Fine mapping of the sunflower resistance locus Pl(ARG) introduced from the wild species Helianthus argophyllus.

Downy mildew, caused by Plasmopara halstedii, is one of the most destructive diseases in cultivated sunflower (Helianthus annuus L.). The dominant resistance locus Pl(ARG) originates from silverleaf sunflower (H. argophyllus Torrey and Gray) and confers resistance to all known races of P. halstedii. We mapped Pl(ARG) on linkage group (LG) 1 of (cms)HA342 × ARG1575-2, a population consisting of 2,145 F(2) individuals. Further, we identified resistance gene candidates (RGCs) that cosegregated with Pl(ARG) as well as closely linked flanking markers. Markers from the target region were mapped with higher resolution in NDBLOS(sel) × KWS04, a population consisting of 2,780 F(2) individuals that does not segregate for Pl(ARG). A large-insert sunflower bacterial artificial chromosome (BAC) library was screened with overgo probes designed for markers RGC52 and RGC151, which cosegregated with Pl(ARG). Two RGC-containing BAC contigs were anchored to the Pl(ARG) region on LG 1.

Laccase-catalyzed carbon-nitrogen bond formation: coupling and derivatization of unprotected L-phenylalanine with different para-hydroquinones.

Unprotected L-phenylalanine was derivatized by an innovative enzymatic method by means of laccases from Pycnoporus cinnabarinus and Myceliophthora thermophila. During the incubation of L-phenylalanine with para-hydroquinones using laccase as biocatalyst, one or two main products were formed. Dependent on the substitution grade of the hydroquinones mono- and diaminated products were detected. Differences of the used laccases are discussed. The described reactions are of interest for the derivatization of amino acids and a synthesis of pharmacological-active amino acid structures in the field of white biotechnology.

Identification and validation of QTL for Sclerotinia midstalk rot resistance in sunflower by selective genotyping.

Midstalk rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is an important cause of yield loss in sunflower (Helianthus annuus L.). Objectives of this study were to: (1) estimate the number, genomic positions and genetic effects of quantitative trait loci (QTL) for resistance to midstalk rot in line TUB-5-3234, derived from an interspecific cross; (2) determine congruency of QTL between this line and other sources of resistance; and (3) make inferences about the efficiency of selective genotyping (SG) in detecting QTL conferring midstalk rot resistance in sunflower. Phenotypic data for three resistance (stem lesion, leaf lesion and speed of fungal growth) and two morphological (leaf length and leaf length with petiole) traits were obtained from 434 F3 families from cross CM625 (susceptible) x TUB-5-3234 (resistant) under artificial infection in field experiments across two environments. The SG was applied by choosing the 60 most resistant and the 60 most susceptible F3 families for stem lesion. For genotyping of the respective F2 plants, 78 simple sequence repeat markers were used. Genotypic variances were highly significant for all traits. Heritabilities and genotypic correlations between reMidstalk rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is an important cause of yield loss in sunflower (Helianthus annuus L.). Objectives of this study were to: (1) estimate the number, genomic positions and genetic effects of quantitative trait loci (QTL) for resistance to midstalk rot in line TUB-5-3234, derived from an interspecific cross; (2) determine congruency of QTL between this line and other sources of resistance; and (3) make inferences about the efficiency of selective genotyping (SG) in detecting QTL conferring midstalk rot resistance in sunflower. Phenotypic data for three resistance (stem lesion, leaf lesion and speed of fungal growth) and two morphological (leaf length and leaf length with petiole) traits were obtained from 434 F3 families from cross CM625 (susceptible) x TUB-5-3234 (resistant) under artificial infection in field experiments across two environments. The SG was applied by choosing the 60 most resistant and the 60 most susceptible F3 families for stem lesion. For genotyping of the respective F2 plants, 78 simple sequence repeat markers were used. Genotypic variances were highly significant for all traits. Heritabilities and genotypic correlations between resistance traits were moderate to high. Three to four putative QTL were detected for each resistance trait explaining between 40.8% and 72.7% of the genotypic variance (PTS). Two QTL for stem lesion showed large genetic effects and corroborated earlier findings from the cross NDBLOSsel (resistant) x CM625 (susceptible). Our results suggest that SG can be efficiently used for QTL detection and the analysis of congruency for resistance genes across populations.

