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190 records · Page 11

Effects of silver ions (Ag+) on contractile ring function and microtubule dynamics during first cleavage in Ilyanassa obsoleta

The terminal phase of cell division involves tight constriction of the cleavage furrow contractile ring, stabilization/elongation of the intercellular bridge, and final separation of the daughter cells. At first cleavage, the fertilized eggs of the mollusk, Ilyanassa obsoleta, form two contractile rings at right angles to each other in the same cytoplasm that constrict to tight necks and partition the egg into a trefoil shape. The cleavage furrow contractile ring (CF) normally constricts around many midbody microtubules (MTs) and results in cleavage; the polar lobe constriction contractile ring (PLC) normally constricts around very few MTs and subsequently relaxes without cleavage. In the presence of Ag+ ions, the PLC 1) begins MT-dependent rapid constriction sooner than controls, 2) encircles more MTs than control egg PLCs, 3) elongates much more than control PLCs, and 4) remains tightly constricted and effectively cleaves the polar lobe from the egg. If Ag(+)-incubated eggs are returned to normal seawater at trefoil, tubulin fluorescence disappears from the PLC neck and the neck relaxes. If nocodazole, a drug that depolymerizes MTs, is added to Ag(+)-incubated eggs during early PLC constriction, the PLC is not stabilized and eventually relaxes. However, if nocodazole is added to Ag(+)-incubated eggs at trefoil, tubulin fluorescence disappears from the PLC neck but the neck remains constricted. These results suggest that Ag+ accelerates and gradually stabilizes the PLC constriction by a mechanism that is initially MT-dependent, but that progressively becomes MT-independent.

Non-NASA Center↗

A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

NASA Discipline Plant Biology↗

Calcium ion dependency of ethylene production in segments of primary roots of Zea mays

We investigated the effect of Ca2+ on ethylene production in 2-cm long apical segments from primary roots of corn (Zea mays L., B73 x Missouri 17) seedlings. The seedlings were raised under different conditions of Ca2+ availability. Low-Ca and high-Ca seedlings were raised by soaking the grains and watering the seedlings with distilled water or 10 mM CaCl2, respectively. Segments from high-Ca roots produced more than twice as much ethylene as segments from low-Ca roots. Indoleacetic acid (IAA; 1 micromole) enhanced ethylene production in segments from both low-Ca and high-Ca roots but auxin-induced promotion of ethylene production was consistently higher in segments from high-Ca roots. Addition of 1-aminocyclopropane-1-carboxylic acid (ACC) to root segments from low-Ca seedlings doubled total ethylene production and the rate of production remained fairly constant during a 24 h period of monitoring. In segments from high-Ca seedlings ACC also increased total ethylene production but most of the ethylene was produced within the first 6 h. The data suggest that Ca2+ enhances the conversion of ACC to ethylene. The terminal 2 mm of the root tip were found to be especially important to ethylene biosynthesis by apical segments and, experiments using 45Ca2+ as tracer indicated that the apical 2 mm of the root is the region of strongest Ca2+ accumulation. Other cations such as Mn2+, Mg2+, and K+ could largely substitute for Ca2+. The significance of these findings is discussed with respect to recent evidence for gravity-induced Ca2+ redistribution and its relationship to the establishment of asymmetric growth during gravitropic curvature.

NASA Discipline Plant Biology↗

4-Coumarate:coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements

When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.

