Evaluation of insertion-deletion markers suitable for genetic diversity studies and marker-trait correlation analyses in cultivated peanut (Arachis hypogaea L.).

Peanut is one of the most important oil crops worldwide. We used insertion-deletion (InDel) markers to assess the genetic diversity and population structure in cultivated peanut. Fifty-four accessions from North China were genotyped using 48 InDel markers. The markers amplified 61 polymorphic loci with 1 to 8 alleles and an average of 2.6 alleles per marker. The polymorphism information content values ranged from 0.0364 to 0.9030, with an average of 0.5038. Population structure and neighbor-joining (NJ) tree analyses suggested that all accessions could be divided into four clusters (A1-A4), using the NJ method. Likewise, four subpopulations (G1-G4) were identified using STRUCTURE analysis. A principal component analysis was also used and results concordant with the other analysis methods were found. A multi-linear stepwise regression analysis revealed that 13 InDel markers correlated with five measured agronomical traits. Our results will provide important information for future peanut molecular breeding and genetic research.

Data mining cDNAs reveals three new single stranded RNA viruses in Nasonia (Hymenoptera: Pteromalidae).

We report three novel small RNA viruses uncovered from cDNA libraries from parasitoid wasps in the genus Nasonia. The genome of this kind of virus is a positive-sense single-stranded RNA with a 3' poly(A), which facilitates cloning from cDNAs. Two of the viruses, NvitV-1 and NvitV-2, possess a RNA-dependent RNA polymerase that associates them with the family Iflaviridae of the order Picornavirales. A third virus, NvitV-3, is most similar to the Nora virus from Drosophila. A reverse transcription-PCR method developed for NvitV-1 indicates that it is a persistent commensal infection of Nasonia.

Species-diagnostic single-nucleotide polymorphism and sequence-tagged site markers for the parasitic wasp genus Nasonia (Hymenoptera: Pteromalidae).

Wasps of the genus Nasonia are important biological control agents of house flies and related filth flies, which are major vectors of human pathogens. Species of Nasonia (Hymenoptera: Pteromalidae) are not easily differentiated from one another by morphological characters, and molecular markers for their reliable identification have been missing so far. Here, we report eight single-nucleotide polymorphism and three sequence-tagged site markers derived from expressed sequenced tag libraries for the two closely related and regionally sympatric species N. giraulti and N. vitripennis. We studied variation of these markers in natural populations of the two species, and we mapped them in the Nasonia genome. The markers are species-diagnostic and evenly spread over all five chromosomes. They are ideal for rapid species identification and hybrid recognition, and they can be used to map economically relevant quantitative trait loci in the Nasonia genome.

Vasoactive intestinal peptide dampens formyl-peptide-induced ROS production and inflammation by targeting a MAPK-p47phox phosphorylation pathway in monocytes.

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47phox, p67phox, p40phox, and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA11,22,28]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the "MAPK-p47phox phosphorylation-NOX2 activation" axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.

Forskolin mimics TSH action on the expression of protein kinase C isozymes in pig thyroid cell cultures.

In porcine thyroid cell cultures, phospholipid-dependent protein kinases (PKCs) have the same characteristics as intact glands. The overall PKC activity, presence of PKC isozymes, chromatographic pattern and endogenous substrates specificity were not modified during the two-day culture period. Three PKC isozymes (cPKC epsilon, nPKC epsilon and aPKC zeta) were identified by immunoblot analysis in the two subcellular fractions: cytosol and particulate extract, both in intact glands and two-day-old cultures. In cells cultured for two days in the presence of TSH (0.1 mU/ml), the overall PKC activity was stimulated (ca. 200%) in the two compartments. This stimulation was parallel to the increase in protein expression of the three PKC isoforms (as demonstrated by immunoblot analysis) and was accompanied by a redistribution of cPKC alpha and nPKC epsilon toward the particulate fraction. In TSH-treated cells, hydroxyapatite chromatography of cytosolic PKC revealed an additional peak of PKC activity eluted at 195 mM potassium phosphate. Its elution molarity did not correspond to the molarity of any known PKC isozyme, and it did not cross-react with antibodies directed against cPKC isozymes--: alpha, beta, or gamma. When TSH was replaced by forskolin (10(-5) M), identical quantitative and qualitative modifications were obtained, suggesting that, in thyroid cells, the cyclic AMP-dependent regulatory cascade could be involved in the control of PKC isoforms expression by TSH.

