Microsatellite identification and characterization in peanut (<Emphasis Type="Italic">A. hypogaea</Emphasis> L.)

A major constraint to the application of biotechnology to the improvement of the allotetraploid peanut, or groundnut (Arachis hypogaea L.), has been the paucity of polymorphism among germplasm lines using biochemical (seed proteins, isozymes) and DNA markers (RFLPs and RAPDs). Six sequence-tagged microsatellite (STMS) markers were previously available that revealed polymorphism in cultivated peanut. Here, we identify and characterize 110 STMS markers that reveal genetic variation in a diverse array of 24 peanut landraces. The simple-sequence repeats (SSRs) were identified with a probe of two 27,648-clone genomic libraries: one constructed using PstI and the other using Sau3AI/BamHI. The most frequent, repeat motifs identified were ATT and GA, which represented 29% and 28%, respectively, of all SSRs identified. These were followed by AT, CTT, and GT. Of the amplifiable primers, 81% of ATT and 70.8% of GA repeats were polymorphic in the cultivated peanut test array. The repeat motif AT showed the maximum number of alleles per locus (5.7). Motifs ATT, GT, and GA had a mean number of alleles per locus of 4.8, 3.8, and 3.6, respectively. The high mean number of alleles per polymorphic locus, combined with their relative frequency in the genome and amenability to probing, make ATT and GA the most useful and appropriate motifs to target to generate further SSR markers for peanut.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by J.S. Heslop-Harrison     

Determination of NG,NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry

A fully validated gas chromatographic-mass spectrometric (GC-MS) method for the accurate and precise quantification of NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography-chemical ionization mass spectrometry, fragment ions at m/z 634 and m/z 640 were obtained for ADMA and for NG,NG-[2H6]-dimethyl-L-arginine ([2H6]-ADMA) as internal standard, respectively. [2H6]-ADMA was synthesized by reaction of L-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 microM. The method was validated in a concentration range of 0-1.2 microM in cell culture medium and 0-2 microM in 50 microl aliquots of human plasma. The precision was > or =97% and the accuracy was determined to be > or =94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.     

Sodium/calcium exchanger (NCX1) macromolecular complex

The sodium-calcium exchanger, NCX1, is a ubiquitously expressed membrane protein essential in calcium homeostasis for many cells including those in mammalian heart and brain. The function of NCX1 depends on subcellular ("local") factors, the phosphorylation state of NCX1, and the subcellular location of NCX1 within the cell. Here we investigate the molecular organization of NCX1 within the cardiac myocyte. We show that NCX1 is dynamically phosphorylated by protein kinase A (PKA)-dependent phosphorylation in vitro. We also provide evidence that the regulation of this phosphorylation is attributed to the existence of an NCX1 macromolecular complex. Specifically, we show that the macromolecular complex includes both the catalytic and regulatory subunits of PKA. However, only the RI regulatory subunit is found in this macromolecular complex, not RII. Other critical regulatory enzymes are also associated with NCX1, including protein kinase C (PKC) and two serine/threonine protein phosphatases, PP1 and PP2A. Importantly, the protein kinase A-anchoring protein, mAKAP, is found and its presence in the macromolecular complex suggests that these regulatory enzymes are coordinately positioned to regulate NCX1 as has been found in diverse cells for a number of channel proteins. Dual immunocytochemical staining showed the colocalization of NCX1 protein with mAKAP and PKA-RI proteins in cardiomyocytes. Finally, leucine/isoleucine zipper motifs have been identified as possible sites of interaction. Our finding of an NCX1 macromolecular complex in heart suggests how NCX1 regulation is achieved in heart and other cells. The existence of the NCX1 macromolecular complex may also provide an explanation for recent controversial findings.     

Fire and site type effects on the long-term carbon and nitrogen balance in pristine Siberian Scots pine forests

Effects of fire and site type on carbon (C) and nitrogen (N) balances were determined by following the change of total and component C and N pools along four chronosequences of fire-prone Siberian Scots pine ecosystems. These differed in the mean return interval of surface fires (unburned – moderately burned, 40 years – heavily burned, 25 years) and site quality (lichen versus Vaccinium site type). Of the Vaccinium site type (higher site quality) only a moderately burned chronosequence was studied. A total of 22 even-aged stands were investigated with stand ages ranging from 2 to 383 years. The C balance was dominated by the opposing dynamics of coarse woody debris (CWD) and biomass and could be divided into three phases: (1) Young stands (up to 40 years)acted as a net source for C of 6-10 mol C m-2 year-1 because the previous generation CWD pool originating from stand-replacing crown fires decayed much faster than biomass increased. During this period the C pool in the unburned lichen type chronosequence decreased from 807 to 480 mol C m-2. (2) Middle aged stands (40-100 years) being in a stage of maximum biomass accumulation were a net sink of 8-10 mol C m-2 year-1. (3)Maturestands (100 to > 350 years) continued to sequester C at a lower rate (0.8-2.5mol C m-2 year-1). Differences in the rates of C sequestration during the two later phases could be explained by the complex interaction between surface fire regime and site type. Recurrent surface fires resulted in enhanced mortality and regularly redistributed C from the living to the CWD pool thereby lowering the rate of C sequestration. Site quality determined the potential to recover from disturbance by fire events. Differences in site type did not correlate with soil and total ecosystem N pool size. However, the N status of needles as well as the N pool of physiologically active tissue was highest in the stands of the Vaccinium type. The woody C pool (biomass + CWD) was sensitive to differences in surface fire regime and site type. It was lowest in the heavily burned lichen type chronosequence (297 ± 108 mol C m-2), intermediate in the unburned and moderately burned lichen type chronosequence (571 ± 179 mol C m-2) and highest in the moderately burned Vaccinium type chronosequence (810 ± 334 mol C m-2). In contrast, the total soil C pool (organic plus mineral layer down to a depth of 25 cm) was independent of stand age, surface fire regimeand site type and fluctuated around a value of 250 mol C m-2. The organic layer C pool oscillated in response to recurring surface fires and its C pool was dependent on time since fire increasing at a rate of about 1.5 mol C m-2 year-during the first 40 years and then reaching a plateau of 170 mol C m-2. The total ecosystem N pool was 7.4 ± 1.5 mol N m-2 on average of which only 25 % were stored in biomass or coarse woody debris. Total ecosystem N was independent of stand age, surface fire regime and site type. No correlation was found between total ecosystem C and N pools. Average total ecosystem C:N ratio was 114 ± 35 mol C mol N-1. A conceptual model illustrating how changes in the regime of stand-replacing crown fires and recurrent surface fires and changes in site quality interact in determining the long-term C balance in Siberian Scots pine forests is presented.     

Phosphatidylinositol-3,5-Bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of A-SMase, L-alpha-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P2), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P2 is 0.53 microM as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells. Even at concentrations of up to 50 microM, PtdIns3,5P2 neither decreased plasma membrane-associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases beta-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.     

Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.