NASA Discipline Plant Biology↗

Curing Composite Materials Using Lower-Energy Electron Beams

In an improved method of fabricating composite-material structures by laying up prepreg tapes (tapes of fiber reinforcement impregnated by uncured matrix materials) and then curing them, one cures the layups by use of beams of electrons having kinetic energies in the range of 200 to 300 keV. In contrast, in a prior method, one used electron beams characterized by kinetic energies up to 20 MeV. The improved method was first suggested by an Italian group in 1993, but had not been demonstrated until recently. With respect to both the prior method and the present improved method, the impetus for the use of electron- beam curing is a desire to avoid the high costs of autoclaves large enough to effect thermal curing of large composite-material structures. Unfortunately, in the prior method, the advantages of electron-beam curing are offset by the need for special walls and ceilings on curing chambers to shield personnel from x rays generated by impacts of energetic electrons. These shields must be thick [typically 2 to 3 ft (about 0.6 to 0.9 m) if made of concrete] and are therefore expensive. They also make it difficult to bring large structures into and out of the curing chambers. Currently, all major companies that fabricate composite-material spacecraft and aircraft structures form their layups by use of automated tape placement (ATP) machines. In the present improved method, an electron-beam gun is attached to an ATP head and used to irradiate the tape as it is pressed onto the workpiece. The electron kinetic energy between 200 and 300 keV is sufficient for penetration of the ply being laid plus one or two of the plies underneath it. Provided that the electron-beam gun is properly positioned, it is possible to administer the required electron dose and, at the same time, to protect personnel with less shielding than is needed in the prior method. Adequate shielding can be provided by concrete walls 6 ft (approximately equal to 1.8 m) high and 16 in. (approximately equal to 41 cm) thick, without a ceiling. The success of the present method depends on the use of a cationic epoxy as the matrix material in the prepreg tape, heating the prepreg tape to a temperature of 50 C immediately prior to layup, and exposing the workpiece to an electron-beam dose of approximately 2 Mrad. Experiments have shown that structures fabricated by the present method have the same mechanical properties as those of nominally identical structures fabricated by the prior method with electron beams of 3 to 4 MeV.

Byrne, Catherine A.↗

Development of Position-Sensitive Magnetic Calorimeters for X-Ray Astronomy

Metallic magnetic calorimeters (MMC) are one of the most promising devices to provide very high energy resolution needed for future astronomical x-ray spectroscopy. MMC detectors can be built to large detector arrays having thousands of pixels. Position-sensitive magnetic (PoSM) microcalorimeters consist of multiple absorbers thermally coupled to one magnetic micro calorimeter. Each absorber element has a different thermal coupling to the MMC, resulting in a distribution of different pulse shapes and enabling position discrimination between the absorber elements. PoSMs therefore achieve the large focal plane area with fewer number of readout channels without compromising spatial sampling. Excellent performance of PoSMs was achieved by optimizing the designs of key parameters such as the thermal conductance among the absorbers, magnetic sensor, and heat sink, as well as the absorber heat capacities. Micro fab ri - cation techniques were developed to construct four-absorber PoSMs, in which each absorber consists of a two-layer composite of bismuth and gold. The energy resolution (FWHM full width at half maximum) was measured to be better than 5 eV at 6 keV x-rays for all four absorbers. Position determination was demonstrated with pulse-shape discrimination, as well as with pulse rise time. X-ray microcalorimeters are usually designed to thermalize as quickly as possible to avoid degradation in energy resolution from position dependence to the pulse shapes. Each pixel consists of an absorber and a temperature sensor, both decoupled from the cold bath through a weak thermal link. Each pixel requires a separate readout channel; for instance, with a SQUID (superconducting quantum interference device). For future astronomy missions where thousands to millions of resolution elements are required, having an individual SQUID readout channel for each pixel becomes difficult. One route to attaining these goals is a position-sensitive detector in which a large continuous or pixilated array of x-ray absorbers shares fewer numbers of temperature sensors. A means of discriminating the signals from different absorber positions, however, needs to be built into the device for each sensor. The design concept for the device is such that the shape of the temperature pulse with time depends on the location of the absorber. This inherent position sensitivity of the signal is then analyzed to determine the location of the event precisely, effectively yielding one device with many sub-pixels. With such devices, the total number of electronic channels required to read out a given number of pixels is significantly reduced. PoSMs were developed that consist of four discrete absorbers connected to a single magnetic sensor. The design concept can be extended to more than four absorbers per sensor. The thermal conductance between the sensor and each absorber is different by design and consequently, the pulse shapes are different depending upon which absorber the xrays are received, allowing position discrimination. A magnetic sensor was used in which a paramagnetic Au:Er temperature-sensitive material is located in a weak magnetic field. Deposition of energy from an x-ray photon causes an increase in temperature, which leads to a change of magnetization of the paramagnetic sensor, which is subsequently read out using a low noise dc-SQUID. The PoSM microcalorimeters are fully microfabricated: the Au:Er sensor is located above the meander, with a thin insulation gap in between. For this position-sensitive device, four electroplated absorbers are thermally linked to the sensor via heat links of different thermal conductance. One pixel is identical to that of a single-pixel design, consisting of an overhanging absorber fabricated directly on top of the sensor. It is therefore very strongly thermally coupled to it. The three other absorbers are supported directly on a silicon-nitride membrane. These absorbers are thermally coupled to the sensor via Ti (5 nm)/Au250 nm) metal links. The strength of the links is parameterized by the number of gold squares making up the link. For detector performance, experimentally different pulse-shapes were demonstrated with 6 keV x-rays, which clearly show different rise times for different absorber positions. For energy resolution measurement, the PoSM was operated at 32 mK with an applied field that was generated using a persistent current of 50 mA. Over the four pixels, energy resolution ranges from 4.4 to 4.7 eV were demonstrated.

