Atomic force microscope imaging of phospholipid bilayer degradation by phospholipase A2.

We have investigated the time course of the degradation of a supported dipalmitoylphosphatidylcholine bilayer by phospholipase A2 in aqueous buffer with an atomic force microscope. Contact mode imaging allows visualization of enzyme activity on the substrate with a lateral resolution of less than 10 nm. Detailed analysis of the micrographs reveals a dependence of enzyme activity on the phospholipid organization and orientation in the bilayer. These experiments suggest that it is possible to observe single enzymes at work in small channels, which are created by the hydrolysis of membrane phospholipids. Indeed, the measured rate of hydrolysis of phospholipids corresponds very well with the enzyme activity found in kinetic studies. It was also possible to correlate the number of enzymes at the surface, as calculated from the binding constant to the number of starting points of the hydrolysis. In addition, the width of the channels was found to be comparable to the diameter of a single phospholipase A2 and thus further supports the single-enzyme hypothesis.     

Effect of acute exposure to 3660 m altitude on orthostatic responses and tolerance.

Orthostatic reflexes were examined at 375 m and after 60 min of exposure in a hypobaric chamber at 3660 m using a 20-min 70 degrees head-up tilt (HUT) test. Mean arterial blood pressure, R wave-R wave interval (RRI), and mean cerebral blood flow velocity (MFV) were examined with coarse-graining spectral analysis. Of 14 subjects, 7 at 375 m and 12 at 3660 m were presyncopal. Immediately on arrival to high altitude, breathing frequency and MFV increased, and endtidal PCO2, RRI, RRI complexity, and the parasympathetic nervous system indicator decreased. MFV was similar in HUT at both altitudes. The sympathetic nervous system indicator increased with tilt at 3660 m, whereas parasympathetic nervous system indicator decreased with tilt at both altitudes. Multiple regression analysis of supine variables from either 375 or 3660 m and the time to presyncope at 3660 m indicated that, after 1 h of exposure, increased presyncope at altitude was the result of 1). ineffective peripheral vasoconstriction, despite increased cardiac sympathetic nervous system activity with HUT, and 2). insufficient cerebral perfusion owing to cerebral vasoconstriction as the result of hypoxic hyperventilation-induced hypocapnia.     

Presymptomatic visualization of plant-virus interactions by thermography.

Salicylic acid (SA), produced by plants as a signal in defense against pathogens, induces metabolic heating mediated by alternative respiration in flowers of thermogenic plants, and, when exogenously applied, increases leaf temperature in nonthermogenic plants. We have postulated that the latter phenomenon would be detectable when SA is synthesized locally in plant leaves. Here, resistance to tobacco mosaic virus (TMV) was monitored thermographically before any disease symptoms became visible on tobacco leaves. Spots of elevated temperature that were confined to the place of infection increased in intensity from 8 h before the onset of visible cell death, and remained detectable as a halo around the ongoing necrosis. Salicylic acid accumulates during the prenecrotic phase in TMV-infected tobacco and is known to induce stomatal closure in certain species. We show that the time course of SA accumulation correlates with the evolution of both localized thermal effect and stomatal closure. Since the contribution of leaf respiration is marginal, we concluded that the thermal effect results predominantly from localized, SA-induced stomatal closure. The presymptomatic temperature increase could be of general significance in incompatible plant-pathogen interactions.     

Sterilization of Plants for Phytoremediation Studies by Bleach Treatment

Bleach treatment of plants was studied as a simple alternative to axenic tissue cultures for demonstrating phytodegradation of aqueous and gas-phase environmental contaminants. Parrotfeather (Myriophyllum aquaticum), spinach (Spinacia oleracea), and wheat (Triticum aestivum) were exposed to 0.525% NaC10 solutions for 15 s, then rinsed in deionized water. Plate counts indicated that 97 to 100% of viable bacteria were removed from parrotfeather and spinach. Transformation rates for 2,4,6-trinitrotoluene (TNT) by bleached and untreated parrotfeather were virtually identical. Similarly, treated and untreated spinach, wheat heads, and wheat leaves removed methyl bromide (MeBr) from air at the same rates. However, wheat root with attendant adhering soil was rendered inactive by bleach treatment. Parrotfeather roots examined by dissecting microscope and by electron microscope showed no significant damage caused by bleach treatment.     

Starvation promotes Dictyostelium development by relieving PufA inhibition of PKA translation through the YakA kinase pathway.

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.     

Linoleic acid hydroperoxide favours hypochlorite- and myeloperoxidase-induced lipid peroxidation

Liposomes composed of soybean phosphatidylcholine were peroxidized using the reagent sodium hypochlorite or the myeloperoxidase-hydrogen peroxide-Cl^- system. Linoleic acid hydroperoxide previously prepared from linoleic acid by means of lipoxidase was incorporated into liposomes. The yield of thiobarbituric acid reactive substances (TBARS) continuously increased with higher amounts of hydroperoxide groups after the initiation of lipid peroxidation by hypochlorous acid producing systems. The accumulation of TBARS was inhibited by scavengers of free radicals such as butylated hydroxytoluene and by the scavengers of hypochlorous acid, taurine and methionine. Lipid peroxidation was also prevented by sodium azide or chloride free medium in the myeloperoxidase-hydrogen peroxide-Cl^- system. Here we show for the first time that the reaction of hypochlorous acid with a biologically relevant hydroperoxide yields free radicals able to cause further oxidation of lipid molecules.     

Subtle re-location of dendritic spine branches containing membrane with fast spiking mechanisms can alter synaptic efficacy

The potential physiological impact of morphological changes in the active dendritic spines, which are believed to be associated with altered synaptic efficacy, was investigated in a computer simulation study using the NEURON package [1]. A compartmental model of a simplified neuron was built, which included 30 complex spines (neck, head, and active zone) and accommodating AMPA-type synaptic inputs with alpha-function conductances. Hodgkin-Huxley type excitable membranes were inserted into the spine heads. It was shown that arranging spines in dense clusters, as opposed to a uniformly random spine distribution, has a negligible effect on the synaptic signal transfer (other model conditions, including synaptic input and spine density, remained unchanged). However, if a proportion (e.g., 3–20%) of the spines partly fuse with their neighbors forming branched spines, this could increase dramatically the cell response to the unchanged synaptic input. Results of this pilot study provide the basis for a more detailed investigation of the relationship between the spine arrangement and synaptic function, considering dual-component synaptic currents and mechanisms controlling ion fluxes in the dendritic compartments.