P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox.

NADPH oxidase is an O2*- -generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67phox, p47phox, p40phox, and the G-protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67phox, p47phox, and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40phox associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40phox. When resting neutrophils were lysed in Triton X-100 or octyl glucoside buffer and separated into detergent-soluble and detergent-insoluble fractions, p40phox and p67phox were mainly associated with the detergent-insoluble fraction (defined as the cytoskeleton), whereas p47phox was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47phox translocation to the cytoskeleton but did not affect the distribution of p40phox or p67phox. Using immunofluorescence confocal microscopy, we found that p40phox colocalized with filamentous actin. In neutrophils from a p67phox-deficient patient with detectable p40phox, p40phox associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X-100 buffer, p40phox, p47phox, and p67phox were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40phox and p47phox was found in the soluble membrane fraction, but no p67phox was detected. These findings show that p40phox, like p67phox and p47phox, binds to the cytoskeleton and membrane skeleton. In addition, p40phox can dissociate from p67phox in activated membranes.

Immunochemical identification and translocation of protein kinase C zeta in human neutrophils.

Western blots of human polymorphonuclear leukocyte (PMN) extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. Two bands corresponding to PKC type zeta with apparent molecular masses of 81 kDa and 76 kDa were identified in the cytosolic fraction of resting cells, in addition to PKC types alpha and beta. PKC zeta was apparently abundant, like PKC beta, whereas PKC delta, -epsilon, and -gamma were not detectable. Following short stimulation (5 min) of PMN with phorbol-12-myristate-13-acetate (1 microgram/ml), physical translocation of PKC zeta from the cytosol to the plasma membrane fraction occurred, although this isoform does not bind phorbol esters. These data show that, in addition to the two calcium-dependent isoenzymes alpha and beta, human PMN express a calcium-independent isoenzyme zeta which translocates in stimulated cells, suggesting a role in the regulation of antibacterial activities.

Aphid biology: expressed genes from alate Toxoptera citricida, the brown citrus aphid.

The brown citrus aphid, Toxoptera citricida (Kirkaldy), is considered the primary vector of citrus tristeza virus, a severe pathogen which causes losses to citrus industries worldwide. The alate (winged) form of this aphid can readily fly long distances with the wind, thus spreading citrus tristeza virus in citrus growing regions. To better understand the biology of the brown citrus aphid and the emergence of genes expressed during wing development, we undertook a large-scale 5' end sequencing project of cDNA clones from alate aphids. Similar large-scale expressed sequence tag (EST) sequencing projects from other insects have provided a vehicle for answering biological questions relating to development and physiology. Although there is a growing database in GenBank of ESTs from insects, most are from Drosophila melanogaster and Anopheles gambiae, with relatively few specifically derived from aphids. However, important morphogenetic processes are exclusively associated with piercing-sucking insect development and sap feeding insect metabolism. In this paper, we describe the first public data set of ESTs from the brown citrus aphid, T. citricida. The cDNA library was derived from alate adults due to their significance in spreading viruses (e.g., citrus tristeza virus). Over 5180 cDNA clones were sequenced, resulting in 4263 high-quality ESTs. Contig alignment of these ESTs resulted in 2124 total assembled sequences, including both contiguous sequences and singlets. Approximately 33% of the ESTs currently have no significant match in either the non-redundant protein or nucleic acid databases. Sequences returning matches with an E-value of < or = -10 using BLASTX, BLASTN, or TBLASTX were annotated based on their putative molecular function and biological process using the Gene Ontology classification system. These data will aid research efforts in the identification of important genes within insects, specifically aphids and other sap feeding insects within the Order Hemiptera.

[Hydantoin fetal syndrome]. / Syndrome foetal à l'hydantoïne.

We report a case of distal finger hypoplasia associated with foetal hydantoin syndrome (FHS). The occurrence of this syndrome is thought to be due to abnormalities of collagen, cytochrome P 450 and arachidonic acid metabolism. The risk of developing FHS could be evaluated by counting the glucocorticoid receptors of lymphocytes or by measuring epoxide hydrolase activity through amniocentesis. FHS can be prevented by taking folates during pregnancy.

