Isoindoline nitroxide-labeled porphyrins as potential fluorescence-suppressed spin probes.

A series of isoindoline nitroxide-labeled porphyrins were synthesized by the reaction of 5-phenyldipyrromethane and 5-(4'-carboethoxy-methyleneoxyphenyl)dipyrromethane with 5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (FTMIO) using the Lindsey method. The corresponding water-soluble spin-labeled porphyrins were also prepared. Subsequently, these compounds were characterized and their in vitro properties were evaluated. The electrochemical assay demonstrated that these isoindoline nitroxide-labeled porphyrins had similar electrochemical and redox properties to 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO). The electron paramagnetic resonance test showed that these porphyrins exhibited hyperfine splittings and characteristic spectra of CTMIO with typical nitroxide g-values and nitrogen isotropic hyperfine coupling constants. The in vitro cytotoxicity assay indicated that these porphyrins possessed low cytotoxicity to human renal tubular epithelial 293T cells (normal cells) and human hepatoma HepG2 cells (tumor cells). Fluorescence spectroscopy revealed that free base isoindoline nitroxide-labeled porphyrins exhibited fluorescence suppression characteristic of nitroxide-fluorophore systems. In vitro fluorescene imaging demonstrated that the reduced isoindoline nitroxide-labeled porphyrins eliminated fluorescence suppression and displayed strong red fluorescence imaging in HepG2 cells. Thus these isoindoline nitroxide-labeled porphyrins may be considered potentially as biological spin probes for fluorescence imaging and EPR spectroscopy.

Effect of Foliarly Applied Spirotetramat on Reproduction of Heterodera avenae on Wheat Roots.

The cereal cyst nematode, Heterodera avenae, has the potential to reduce yields of cereal crops in the Pacific Northwest. Spirotetramat (Movento) is a foliar-applied insecticide with ambimobile translocation that reduces fecundity of sucking insects which feed on roots as well as foliage. Spirotetramat (88 g/ha) was applied to foliage during 2010 in two wheat fields infested by H. avenae near St. Anthony, ID and Palouse, WA. In Idaho, two applications at 2-week intervals during late spring to plants already exhibiting swollen white females reduced the postharvest density of H. avenae eggs plus juveniles by 35% (P = 0.03) compared to the nontreated control. In Washington, a single application before white females became apparent reduced the nematode density by 78% (P = 0.01). Grain yields and test weights were not significantly affected by application of spirotetramat at either location. In addition, symptomatic plants from the Idaho field were transplanted into greenhouse pots and treated with spirotetramat. One application (110 g/ha) reduced numbers of eggs plus juveniles/plant by 78% (P = 0.02). Spirotetramat effectively reduced H. avenae populations and warrants further evaluation as a substitute for crop rotations or long fallow periods that reduce nematode population densities in infested fields.

First Record of the Cyst Nematode Heterodera filipjevi on Wheat in Oregon.