Bandler, SImon↗

Glycine betaine accumulation, ionic and water relations of red-beet at contrasting levels of sodium supply

Exposure of plants to sodium (Na) and salinity may increase glycine betaine accumulation in tissues. To study this, red-beet cvs. Scarlet Supreme and Ruby Queen, were grown for 42 days in a growth chamber using a re-circulating nutrient film technique with 0.25 mmol/L K and either 4.75 mmol/L (control) or 54.75 mmol/L (saline) Na (as NaCl). Plants were harvested at weekly intervals and measurements were taken on leaf water relations, leaf photosynthetic rates, chlorophyll fluorescence, chlorophyll levels, glycine betaine levels, and tissue elemental composition. Glycine betaine accumulation increased under salinity and this accumulation correlated with higher tissue levels of Na in both cultivars. Na accounted for 80 to 90% of the total cation uptake under the saline treatment. At final harvest (42 days), K concentrations in laminae ranged from approximately 65-95 micromoles g-1 dry matter (DM), whereas Na in shoot tissue ranged from approximately 3000-4000 micromoles g-1. Leaf sap osmotic potential at full turgor [psi(s100)] increased as lamina Na content increased. Glycine betaine levels of leaf laminae showed a linear relationship with leaf sap [psi(s100)]. Chlorophyll levels, leaf photosynthetic rates, and chlorophyll fluorescence were not affected by Na levels. These results suggest that the metabolic tolerance to high levels of tissue Na in red-beet could be due to its ability to synthesize and regulate glycine betaine production, and to control partitioning of Na and glycine betaine between the vacuole and the cytoplasm.

NASA Center KSC↗

Possible prebiotic significance of polyamines in the condensation, protection, encapsulation, and biological properties of DNA

Some properties of DNA condensed with spermidine have been compared with the properties of DNA condensed with Co3+(NH3)6 to determine whether condensation of DNA with these trivalent cations protects DNA against the action of DNase I and increases transcription and encapsulation of DNA into liposomes. It was shown that DNA condensed with Co3+(NH3)6 was resistant to the action of the endonuclease DNase I such as DNA condensed with spermidine was. However, DNA condensed with Co3+(NH3)6 was significantly less active in transcription with the E. coli RNA polymerase than DNA-spermidine condensed forms. In addition, it was demonstrated that both compacted forms of DNA were more efficiently encapsulated into neutral liposomes; however, negatively, charged liposomes were scarcely formed in the presence of DNA condensed with Co3+(NH3)6. These experiments and the well documented properties of polyamines increasing the resistance to radiations and hydrolysis of nucleic acids, as well as their biological activities, such as replication, transcription, and translation, together with the low concentration of Co3+ in the environment, lead us to propose spermidine as a plausible prebiotic DNA condensing agent rather than Co3+ and the basic proteins proposed by other authors. Then, we consider the possible role and relevance of the polyamine-nucleic acids complexes in the evolution of life.