[Hydroxychloroquine-puvatherapy photoinduced acute generalized exanthematous pustulosis]. / Pustulose exanthématique aiguë généralisée photodéclenchée par l'association hydroxychloroquine-puvathérapie.

INTRODUCTION: Acute generalized exanthematous pustulosis usually occurs as a typical skin reaction to drugs. We observed a case with a photodistribution induced by hydroxychloroquine and/or PUVA. CASE REPORT: A male subject had been treated for actinic pseudolymphoma since 1988. General corticosteroids had been given initially and were followed by PUVA and azathioprine. A new episode with erythema involving the trunk and the proximal portion of the limbs was treated with corticosteroids and hydroxychloroquine. The symptomatology regressed but pustular erythema developed in exposed areas two days after a PUVA session on the upper part of the body. The eruption did not involve the zones of the phototests one month earlier. The lesions resolved rapidly after withdrawal of hydroxychloroquine and PUVA. DISCUSSION: Photo-induced acute generalized exanthematous pustulosis with a photodistribution has not been reported previously. The imputability of hydroxychloroquine and PUVA, and their association is suggested. The appearance of pustular lesion on exposed areas and the protection resulting from the phototests would lead to several hypotheses. General corticosteroids were ineffective in preventing and in treating acute generalized exanthematous pustulosis.

[Multocida Pasteurella and leg ulcer]. / Pasteurella multocida et ulcération de jambe.

INTRODUCTION: We observed two cases of Pasteurella and discuss the role of bacterial sampling in ulcerations of the lower limb. CASE REPORTS: In the first case, Pasteurella was discovered in an ulceration of the lower limb and was cured with no particularly specific care. In the second case, Pasteurella had been inoculated by scratching an ulceration and could not be cured without specific treatment. DISCUSSION: No specific pathological consequence in chronic carriers could be possible, a situation which has often been reported in the literature. Pasteurella is an unusual specific cause of ulcerations of the lower limb.

[Multicentric histiocytosis with hematological involvement]. / Histiocytose multicentrique avec atteinte hématologique.

INTRODUCTION: The aim of this work was to present a case of multicentric histiocytosis with haematologic involvement. CASE REPORT: A 68-year-old man presented with poor general health and a nodular eruption of the skin and larynx. On clinical examination there was an enlarged spleen and laboratory results revealed an inflammatory syndrome, platelet count 60,000 and myelemia with 10 p. 100 immature elements in a white cell count of 14,000. Pathology and ultrastructure examinations led to the diagnosis of multicentric histiocytosis. Bone marrow aspiration was normal. Pancytopenia then developed with bone marrow hypoplasia without infiltration. Corticosteroids then cyclophosphamide were uneffective for either the skin disease or the hematologic disorder. The patient developed severe buccal aphthosis which responded well to thalidomide. This treatment reduced the size and the number of skin nodules. Finally, renal failure of unknown origin was observed. DISCUSSION: Electron microscopy is essential for positive and differential diagnosis of atypical multicentric histiocytosis. Hematological disorders associated with multicentric histiocytosis may either be specific or totally independent.

Redistribution of protein kinase C isoforms in human neutrophils stimulated by formyl peptides and phorbol myristate acetate.

The redistribution of protein kinase C (PKC) isoforms between the cytosolic and plasma membrane fractions of stimulated human polymorphonuclear leukocytes (PMN) was analysed by means of western blotting with antibodies against PKC beta I, beta II and Zeta. Treatment of PMN with 1 microM formyl-methionyl-leucyl-phenylalanine (fMLP) induced a rapid (5-10 sec) and sustained (at least 10 min) increase in the membrane association of PKC beta I, beta II, and the two immunoreactive proteins (76-81 kDa) recognized by the antibody directed against PKC zeta. Optimal translocation of PKC isoforms to the plasma membrane occurred in the presence of 10(-6) M fMLP and was not associated with a detectable fall in cytosolic PKC. In the absence of external calcium, the translocation of all PKC isoforms induced by fMLP was rapid (5 sec) but the membrane association of PKC was lost within one minute. Unlike fMLP, phorbol myristate acetate (PMA) induced a concentration-dependent translocation of the PKC isoforms, which persisted in the membrane in the absence of external calcium. These data provide the first evidence of redistribution of PKC isoforms by a chemoattractant. They further indicate that external calcium plays a crucial role in the persistence of the membrane association of PKC beta I, beta II and zeta induced by formyl peptides.

