Basis for Phospholipid Incorporation into Peripheral Nerve Myelin

Abstract: To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23–24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P₀ (Golgi) and myelin basic proteins (more direct).     

Microinjection of pp60v-src into Xenopus oocytes increases phosphorylation of ribosomal protein S6 and accelerates the rate of progesterone-induced meiotic maturation.

Microinjection of purified pp60v-src into Xenopus oocytes caused the phosphorylation of ribosomal protein S6 on serine residues and also increased total protein phosphorylation, with almost a two-fold increase in the percentage of phosphotyrosine present. In addition, pp60v-src accelerated the time course of progesterone-induced oocyte maturation, suggesting that the biochemical pathway influenced by pp60v-src is related to that induced by progesterone.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Biodegradation of Trichloroethylene and Dichloromethane in Contaminated Soil and Groundwater

Soil column and serum bottle microcosm experiments were conducted to investigate the potential for in situ anaerobic bioremediation of trichloroethy lene (TCE) and dichloromethane (DCM) at the Pinellas site near Largo, Florida. Soil columns with continuous groundwater recycle were used to evaluate treatment with complex nutrients (casamino acids, methanol, lactate, sulfate); benzoate and sulfate; and methanol. The complex nutrients drove microbial dechlorination of TCE to ethene, whereas the benzoate/sulfate and methanol supported microbial dechlorination of TCE only to cis-1 ,2-dichloroethylene (cDCE). Microbial sulfate depletion in the benzoate/sulfate column allowed further dechlorination of cDCE to vinyl chloride. Serum bottle microcosms were used to investigate TCE dechlorination and DCM biodegradation in Pinellas soil slurries bioaugmented with liquid from the soil columns possessing TCE-dechlorinating activity and DCM biodegradation by indigenous microorganisms. Bioaugmented soil microcosms showed immediate TCE dechlorination in the microcosms with methanol or complex nutrients, but no dechlorination in the benzoate/sulfate microcosm. DCM biodegradation by indigenous microorganisms occurred in soil microcosms amended with either benzoate/sulfate or methanol, but not with complex nutrients. Bioaugmentation stimulated DCM biodegradation in both complex nutrient and methanol-amended microcosms, but appeared to inhibit DCM biodegradation in benzoate/sulfate-amended microcosms. TCE dechlorination occurred before DCM biodegradation in bioaugmented microcosms when both compounds were present.     

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development.     

A versatile and sensitive tritium-based radioassay for measuring hydrogenase activity in aquatic sediments

We present a method for the measurement of hydrogenase (H(2)ase) activity in aquatic sediments. The assay is based on the H(2)ase-mediated isotopic exchange between dissolved molecular hydrogen (H(2)) and water. A slurry of sediment material is incubated with a tritiated hydrogen (HT) headspace in a glass syringe on a rotary shaker. The method includes a procedure for preparing HT from radiolabeled sodium borohydride, which is a useful alternative to purchasing HT directly. A method for measuring HT specific activity based on liquid scintillation counting is also presented. Validation tests were run using live and frozen cultures of Clostridium pasteurianum and Desulfovibrio vulgaris, and freshly collected marine sediments. Adherence to Michaelis-Menten kinetics was demonstrated. An interassay coefficient of variation of 15% was determined using frozen C. pasteurianum cultures as reference material. Serial dilutions of cultures and sediments showed that measured H(2)ase activity scales with cell concentration, and indicate that the method can detect C. pasteurianum cell concentrations of between 300 and 3000 cells/ml. This technique allows measurement of H(2)ase activity in a variety of environmental samples, and will be particularly useful in the study of deep marine sediments with low microbial activity.