QTL mapping of resistance to Sclerotinia midstalk rot in RIL of sunflower population NDBLOSsel x CM625.

Midstalk rot caused by Sclerotinia sclerotiorum is an important disease of sunflower in its main areas of cultivation. The objectives of this study were to (1) verify quantitative trait loci (QTL) for midstalk-rot resistance found in F3 families of the NDBLOSsel x CM625 population in recombinant inbred lines (RIL) derived from the same cross; (2) re-estimate their position and genetic effects; (3) draw inferences about the predictive quality of QTL for midstalk-rot resistance identified in the F3 families as compared to those in the RIL. Phenotypic data for three resistance (leaf lesion, stem lesion, and speed of fungal growth) and two morphological traits (leaf length and leaf length with petiole) were obtained from 317 RIL following artificial infection in field experiments across two environments. For genotyping the 248 RIL, we selected 41 simple sequence repeat (SSR) markers based on their association with QTL for Sclerotinia midstalk-rot resistance in an earlier study. The resistance traits showed intermediate to high heritabilities (0.51 < h2 <0.79) and were significantly correlated with each other (0.45 < rg < 0.78). Genotypic correlations between F3 families and the RIL were highly significant and ranged between 0.50 for leaf length and 0.64 for stem lesion. For stem lesion, two genomic regions on linkage group (LG) 8 and LG16 explaining 26.5% of the genotypic variance for Sclerotinia midstalk-rot resistance were consistent across generations. For this trait, the genotypic correlation between the observed performance and its prediction based on QTL positions and effects in F3 families was surprisingly high (rg(MiF3, YiRIL). The genetic effects and predictive quality of these two QTL are promising for application in marker-assisted selection to Sclerotinia midstalk-rot resistance.

Improving the prediction of hepatocellular carcinoma in cirrhotic patients with an arterially-enhancing liver mass.

In the United States, cirrhotic patients with known or suspected hepatocellular carcinoma (HCC) are prioritized for liver transplantation. Noninvasive criteria for the diagnosis of HCC rely on arterial enhancement of a mass. The aim of this study was to determine whether clinical, laboratory, and / or radiologic data can improve the prediction of HCC in cirrhotic patients with an arterially-enhancing mass. Between May 2002 and June 2003, dynamic gadolinium-enhanced magnetic resonance imaging (MRI) of consecutive patients with liver cirrhosis and a solid mass were reviewed by 2 radiologists blinded to the clinical diagnosis. Clinical, laboratory, and radiologic data were recorded for all patients. A total of 94 patients with cirrhosis and an arterially-enhancing liver mass were studied, 66 (70%) of whom had HCC. Alpha-fetoprotein (AFP) >20 ng/mL (P = .029), tumor size >2 cm (P = .0018), and delayed hypointensity (P = .0001) were independent predictors of HCC. Delayed hypointensity of an arterially-enhancing mass had a sensitivity of 89% and a specificity of 96% for HCC. The presence of delayed hypointensity was the only independent predictor of HCC among patients with arterially-enhancing lesions <2 cm (odds ratio, 6.3; 95% confidence interval [CI], 1.8-13), with a sensitivity of 80% and a specificity of 95%. In conclusion, delayed hypointensity of an arterially-enhancing mass was the strongest independent predictor of HCC, regardless of the size of the lesion. If additional studies confirm our results, the noninvasive criteria utilized to make a diagnosis of HCC should be revised.

Quantitative Trait Loci Analysis of Resistance to Sclerotinia sclerotiorum in Sunflower.

ABSTRACT A quantitative trait loci (QTL) analysis of resistance to Sclerotinia sclerotiorum was carried out with 283 sunflower (Helianthus annuus) F(2:3) families derived from a cross between a resistant (SWS-B-04) and a highly susceptible sunflower inbred line. For that purpose, a genetic map based on 195 amplified fragment length polymorphism and 20 simple sequence repeat markers was constructed. The map has a size of 2,273.5 centimorgans and comprises 17 linkage groups, 12 of which could be associated to already defined linkage groups. The heads of sunflower F(3) families were artificially inoculated by using sclerotinia mycelium in three field environments. The lesion length was measured in centimeters 1 week postinoculation and head rot was scored according to a 1-to-8 head rot scale 2 weeks postinoculation. Using the composite interval mapping procedure, three QTL for lesion length and two QTL for head rot could be identified. These QTL explain 10.6 to 17.1% of the total phenotypic variance.