Plant and soil samples from an irrigated winter wheat (Triticum aestivum) field near Imbler (Union County), OR were evaluated for root diseases during April 2007. The field exhibited patches with as much as 90% plant mortality. Previous crops were winter wheat (2004), chickpea (Cicer arietinum, 2005), and spring wheat (cv. Jefferson, 2006). Stubble was baled and removed, and the field was cultivated before replanting to winter wheat cv. Chukar in October. Patches of stunted seedlings (three- to five-leaf stage) appeared in March 2007. Stunted seedlings exhibited chlorotic or necrotic lower leaves, healthy younger leaves, few or no tillers, rotting of lower culms and crowns, and light brown roots with little or no branching. Signs and symptoms of fungal pathogens (Pythium spp., Gaeumannomyces graminis var. tritici, Rhizoctonia solani AG-8, and Typhula incarnata) were present on affected plants. Most small grain fields in Union County are infested with Heterodera avenae (4) but none of the roots, on either healthy or stunted plants, exhibited the bushy branching pattern typical of sites where H. avenae females penetrate and encyst. Extraction of motile nematodes (Whitehead tray method) from soil revealed high populations of Pratylenchus neglectus (6,560/kg of soil), Tylenchorhynchus spp. (2,369/kg of soil), and a species initially thought to be H. avenae (3,098 juveniles/kg of soil). Cysts were also extracted. During PCR-restriction fragment length polymorphism identification (1) of H. avenae collected in Oregon, Washington, and Idaho, four restriction enzymes applied to amplified DNA of cysts from the Imbler field consistently revealed a pattern identical to that of a H. filipjevi DNA standard and distinct from patterns of H. avenae, H. schachtii, and H. latipons. DNA standards were obtained from R. Rivoal, INRA, Rennes, France. Morphological evidence confirmed that the specimens were H. filipjevi, a member of the 'H. avenae Group' of cereal cyst nematodes (2,3). Measurements of second-stage juveniles (n = 15) included length of body (range = 530 to 570 µm, mean = 549, st. dev. = 13.0), stylet (22.5 to 24.5, 23.2, 0.6) with anchor-shaped basal knobs, tail (52.5 to 62.5, 57.4, 2.7), and hyaline tail terminal (30 to 38, 33.5, 2.6). The lateral field had four lines of which the inner two were more distinct. Shapes of the tail, tail terminus, and stylet knobs were also consistent with H. filipjevi. Cysts (n = 10) were lemon shaped and light brown. The cyst wall had a zigzag pattern. The vulval cone was bifenestrate with horseshoe-shaped semifenestra. The cysts were characterized by body length including neck (range = 718 to 940 µm, mean = 809.7, st. dev. = 61.8), body width (395 to 619, 504, 71.2), L/W ratio = (1.1 to 2.2, 1.4, 0.3), neck length (75 to 140, 103.2, 22.1) and width (50 to 95, 71.4, 10.9), fenestra length (50 to 65 µm, 56.5, 6.6) and width (27 to 40, 29.0, 3.8), heavy underbridge (60 to 80, 69, 8.5), vulval slit (7.5 to 8.5, 7.8, 0.4), and many bullae. As described for H. filipjevi, cysts hatched much more readily and at lower temperatures than populations of H. avenae. Detection of H. filipjevi in Oregon represents a new record for the occurrence of this species in the United States and for North America. The pathotype and resistance genes for incorporation into wheat, barley, and oat are being identified. References: (1) S. Bekal et al. Genome 40:479, 1997. (2) Z. A. Handoo. J. Nematol. 34:250, 2002. (3) R. Holgado et al. J. Nematol. Morphol. Syst. 7:77, 2004. (4) R. W. Smiley et al. J. Nematol. 37:297, 2005.

Molecular mapping of a recessive gene for resistance to stripe rust in barley.

Barley stripe rust, caused by Puccinia striiformis f. sp. hordei, is one of the most important barley (Hordeum vulgare) diseases in the United States. The disease is best controlled using resistant cultivars. Barley genotype Grannenlose Zweizeilige (GZ) has a recessive gene (rpsGZ) that is effective against all races of P. striiformis f. sp. hordei identified so far in the USA. To develop a molecular map for mapping the gene, F(8 )recombinant inbred lines (RILs) were developed from the Steptoe X GZ cross through single-seed descent. Seedlings of the parents and RILs were evaluated for resistance to races PSH-14 and PSH-54 of P. striiformis f. sp. hordei under controlled greenhouse conditions. Genomic DNA was extracted from the parents and 182 F(8 )RILs and used for linkage analysis. The resistance gene analog polymorphism (RGAP) technique was used to identify molecular markers for rpsGZ. A linkage group for the gene was constructed with 12 RGAP markers, of which two markers co-segregated with the resistance locus, and two markers were closely linked to the locus with a genetic distance of 0.9 and 2.0 cM, respectively. These four markers were present only in the susceptible parent. The closest marker to the resistance allele was 11.7 cM away. Analyses of two sets of barley chromosome addition lines of wheat with the two RGAP markers that were cosegregating with the susceptibility allele showed that rpsGZ and the markers were located on the long arm of barley chromosome 4H. Further, tests with four simple sequence repeat (SSR) markers confirmed the chromosomal location of the rpsGZ gene and also integrated the RGAP markers into the known SSR-based linkage map of barley. The closest SSR marker EBmac0679 had a genetic distance of 7.5 cM with the gene in the integrated linkage map constructed with the 12 RGAP markers and 4 SSR markers. The information on chromosomal location and molecular markers for rpsGZ should be useful for incorporating this gene into commercial cultivars and combining it with other resistance genes for durable resistance.

