Effects of Host Age and Density on the Performance of Apanteles hemara (Hymenoptera: Braconidae), a Larval Endoparasitoid of Spoladea recurvalis (Lepidoptera: Crambidae).

The amaranth leaf-webber, Spoladea recurvalis (Fabricius; Lepidoptera: Crambidae), is a serious pest of Amaranthus sp. in Africa and Asia. Apanteles hemara (Nixon; Hymenoptera: Braconidae) is by far the most important larval endoparasitoid of the amaranth leaf-webber. We examined the effects of host density and age on the biological characteristics of A. hemara. The regression model of the number of hosts supplied to A. hemara against the number of larvae parasitized resulted in a curve corresponding to type II functional response, with a significant increase in the number of hosts parasitized up to the density of 30 hosts before being constant up to 40 hosts. In contrast, the parasitism rate decreased linearly with increasing host densities. Development time, sex ratio, and adult longevity were not significantly affected by host density. The immature parasitoid mortality was significantly higher at higher host densities. Apanteles hemara did not parasitize 7-d-old larvae and beyond, while parasitism was significantly higher among 1- to 2-d-old compared with 3- to 4-d-old larvae. Immature parasitoid mortality was 2.6 times higher in 1- to 2-d-old larvae compared with 5- to 6-d-old larvae. The developmental period of the parasitoid from egg to adult was longest among 1- to 2-d-old larvae and least among 5- to 6-d-old larvae. Nonreproductive mortality was markedly higher among 1- to 2-d-old larvae compared with the older larvae. Adult female A. hemara were significantly larger on 3- to 4-d-old larvae compared with either 1- to 2-d-old or 5- to 6-d-old larvae. We discuss the implications of our results for the interpretation of functional response in parasitoids, mass rearing, conservation, and augmentative biological control of S. recurvalis.

Effect of Partially Screened Nuclei on Fast-Electron Dynamics.

We analyze the dynamics of fast electrons in plasmas containing partially ionized impurity atoms, where the screening effect of bound electrons must be included. We derive analytical expressions for the deflection and slowing-down frequencies, and show that they are increased significantly compared to the results obtained with complete screening, already at subrelativistic electron energies. Furthermore, we show that the modifications to the deflection and slowing down frequencies are of equal importance in describing the runaway current evolution. Our results greatly affect fast-electron dynamics and have important implications, e.g., for the efficacy of mitigation strategies for runaway electrons in tokamak devices, and energy loss during relativistic breakdown in atmospheric discharges.

Clinical value of R-spondins in triple-negative and metaplastic breast cancers.

BACKGROUND: RSPO ligands, activators of the Wnt/ß-catenin pathway, are overexpressed in different cancers. The objective of this study was to investigate the role of RSPOs in breast cancer (BC). METHODS: Expression of RSPO and markers of various cancer pathways were measured in breast tumours and cell lines by qRT-PCR. The effect of RSPO on the Wnt/ß-catenin pathway activity was determined by luciferase assay, western blotting, and qRT-PCR. The effect of RSPO2 inhibition on proliferation was determined by using RSPO2 siRNAs. The effect of IWR-1, an inhibitor of the Wnt/ß-catenin pathway, was examined on the growth of an RSPO2-positive patient-derived xenograft (PDX) model of metaplastic triple-negative BC. RESULTS: We detected RSPO2 and RSPO4 overexpression levels in BC, particularly in triple-negative BC (TNBC), metaplastic BC, and triple-negative cell lines. Various mechanisms could account for this overexpression: presence of fusion transcripts involving RSPO, and amplification or hypomethylation of RSPO genes. Patients with RSPO2-overexpressing tumours have a poorer metastasis-free survival (P=3.6 × 10-4). RSPO2 and RSPO4 stimulate Wnt/ß-catenin pathway activity. Inhibition of RSPO expression in a TN cell line inhibits cell growth, and IWR-1 significantly inhibits the growth of an RSPO2-overexpressing PDX. CONCLUSIONS: RSPO overexpression could therefore be a new prognostic biomarker and therapeutic target for TNBC.

The pathogenicity of Beauveria bassiana: what happens after an endophytic phase in plants?