NADPH dehydrogenase activity of p67PHOX, a cytosolic subunit of the leukocyte NADPH oxidase.

The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b558) and at least four cytosolic components: p47PHOX, p67PHOX, p40PHOX, and Rac, a small GTPase. All the oxidase components except p40PHOX are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67PHOX from baculovirus-infected Sf9 cells could mediate the dehydrogenation of NADPH. NADPH dehydrogenation was not dependent on FAD and was insensitive to superoxide dismutase. Several control experiments showed that NADPH dehydrogenation was accomplished by p67PHOX, not by a trace contaminant in the p67PHOX preparation. The NADPH dehydrogenase activity of p67PHOX was proportional to enzyme concentration, and showed saturation kinetics with NADPH (Km 92 +/- 5 microM), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67PHOX (Km 123 +/- 38 microM). Taken together, these results show that p67PHOX is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67PHOX might participate directly in electron transfer between NADPH and the oxidase flavin.

Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67PHOX and cytochrome b558.

Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47(PHOX), p67(PHOX), p40(PHOX), and Rac1 or Rac2, with the membrane-bound cytochrome b(558). Whereas the interaction of p47(PHOX) with cytochrome b(558) is well established, an interaction between p67(PHOX) and cytochrome b(558) has never been investigated. We report here a direct interaction between p67(PHOX) and cytochrome b(558). First, labeled p67(PHOX) recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b(558)-deficient patient. Second, p67(PHOX) binds to cytochrome b(558) that has been bound to nitrocellulose. Third, GTP-p67(PHOX) bound to glutathione agarose is able to pull down cytochrome b(558.) Rac1-GTP or Rac1-GDP increased the binding of p67(PHOX) to cytochrome b(558), suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67(PHOX) and cytochrome b(558).

Binding of nicotinamide adenine dinucleotide phosphate to the tetratricopeptide repeat domains at the N-terminus of p67PHOX, a subunit of the leukocyte nicotinamide adenine dinucleotide phosphate oxidase.

The nicotinamide adenine dinucleotide phosphate (NADPH) binding site of the NADPH oxidase complex is believed to be located on the beta, subunit of cytochrome b558. However, our previous studies showed that p67PHOX also contains an NADPH binding site that is essential for normal oxidase activity and that p67PHOX is able to mediate a slow electron transfer from a reduced pyridine nucleotide to an artificial electron acceptor. Using both affinity labeling and fluorescence quenching, we have obtained further evidence that p67PHOX is able to bind NADPH. We have used a number of truncated forms of p67PHOX, including p67PHOX(1-243), p67PHOX(1-210), p67PHOX(1-199), and p67PHOX(244-526) (where the numbers represent the initial and final amino acids in the truncated p67PHOX) in order to localize the binding site. We found that NADPH could bind to p67PHOX(1-243), p67PHOX(1-210), and p67PHOX(1-199) but not to p67PHOX(244-526). The p67PHOX(1-199) fragment consists largely of four tetratricopeptide (TPR) domains. We showed further that Rac2-GTP gamma S and to a lesser extent Rac2-GDP beta S could modulate the binding of NADPH to p67PHOX.

Selenium concentrations and glutathione peroxidase activities in a population exposed to selenium via drinking water.

Selenium concentrations in blood, urine, hair, and tap water were determined in samples obtained from individuals exposed to varying amounts of the element in water form home wells. Glutathione peroxidase activities were also determined on the blood samples. Correlations of blood Se with the enzyme activity were not statistically significant. Correlations of water Se, urine Se, and hair Se with glutathione peroxidase activity were also not statistically significant. It is concluded that a relationship between Se and glutathione peroxidase activity does not exist when Se status is adequate.

Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst.

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.

Phosphorylation of the respiratory burst oxidase subunit p67(phox) during human neutrophil activation. Regulation by protein kinase C-dependent and independent pathways.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O-2) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67(phox). In this study, we found that activation of human neutrophils with formyl-methionyl-leucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67(phox). Using an anti-p67(phox) antibody or an anti-p47(phox) antibody, we showed that phosphorylated p67(phox) and p47(phox) form a complex. Phosphoamino acid analysis of the phosphorylated p67(phox) revealed only 32P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67(phox) is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67(phox) in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67(phox) was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67(phox) is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.