QTL mapping of Sclerotinia midstalk-rot resistance in sunflower.

In many sunflower-growing regions of the world, Sclerotinia sclerotiorum (Lib.) de Bary is the major disease of sunflower (Helianthus annuus L.). In this study, we mapped and characterized quantitative trait loci (QTL) involved in resistance to S. sclerotiorum midstalk rot and two morphological traits. A total of 351 F3 families developed from a cross between a resistant inbred line from the germplasm pool NDBLOS and the susceptible line CM625 were assayed for their parental F2 genotype at 117 codominant simple sequence repeat markers. Disease resistance of the F3 families was screened under artificial infection in field experiments across two sowing times in 1999. For the three resistance traits (leaf lesion, stem lesion, and speed of fungal growth) and the two morphological traits, genotypic variances were highly significant. Heritabilities were moderate to high (h2=0.55-0.89). Genotypic correlations between resistance traits were highly significant (P<0.01) but moderate. QTL were detected for all three resistance traits, but estimated effects at most QTL were small. Simultaneously, they explained between 24.4% and 33.7% of the genotypic variance for resistance against S. sclerotiorum. Five of the 15 genomic regions carrying a QTL for either of the three resistance traits also carried a QTL for one of the two morphological traits. The prospects of marker-assisted selection (MAS) for resistance to S. sclerotiorum are limited due to the complex genetic architecture of the trait. MAS can be superior to classical phenotypic selection only with low marker costs and fast selection cycles.

PlArg from Helianthus argophyllus is unlinked to other known downy mildew resistance genes in sunflower.

The PlArg locus in the sunflower (Helianthus annuus L.) inbred line Arg1575-2 conferring resistance to at least four tested races (300, 700, 730, 770) of downy mildew (Plasmopara halstedii) was localized by the use of simple sequence repeat (SSR) markers. Bulked segregant analysis (BSA) was conducted on 126 individuals of an F2 progeny from a cross between a downy mildew susceptible line, CmsHA342, and Arg1575-2. Twelve SSR markers linked to the PlArg locus were identified. All markers were located proximal to PlArg on linkage group LG1 based on the map of Yu et al. (2003) in a window of 9.3 cM. Since PlArg was mapped to a linkage group different from all other Pl genes previously mapped with SSRs, it can be concluded that PlArg provides a new source of resistance against P. halstedii in sunflower.

Developmental decline in components of signal transduction pathways regulating protein synthesis in pig muscle.

Our previous studies showed that the feeding-induced stimulation of protein synthesis in skeletal muscle of neonatal pigs is accompanied by enhanced phosphorylation of the eukaryotic initiation factor (eIF)4E-binding protein (4E-BP1) and the ribosomal protein S6 kinase (S6K1). These effects of feeding are substantially reduced with development. The goal of the present investigation was to delineate the basis for the reduced responsiveness to feeding observed in the older animals. In these studies, the content and activity of protein kinases located upstream of S6K1 and 4E-BP1 in signal transduction pathways activated by amino acids, insulin, and insulin-like growth factor I were examined in 7- and 26-day-old pigs that were either fasted overnight or fed porcine milk after an overnight fast. Feeding stimulated phosphatidylinositol (PI) 3-kinase activity to the same extent in muscle of 7- and 26-day-old pigs, suggesting that PI 3-kinase is not limiting in muscle of older animals. In contrast, protein kinase B (PKB) activity was significantly less in muscle from 26- vs. 7-day-old pigs, regardless of nutritional status, suggesting that its activity is regulated by mechanisms distinct from PI 3-kinase. In part, the reduced PKB responsiveness can be attributed to a developmental decline in PKB content. Likewise, muscle content of the protein kinase termed mammalian target of rapamycin (mTOR) in 26-day-old pigs was <25% of that in 7-day-old animals. Finally, in agreement with our earlier work showing that S6K1 phosphorylation is reduced in older animals, S6K1 activity was stimulated to a lesser extent in 26- compared with 7-day-old pigs. Overall, the results suggest that the blunted protein synthetic response observed in 26- vs. 7-day-old neonatal pigs is due in part to decreased content and/or activity of signaling components downstream of PI 3-kinase, e.g., PKB, mTOR, and S6K1.