NASA Discipline Exobiology↗

Parathyroid hormone enhances fluid shear-induced [Ca2+]i signaling in osteoblastic cells through activation of mechanosensitive and voltage-sensitive Ca2+ channels

Osteoblasts respond to both fluid shear and parathyroid hormone (PTH) with a rapid increase in intracellular calcium concentration ([Ca2+]i). Because both stimuli modulate the kinetics of the mechanosensitive cation channel (MSCC), we postulated PTH would enhance the [Ca2+]i response to fluid shear by increasing the sensitivity of MSCCs. After a 3-minute preflow at 1 dyne/cm2, MC3T3-E1 cells were subjected to various levels of shear and changes in [Ca2+]i were assessed using Fura-2. Pretreatment with 50 nM bovine PTH(1-34) [bPTH(1-34)] significantly enhanced the shear magnitude-dependent increase in [Ca2+]i. Gadolinium (Gd3+), an MSCC blocker, significantly inhibited the mean peak [Ca2+]i response to shear and shear + bPTH(1-34). Nifedipine (Nif), an L-type voltage-sensitive Ca2+ channel (VSCC) blocker, also significantly reduced the [Ca2+]i response to shear + bPTH(1-34), but not to shear alone, suggesting VSCC activation plays an interactive role in the action of these stimuli together. Activation of either the protein kinase C (PKC) or protein kinase A (PKA) pathways with specific agonists indicated that PKC activation did not alter the Ca2+ response to shear, whereas PKA activation significantly increased the [Ca2+]i response to lower magnitudes of shear. bPTH(1-34), which activates both pathways, induced the greatest [Ca2+]i response at each level of shear, suggesting an interaction of these pathways in this response. These data indicate that PTH significantly enhances the [Ca2+]i response to shear primarily via PKA modulation of the MSCC and VSCC.

Non-NASA Center↗

Effect of osteoblastic culture conditions on the structure of poly(DL-lactic-co-glycolic acid) foam scaffolds

Poly(DL-lactic-co-glycolic acid) (PLGA) foams are an osteoconductive support that holds promise for the development of bone tissue in vitro and implantation into orthopedic defects. Because it is desirable that foams maintain their shape and size, we examined a variety of foams cultured in vitro with osteoblastic cells. Foams were prepared with different porosities and pore sizes by the method of solvent casting/porogen leaching using 80, 85, and 90 wt% NaCl sieved with particle sizes of 150-300 and 300-500 microm and characterized by mercury intrusion porosimetry. Foams seeded with cells were found to have volumes after 7 days in static culture that decreased with increasing porosity: the least porous exhibited no change in volume while the most porous foams decreased by 39 +/- 10%. In addition, a correlation was observed between decreasing foam volume after 7 days in culture and decreasing internal surface area of the foams prior to seeding. Furthermore, foams prepared with the 300-500 microm porogen had lower porosities, greater mean wall thicknesses between adjacent pores, and larger volumes after 7 days in culture than those prepared with the smaller porogen. Two culture conditions for maintaining cells, static and agitated (in a rotary vessel), were found to have similar influences on foam size, cell density, and osteoblastic function for 7 and 14 days in culture. Finally, we examined unseeded foams in aqueous solutions of pH 3.0, 5.0, and 7.4 and found no significant decrease in foam size with degradation. This study demonstrates that adherent osteoblastic cells may collapse very porous PLGA foams prepared by solvent casting/particulate leaching: a potentially undesirable property for repair of orthopedic defects.

NASA Program Fundamental Space Biology↗