A recently silenced, duplicate PgiC locus in Clarkia

Previous electrophoretic analysis showed that 17 diploid species of the wildflower Clarkia (Onagraceae) have two cytosolic isozymes of phosphoglucose isomerase (PGIC; EC 5.3.1.9), whereas 15 other diploid species have a single PGIC. Molecular studies revealed that the two isozymes in the former species are encoded by duplicate genes, PgiC1 and PgiC2, whereas the single isozyme in the latter is always encoded by PgiC1. Phylogenetic analysis of the nucleotide sequences implied that PgiC2 was silenced four times independently in the genus. Here we describe a psi PgiC2 from C. mildrediae, a species in which only PgiC1 is expressed. The discovery of the psi PgiC2 is significant because it confirms a formal prediction of the phylogenetic analysis. The psi PgiC2 includes 5,039 nucleotides corresponding to 18 of the 23 exons of PgiC, as well as the intervening introns and 3' nontranslated region. The absence of an increase of nucleotide substitutions in its "exons" suggests that the gene was silenced recently. The present study appears to be the first to establish that a specific duplicate gene locus regularly expressed in a group of related plant species has been silenced in one of them. The multiple independent silencings of PgiC2 suggest that it remained functional but inessential in ancestral lineages. We discuss the possibility that PgiC2 may have been preserved in these lineages by selection against mutants causing defective PGIC1- PGIC2 heterodimers.      

Pharmacological basis for the use of nimodipine in central nervous system disorders

Nimodipine, a Ca2+ antagonist with cerebrovasodilatory and anti-ischemic effects, binds to rat, guinea pig, and human brain membranes with high affinity (less than 1 nM). Only at higher concentrations has nimodipine been reported to block the release of some neurotransmitters and hormones from neuronal tissue. Nimodipine has no consistent effect on brain oxygen consumption or cortical ATP or phosphocreatine levels, although the ischemia-induced fall of brain ATP levels in gerbils or the lowering of intracellular brain pH in rabbits with focal cerebral ischemia were antagonized by the drug. In rats and baboons with middle cerebral artery occlusion, nimodipine was found to reduce neurological deficits without an increase in intracranial pressure or brain edema. Electrophysiological studies with nimodipine suggested a direct neuronal action. In rabbit dorsal root ganglion cells, concentrations as low as 20 nM were reported to block inward Ca2+ currents. Recent studies have suggested that nimodipine may also improve memory in brain-damaged or old rats, restore sensorimotor function and abnormal walking patterns of old rats, and accelerate acquisition of associative learning in aging rabbits. Blockade of age-related changes in Ca2+ fluxes in rat hippocampal neurones by nimodipine in vitro pointed to neuronal plasma membrane as the site of nimodipine action. The therapeutic usefulness of nimodipine appears not to be limited to cerebral ischemia, but may include dementia, age-related degenerative diseases, epilepsy, and ethanol intoxication.     

In vitro biosynthesis of [Thr2,Leu5,D-Hiv8,Leu10]-cyclosporin, a cyclosporin-related peptolide, with immunosuppressive activity by a multienzyme polypeptide

A new cyclic peptolide (SDZ 214-103), which is produced by the fungus Cylindrotrichumoligospermum (Corda) BONORDEN (Dreyfuss, M. M., Schreier, M. H., Tscherter, H., and Wenger, R. (June 15, 1988) European Patent Application 0 296 123 A2) and is closely related to cyclosporin A (CyA), has as the main structural difference D-2-hydroxyisovaleric acid in ester linkage at position 8 instead of D-alanine in the cyclosporins. This peptolide exerts similar biological activities to CyA. We were able to prepare an enzyme fraction of crude extracts of the mycelium, which is capable of synthesizing the peptolide with consumption of the constitutive amino acids, D-2-hydroxyisovaleric acid, ATP, and S-adenosyl-L-methionine. The in vitro product co-chromatographs with authentic peptolide on thin layer chromatography and high performance liquid chromatography and shows similar immunosuppressive activity in vitro. The enzyme does not synthesize CyA, whereas cyclosporin synthetase does not synthesize the peptolide. Peptolide synthetase has a high molecular weight (in the same range as cyclosporin synthetase) and also does not appear to be glycosylated. The enzyme cross-reacts with antibodies directed specifically against cyclosporin synthetase.     

Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation

Vitamin E disappearance is accelerated in cigarette smokers due to their increased oxidative stress and is inversely correlated with plasma vitamin C concentrations. Therefore, we hypothesized that ascorbic acid supplementation (500 mg, twice daily; 2 weeks) would normalize smokers' plasma alpha- and gamma-tocopherol disappearance rates and conducted a double-blind, placebo-controlled, randomized crossover investigation in smokers (n=11) and nonsmokers (n=13) given a single dose of deuterium-labeled alpha- and gamma-tocopherols (50 mg each d6-RRR-alpha and d2-RRR-gamma-tocopheryl acetate). During the placebo trial, smokers, compared with nonsmokers, had significantly (P<0.05) greater alpha- and gamma-tocopherol fractional disappearance rates and shorter half-lives. Ascorbic acid supplementation doubled (P<0.0001) plasma ascorbic acid concentrations in both groups and attenuated smokers', but not nonsmokers', plasma alpha- and gamma-tocopherol (P<0.05) fractional disappearance rates by 25% and 45%, respectively. Likewise, smokers' plasma deuterium-labeled alpha- and gamma-tocopherol concentrations were significantly higher (P<0.05) at 72 h during ascorbic acid supplementation compared with placebo. Ascorbic acid supplementation did not significantly change (P>0.05) time of maximal or maximal-labeled alpha- and gamma-tocopherol concentrations. Smokers' plasma F2alpha-isoprostanes were approximately 26% higher than nonsmokers (P>0.05) and were not affected by ascorbic acid supplementation in either group (P>0.05). In summary, cigarette smoking increased plasma alpha- and gamma-tocopherol fractional disappearance rates, suggesting that the oxidative stress from smoking oxidizes tocopherols and that plasma ascorbic acid reduces alpha- and gamma-tocopheroxyl radicals to nonoxidized forms, thereby decreasing vitamin E disappearance in humans.     

Evaluation of a mechanical seed planter for transplanting Zostera marina (eelgrass) seeds

Few seagrass transplant projects worldwide have relied on seeds, and those projects using Zostera marina (eelgrass) seeds have generally found low rates of seedling establishment (<10%). We compared seedling establishment achieved by a mechanical seed planter with seeds broadcast on the sediment surface by hand. The planter injected seeds into the sediment by pumping the seeds, suspended in a gelatin-based matrix, to a benthic sled with eight planting nozzles. As a control for the gel, seeds were also injected into the sediment without gel using a hand-held pipette. Seeds were planted at a density of 300 m⁻² into six replicate plots at each of three sites in the Chesapeake Bay region in September 2005, with seedling establishment measured in April 2006. Burying seeds, either with or without gel, had an overall positive effect on seedling establishment, but the effectiveness and the best method varied among sites. Mean seedling establishment for machine-planted seeds was significantly greater than for broadcast seeds at the Piankatank River site (4% vs. 1%), but not at the York (1.2% vs. 1.4%) or Spider Crab Bay (10.1% vs. 7.4%) sites. The effect of the gel was inconsistent among sites, with the highest seedling establishment (18.8%), resulting from seeds injected by pipette without gel at the Spider Crab site. Seed burial shows promise for increasing seedling establishment relative to seed broadcasting in the Chesapeake region, but further investigation of seed–sediment interactions at specific restoration sites is necessary. Low seedling establishment rates remain a bottleneck for seed-based eelgrass restoration.     

Quantitation of plasma total 15-series F2-isoprostanes by sequential solid phase and liquid-liquid extraction

F₂-isoprostanes are stereo- and regioisomers of prostaglandin F_2α (PGF_2α) and are used as biomarkers for lipid peroxidation. We modified our liquid-liquid extraction (LLE) procedure for F₂-isoprostane analysis (Anal. Biochem. 350 (2006) 41-51) to use a combination of solid phase extraction (SPE) and LLE to produce a cleaner extract that can be easily concentrated. In addition, shortening the high-performance liquid chromatography (HPLC) separation increased peak heights in HPLC-tandem mass spectrometry (HPLC-MS/MS) analysis. Both changes increased the overall sensitivity of the assay. MS/MS analysis served to confirm the identity of specific peaks that may be better biomarkers than the commonly measured 8-iso-PGF_2α.