The antidepressant effect of an antiulcer pentadecapeptide BPC 157 in Porsolt's test and chronic unpredictable stress in rats. A comparison with antidepressants.

Various antidepressants have antiulcer activity. Likewise, the models currently used in ulcers and depression disorders research have a considerable degree of similarity. Therefore, the possibility that depression disorders could be effectively influenced by a primary antiulcer agent with a cyto/organoprotective activity, such as the novel stomach pentadecapeptide BPC 157, was investigated in two rat depression assays. First, a forced swimming test (a Porsolt's procedure) was used. As a more severe procedure, chronic unpredictable stress (after 5 d of unpredictable stress protocol, once daily drug application during stress procedure, open field-immobility test assessment at fourth or sixth day of medication) was used. In a forced swimming test, a reduction of the immobility time in BPC 157 (10 microg, 10 ng x kg(-1) i.p.) treated rats corresponds to the activity of the 15 mg or 40 mg (i.p.) of conventional antidepressants, imipramine or nialamide, respectively, given according to the original Porsolt's protocol. In chronic unpredictable stress procedure, particular aggravation of experimental conditions markedly affected the conventional antidepressant activity, whereas BPC 157 effectiveness was continuously present. The effect of daily imipramine (30 mg) medication could be seen only after a more prolonged period, but not after a shorter period (i.e., 4-d protocol). In these conditions, no delay in the effectiveness was noted in BPC 157 medication and a reduction of the immobility of chronically stressed rats was noted after both 4 and 6 d of BPC 157 (10 microg, 10 ng) medication.

Pentadecapeptide BPC 157 attenuates gastric lesions induced by alloxan in rats and mice.

A diabetogenic alloxan regimen produced lesions in all stomachs of treated animals, either rats (200 mg x kg(-1) s.c.) or mice (400 mg x kg(-1) i.p.). In control animals, the lesions, when developed (i.e. 24 h following application), appear to be quite sustained, and consistently present also after 1 or 2 weeks. The application of the pentadecapeptide BPC 157 (10 microg or 10 ng x kg(-1) i.p. coadministered together with alloxan) would significantly attenuate these lesions' appearance. This beneficial effect seems to be present in either rats or mice and in either of the tested intervals. Importantly, the beneficial effect seems to be shared by both microgram and nanogram regimens.

A behavioural study of the effect of pentadecapeptide BPC 157 in Parkinson's disease models in mice and gastric lesions induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydrophyridine.

The effect of a stomach pentadecapeptide, BPC 157, on Parkinson's disease in mice was investigated, along with its salutary activity on stomach lesions induced by parkinsongenic agents. Parkinsongenic agents, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30.0 mg x kg(-1)b.w. i.p. once daily for 6d, and after 4d once 50.0 mg x kg(-1)b.w. i.p.) or reserpine (5.0 mg x kg(-1)b.w. i.p.) were applied i.p. BPC 157 (1.50 microg or 15.0 ng x kg(-1)b.w. i.p.) was applied 15 min before or alternatively 15 min after each MPTP administration. In reserpine studies, BPC 157 (10.0 microg or 10.0 ng x kg(-1)b.w. i.p.) was given either 15 min before reserpine or in the already established complete catalepsy 24 h thereafter. BPC 157 strongly improved the MPTP-impaired somatosensory orientation and reduced the MPTP-induced hyperactivity, and most importantly, MPTP-motor abnormalities (tremor, akinesia, catalepsy -otherwise very prominent in saline control), leading to almost complete abolition of otherwise regularly lethal course of MPTP treatment in controls. Likewise, in reserpine experiments, BPC 157 strongly prevented the development of otherwise very prominent catalepsy and when applied 24 h thereafter reversed the established catalepsy. In addition, a reduction of reserpine-hypothermy (BPC 157 pre-treatment) and reversal of further prominent temperature fall (BPC 157 post-treatment) have been consistently observed. Taking together these data, as the two most suitable animal models were consistently used and since the high effectiveness was demonstrated in pre- and post-treatment, microg and ng regimens, BPC 157 as an organoprotector should be further therapeutically investigated. Additionally, given in either regimen, pentadecapeptide BPC 157 strongly attenuated the stomach lesions in mice that otherwise consistently appeared in mice treated with the parkinsogenic neurotoxin MPTP.