The banana weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) is a serious constraint to banana (Musa spp.) production throughout the world. The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) offers a potential weevil management option, but conventional delivery mechanisms have limited its success. As an endophyte, however, B. bassiana can be efficiently delivered to banana planting materials for the potential management of C. sordidus. However, entomopathogens can change morphology and efficacy against their target host when successively sub-cultured on artificial media or when exposed to certain physical and chemical environmental conditions. Whether such changes occur in B. bassiana after an endophytic phase inside a banana plant remains unknown. The primary aim of our study was to evaluate the viability, growth, sporulation and pathogenicity of endophytic B. bassiana. To attain this, two sets of experiments, namely morphological characterization and larval bioassays, were conducted under laboratory conditions. In these experiments, growth and pathogenicity of the wild-type B. bassiana strain G41, obtained originally from banana farms, was compared with the endophytic B. bassiana strain G41, re-isolated from the rhizome of B. bassiana-inoculated banana plants at one month post-inoculation. Morphological characterization, conidial germination, colony growth and sporulation rate was assessed on SDAY media while pathogenicity was determined 15 days after immersing the larvae of C. sordidus in different conidial doses. No differences were observed in colony appearance and growth rate between the endophytic and wild-type strain. Percentage conidial germination for the endophytic strain (91.4-94.0%) was higher than for the wild-type (86.6-89.7%). LD50 equated 1.76 x 10(5) and 0.71 x 10(5) conidia/ml for the wild-type and endophytic B. bassiana strains, respectively, but did not differ between strains. Our study demonstrated that, after an endophytic phase inside the banana plant, B. bassiana retains it morphology and pathogenicity against the banana weevil larvae; and thus can offer protection against the damaging larvae feeding inside the rhizome.

Specificity of 14-3-3 isoform dimer interactions and phosphorylation.

Proteins that interact with 14-3-3 isoforms are involved in regulation of the cell cycle, intracellular trafficking/targeting, signal transduction, cytoskeletal structure and transcription. Recent novel roles for 14-3-3 isoforms include nuclear trafficking the direct interaction with cruciform DNA and with a number of receptors, small G-proteins and their regulators. Recent findings also show that the mechanism of interaction is also more complex than the initial finding of the novel phosphoserine/threonine motif. Non-phosphorylated binding motifs that can also be of high affinity may show a more isoform-dependent interaction and binding of a protein through two distinct binding motifs to a dimeric 14-3-3 may also be essential for full interaction. Phosphorylation of specific 14-3-3 isoforms can also regulate interactions. In many cases, they show a distinct preference for a particular isoform(s) of 14-3-3. A specific repertoire of dimer formation may influence which of the 14-3-3-interacting proteins could be brought together. Mammalian and yeast 14-3-3 isoforms show a preference for dimerization with specific partners in vivo.

Casein kinase I associates with members of the centaurin-alpha family of phosphatidylinositol 3,4,5-trisphosphate-binding proteins.

Mammalian casein kinases I (CKI) belong to a family of serine/threonine protein kinases involved in diverse cellular processes including cell cycle progression, membrane trafficking, circadian rhythms, and Wnt signaling. Here we show that CKIalpha co-purifies with centaurin-alpha(1) in brain and that they interact in vitro and form a complex in cells. In addition, we show that the association is direct and occurs through the kinase domain of CKI within a loop comprising residues 217-233. These residues are well conserved in all members of the CKI family, and we show that centaurin-alpha(1) associates in vitro with all mammalian CKI isoforms. To date, CKIalpha represents the first protein partner identified for centaurin-alpha(1). However, our data suggest that centaurin-alpha(1) is not a substrate for CKIalpha and has no effect on CKIalpha activity. Centaurin-alpha(1) has been identified as a phosphatidylinositol 3,4,5-trisphosphate-binding protein. Centaurin-alpha(1) contains a cysteine-rich domain that is shared by members of a newly identified family of ADP-ribosylation factor guanosine trisphosphatase-activating proteins. These proteins are involved in membrane trafficking and actin cytoskeleton rearrangement, thus supporting a role for CKIalpha in these biological events.

The gradual transition to a team-based environment: the success story of a medium-sized manufacturing facility.

This article will review the case in which a medium-sized manufacturing facility in upstate New York underwent the transition to a team-based environment. As with any change, this transition involved a decision process, a design process, and an implementation process. The transition took place gradually over a period of several years, allowing each stage to be studied in some detail. The degree to which these approaches are applicable to an organization will depend in part on how similar that organization is to the study facility; therefore, this article will begin with a background discussion of the nature of that facility.

Annexin V inhibits protein kinase C activity via a mechanism of phospholipid sequestration.

In this study, we assessed the role of annexin V, a Ca2+-dependent phospholipid-binding protein, as a regulator of protein kinase C (PKC) and characterized its mechanism of inhibition. Several mutants obtained by oligonucleotide site-directed mutagenesis were tested in vitro on PKC activity in cytosolic fractions from Jurkat cells and on purified PKCalpha. Annexin V inhibited phosphorylation of annexin II by endogenous PKC and phosphorylation of myelin basic protein by PKCalpha. In both systems, the use of single Ca2+-binding-site mutants of annexin V led to a partial reversal of inhibition, and the Ca2+-binding site located in the first domain of annexin V was found to have the most important role. An increase in the number of mutated Ca2+-binding sites led to a greater loss of inhibition. These results corroborated those showing the progressive loss of binding of these mutants to phospholipid liposomes. In conclusion, we show that PKC inhibition by annexin V is the consequence of a mechanism involving phospholipid sequestration by annexin V, and that the Ca2+-binding site located in domain 1 of annexin V plays a predominant role in this process. In addition, we show that the R122AIK site, which may act analogously to a PKC-inhibitory pseudosubstrate site, is not involved in PKC inhibition, and that a peptide corresponding to the C-terminal tail of annexin V inhibits PKC activity but to a lesser extent than annexin V itself.

