. We show that myosin IIB is required for actin-cytoskeletal organization in both superficial and deep layers of the Xenopus neural plate. In the superficial layer, myosin IIB is needed for apical actin accumulation, which underlies constriction of the neuroepithelial cells, and that ultimately drive neural plate bending, whereas in the deep neural cells myosin IIB organizes a cortical actin cytoskeleton, which we describe for the first time, and that is necessary for both normal neural cell cortical tension and shape and for autonomous CE of the neural tissue. We also show that myosin IIB is required for resistance to deformation (“stiffness”) in the neural plate, indicating that the cytoskeleton-organizing roles of this protein translate in regulation of the biomechanical properties of the neural plate at the tissue-level.     

Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver

1. Rat liver slices were employed to study the relative rates of incorporation of a mixture of [2-(3)H]- or [1,3-(3)H]-glycerol and [1-(14)C]glycerol into lipids. 2. With 0.1mm-glycerol approx. 82% of the newly synthesized lipid, calculated from (14)C incorporation, was present as neutral lipid, 13% as phosphatidylcholine and 5% as phosphatidylethanolamine. Increasing the glycerol concentration to 40mm caused a decrease in the percentage of neutral lipid to 59% and a corresponding increase in the percentage of phosphatidylcholine to 36% of the newly synthesized lipid. 3. The (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in glycerolipid was considerably higher than that in precursor glycerol throughout the range of experimental conditions. In contrast the incorporation of a mixture of [1,3-(3)H]glycerol and [1-(14)C]glycerol into lipid occurred with little or no change in the (3)H/(14)C ratio. 4. Respiring rat liver mitochondria were found to oxidize a mixture of sn-[2-(3)H]- and sn-[1-(14)C]-glycerol 3-phosphate with a resultant increase in the (3)H/(14)C ratio of the remaining sn-glycerol 3-phosphate. This increase is due to a (3)H isotope effect of the mitochondrial sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5), which discriminates against sn-[2-(3)H]glycerol 3-phosphate during oxidation. 5. A method is described for the simultaneous determination of the relative contributions of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver slices. The method involves measurement of the (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in both sn-glycerol 3-phosphate and glycerolipid after incubation of rat liver slices with a mixture of [2-(3)H]glycerol and [1-(14)C]glycerol for various times. 6. By using this method it was shown that 40-50% of the glycerol incorporated into lipid by rat liver slices proceeded via the sn-glycerol 3-phosphate pathway and 50-60% was incorporated via dihydroxyacetone phosphate.     

Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts

Abstract: Basic fibroblast growth factor (FGF-2) is normally expressed as a cell-associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF-2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF-2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18-kDa form of FGF-2 in primary fibroblasts as a cell-associated (FGF-2-B) or as a secreted (FGF-2-S) protein. FGF-2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF-2-S cells. No FGF-2 is detected in control (untransfected) cells. FGF-2-S cells also grow faster than the control or FGF-2-B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF-2 is active when engineered to be expressed as a cell-associated form or secreted from cells.     

Extent of intracellular pH changes during H+ extrusion by maize root-tip cells

³¹P-Nuclear-magnetic-resonance spectra of maize (Zea mays L.) root tips, that had been induced to extrude large amounts of H⁺ in response to fusicoccin (FC) in the presence of potassium salts, indicate that the cytoplasmic pH does not become higher than that of controls. In fact, the cytoplasmic pH may become slightly (approx. 0.1 pH unit) lower in cells extruding H⁺. Estimations of the buffer capacity of the cells show that without active intracellular pH regulation, H⁺ extrusion caused by FC would cause the intracellular pH to rise by at least 0.6 pH unit h^-1. Our results indicate that intracellular pH is tightly regulated even during extreme rates of acid extrusion, and that a rise in cytoplasmic pH is not the signal linking H⁺ extrusion with enhanced organic-acid synthesis or other intracellular responses to H⁺ pumping.Abbreviations FC fusicoccin - P_i inorganic phosphate - NMR nuclear magnetic resonance - chemical shift - MDP methylene diphosphonic acid     

Efficient plant regeneration of native spearmint (Mentha spicata L.)

Summary Protocols and media constituents for efficient in vitro plant regeneration of Native Spearmint (Mentha spicata L. cultivar ‘Native Spearmint’) have been defined. Adventitious shoots were initiated either directly from morphogenetically competent cells of explants or primary callus. Leaf explants from at least 2-mo.-old in vitro-maintained shoots exhibited the greatest morphogenetic capacity. Explants derived from basal portions of leaves at the bottom of the shoot were most responsive, with up to a 100% regeneration frequency and greater than nine shoots per explant. Highest frequency of meristemoids and morphogenetic callus were initiated from explants cultured onto a basal medium containing Murashige and Skoog (MS) salts, supplemented with 4 mg thidiazuron (TDZ) per L and 25% (vol/vol) coconut water (CW) for 10 to 14 d in darkness. Bud and shoot development required removal of both TDZ and CW from the medium. Shoot propagules were transferred to basal medium supplemented with 0.01 mg α-naphthaleneacetic acid (NAA) per L and grown under low light for about 2 wk to facilitate shoot elongation. Individual shoots about 1 cm tall were dissected and retransferred onto the same medium. Root initiation began within 4 to 6 d and a functional root system developed within 2 to 3 wk. These plantlets were transferred to soil and acclimated successfully for growth and development in a greenhouse. This is the first report of an efficient regeneration system for Native Spearmint based on adventitious organogenesis.     

Proteins of the sieve-tube exudate of Cucurbita maxima

Summary Sieve-tube exudate which appears on cut surfaces of stems of Cucurbita maxima as distinct droplets has been depicted in electron micrographs of longitudinal sections of the phloem. The exudate, which was produced from mature sieve tubes only, contained filaments of P-protein, but no mitochondria or vesicles of endoplasmic reticulum. The water-soluble part of the exudate contained at least 12 proteins, as shown by disc-electrophoresis. Enzymic activity was found for peroxidases, acid phosphatases, and aldolases. Color tests and assays for other enzymes, including ATPase, fructokinase, several dehydrogenases, and UDP-glucose: D-fructose-2-glucosyl transferase, gave negative results. With repeated cutting of a stem, the protein content of the exudate increased, while the amount of exudate decreased.Supported by Deutsche Forschungsgemeinschaft and Stiftung Volkswagenwerk. During part of this investigation the senior author held a U.S. National Science Foundation Senior Foreign Scientist Fellowship at the University of Wisconsin.     

Distribution and biochemical properties of an M1-family aminopeptidase in Plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism

Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the food vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68- and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the food vacuole lumen. Determination of steady-state kinetic parameters for both aminoacyl-β-naphthylamide and unmodified dipeptide substrates over the pH range 5.0-8.5 reveals that k(cat) is relatively insensitive to pH, whereas K(m) increases at pH values below 6.5. At the pH of the food vacuole lumen (5.0-5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the food vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.     

β-Amylase fromBacillus megaterium

Bacillus megaterium strain B₆ producing extracellular β-amylase was isolated and grown in a medium supplemented with waste potato starch. It showed highest enzyme synthesis in the early stationary phase. The partially purified β-amylase had a temperature optimum at 60°C and a pH optimum at 6.9 and was not affected by Schardinger dextrins. These properties would allow its application in sugar industry.