The influence of semantic and perceptual encoding on recognition memory in Alzheimer's disease.

Previous results from a population of patients with Alzheimer's disease (Dalla Barba and Goldblum, 1996) demonstrated that the ability of patients to make a semantic association between two items was significantly and positively correlated to their performance on a yes/no recognition task for the same items and that patients who were impaired on the semantic task did significantly worse on the recognition task than patients who were unimpaired on the semantic task. These findings gave support to a hierarchical model of organization of human memory in which episodic memory depends on the integrity of semantic memory. The present study further investigates the relationship between semantic memory deficits and episodic recognition memory in 15 patients with Alzheimer's disease and 15 controls, as a function of their semantic and perceptual encoding abilities and of their cognitive impairment in other domains. The results confirmed the previous findings and showed that, although patients heavily relied on perceptual analysis, this type of encoding did not enhance their recognition memory. Correlations analyses showed that some patients who were not impaired in the semantic association, but with particularly low scores on a verbal fluency task presented with a pattern, in recognition memory tasks, that suggests a possible early involvement of frontal lobes in this subgroup of patients.

Diagnosis of "sporadic" Huntington's disease.

The diagnosis of Huntington's disease (HD) in patients with progressive chorea and mental impairment, but without similarly affected relatives, remains uncertain and impedes genetic counseling. Twenty patients with suspected HD, but with no family history of the disease underwent molecular analysis of the CAG repeat in the IT15 gene for HD. Eighteen patients displayed the HD expanded allele and two had CAG repeats in the normal range. Neuropsychological tests could be performed in 12 of the 20 patients. Of these 10 with the expanded allele presented the deficits typical of HD, but not the two patients without the HD mutation. This study shows that a neuropsychological pattern is specific to patients with the expanded CAG and that most isolated patients with suspected HD are in fact affected.

Molecular analysis of the cms-inducing MAX1 cytoplasm in sunflower.

DNA from different male sterility-inducing sunflower cytoplasms was investigated in order to determine whether the cytoplasmic male sterility-inducing insertion of the PET1 mitochondrial DNA (mtDNA) is present in other cytoplasms. In one of these cytoplasms (MAX1) the mtDNA shows 93% sequence homology to the orfH522 of the PET1 mtDNA, which is probably responsible for cytoplasmic male sterility (cms) in the latter cytoplasm. In contrast to the situation in the PET1 mitochondrial genome, no transcription of the orfH522-related sequence could be detected in lines with the MAX1 cytoplasm. The organization of the MAX1 mtDNA and the mtDNA of a fertile line is shown to be widely different. In the study described here, homology to the mtDNA insertion was also detected in a fertile Helianthus maximiliani population, whereas DNA of four other H. maximiliani populations showed no hybridization signals.

Cloning and nucleotide sequence of the csp1 gene encoding PS1, one of the two major secreted proteins of Corynebacterium glutamicum: the deduced N-terminal region of PS1 is similar to the Mycobacterium antigen 85 complex.

Two proteins, PS1 and PS2, were detected in the culture medium of Corynebacterium glutamicum and are the major proteins secreted by this bacterium. No enzymatic activity was identified for either of the two proteins. Immunologically cross-reacting proteins were found in a variety of C. glutamicum strains but not in the coryneform Arthrobacter aureus. The gene encoding PS1, csp1, was cloned in lambda gt11 using polyclonal antibodies raised against PS1 to screen for producing clones. The csp1 gene was expressed in Escherichia coli, presumably from its own promoter, and directed the synthesis of two proteins recognized by anti-PS1 antibodies. The major protein band, of lower M(r), was detected in the periplasmic fraction. It had the same M(r) as the PS1 protein band detected in the supernatant of C. glutamicum cultures and presumably corresponds to the mature form of PS1. The minor protein band appears to be the precursor form of PS1. The nucleotide sequence of the csp1 gene was determined and contained an open reading frame encoding a polypeptide with a calculated molecular weight of 70,874, with a putative signal peptide with a molecular weight of 4411. This is consistent with the M(r) determined for PS1 from C. glutamicum culture supernatant and E. coli whole-cell extracts. The NH2-half of the deduced amino acid is similar (about 33% identical residues and 52% including similar residues) to the secreted antigen 85 protein complex of Mycobacterium. The csp1 gene in C. glutamicum was disrupted without any apparent effect on growth or viability.