Resistance gene-analog polymorphism markers co-segregating with the YR5 gene for resistance to wheat stripe rust.

The Yr5 gene confers resistance to all races of the stripe rust pathogen ( Puccinia striiformis f. sp. tritici) of wheat in the United States. To develop molecular markers for Yr5, a BC(7):F(3) population was developed by backcrossing the Yr5 donor ' Triticum spelta album' (TSA) with the recurrent parent 'Avocet Susceptible' (AVS). Seedlings of the Yr5 near-isogenic lines (AVS/6* Yr5), AVS, TSA, and the BC(7):F(3) lines were tested with North American races of P. striiformis f. sp. tritici under controlled greenhouse conditions. The single gene was confirmed by a 1:2:1 segregation ratio for homozygous-resistant, heterozygous and homozygous-susceptible BC(7):F(3) lines. Genomic DNA was extracted from the parents (the Yr5 near-isogenic line and AVS) and 202 BC(7):F(3) lines. The resistance gene-analog polymorphism (RGAP) technique was used to identify molecular markers. The parents and the homozygous-resistant and homozygous-susceptible BC(7):F(3) bulks were used to identify putative RGAP markers for Yr5. Association of the markers with Yr5 was determined using segregation analysis with DNA from the individual BC(7):F(3) lines. Of 16 RGAP markers confirmed by segregation analysis with 109 BC(7):F(3) lines, and nine of the markers confirmed with an additional 93 BC(7):F(3) lines, three markers co-segregated with the resistance allele and three markers co-segregated with the susceptibility allele at the Yr5 locus. The other four markers were tightly linked to the locus. Analysis of a set of Chinese Spring nulli-tetrasomic lines with three markers that co-segregated with, or were linked to, the susceptibility allele confirmed that the Yr5 locus is on chromosome 2B. Of five RGAP markers that were cloned and sequenced, markers Xwgp-17 and Xwgp-18 that co-segregated with the Yr5 locus were co-dominant and had 98% homology with each other in both DNA and translated amino-acid sequences. The two markers had 97% homology with a resistance gene-like sequence from Aegilops ventricosa and had significant homology with many known plant resistance genes, resistance gene analogs and expressed sequence tags (ESTs) from wheat and other plant species. The markers Xwgp-17 and Xwgp-18 also had significant homology with the NB-ARC domain that is in several genes for plant resistance to diseases, nematode cell death and human apoptotic signaling. These markers should be useful to clone Yr5 and combine Yr5 with other genes for durable and superior resistance for the control of stripe rust.

[Analgesic action of microinjection of neurokinin A into the lateral reticular nucleus and nucleus raphe magnus in rats].

Using the microinjection technique, the analgesic effect of neurokinin A (NKA) microinjected into the lateral reticular nucleus (LRN) and nucleus raphe magnus (NRM) was investigated in lightly pentobarbital-anesthetized rats using tail flick latency (TFL) as an index. Microinjection of NKA (0.5 microgram/0.5 microliter) into LRN significantly increased TFL lasting for 10 min (n = 12, P < 0.001). Microinjection of the same amount of NKA into NRM also produced evident increase in TFL for 5 min (n = 13, P < 0.001). The results indicate that NKA modulates pain reaction in both LRN and NRM in rats.