In adrenocortical tissue, annexins II and VI are attached to clathrin coated vesicles in a calcium-independent manner.

We have previously characterized three populations of clathrin coated vesicles (CCVs) isolated from bovine adrenocortical tissue and designated them as large, medium and small coated vesicles, i.e., LCV, MCV and SCV, respectively. Here, we show that annexins II and VI, two of the annexins involved in membrane traffic, are present in the three populations of CCVs but with different distributions between coat proteins (CP) and lipidic vesicle membrane. Annexin VI is only associated with the membrane, whatever the CCV population. In contrast, annexin II is differently distributed between coat and membrane, depending on the CCV population. Both annexins are bound to membranes in a calcium-independent manner and solubilization studies in Triton X114 (TX114) suggest that they interact poorly with lipids by hydrophobic interactions. Ligand blotting experiments show that both annexins bind to CCV proteins: annexin II to a 200-kDa component in all CCVs and annexin VI to a 100-kDa component in LCV and SCV identified as dynamin, a GTPase essential for endocytic CCV pinching off. Dynamin is tightly associated to annexin VI only in LCVs, the endocytic [transferrin (Tf) positive] vesicles. Our data suggest that annexins II and VI could define specific protein-lipid interaction microdomains that could play a role in the different functions of the CCVs.

14-3-3 is phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in vivo regulates Raf/14-3-3 interaction.

14-3-3 proteins mediate interactions between proteins involved in signal transduction and cell cycle regulation. Phosphorylation of target proteins as well as 14-3-3 are important for protein-protein interactions. Here, we describe the purification of a protein kinase from porcine brain that phosphorylates 14-3-3 zeta on Thr-233. This protein kinase has been identified as casein kinase Ialpha (CKIalpha) by peptide mapping analysis and sequencing. Among mammalian 14-3-3, only 14-3-3 tau possesses a phosphorylatable residue at the same position (Ser-233), and we show that this residue is also phosphorylated by CKI. In addition, we show that 14-3-3 zeta is exclusively phosphorylated on Thr-233 in human embryonic kidney 293 cells. The residue 233 is located within a region shown to be important for the association of 14-3-3 to target proteins. We showed previously that, in 293 cells, only the unphosphorylated form of 14-3-3 zeta associates with the regulatory domain of c-Raf. We have now shown that in vivo phosphorylation of 14-3-3 zeta at the CKIalpha site (Thr-233) negatively regulates its binding to c-Raf, and may be important in Raf-mediated signal transduction.

Structure and sites of phosphorylation of 14-3-3 protein: role in coordinating signal transduction pathways.

The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of 'adaptor' protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition. It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions. 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX1,2SpXP. The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases. The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins. We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms.

Inhibition of cytosolic phospholipase A2 by annexin V in differentiated permeabilized HL-60 cells. Evidence of crucial importance of domain I type II Ca2+-binding site in the mechanism of inhibition.

Annexin V belongs to a family of proteins that interact with phospholipids in a Ca2+-dependent manner. This protein has been demonstrated to have anti-phospholipase A2 activity. However, this effect has never yet been reported with the 85-kDa cytosolic PLA2 (cPLA2). We studied, in a model of differentiated and streptolysin O-permeabilized HL-60 cells, the effect of annexin V on cPLA2 activity after stimulation by calcium, GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), formyl-Met-Leu-Phe, or phorbol 12-myristate 13-acetate. Both recombinant and human placental purified annexin V inhibit cPLA2 activity whatever the stimulus used. The decrease of arachidonic acid release is of 40 and 50%, respectively, at [Ca2+] of 3 and 10 microM. The mechanism of inhibition was also analyzed. cPLA2 requires calcium and protein kinase C (PKC) or mitogen-activated protein kinase phosphorylation for its activation. As annexin V was shown to be an endogenous inhibitor of PKC, PKC-stimulated cPLA2 activity was analyzed. Using GF109203x, a specific PKC inhibitor, we demonstrated that this pathway is of minor importance in our model. cPLA2 inhibition by annexin V is not linked to PKC inhibition. To test the hypothesis of phospholipid depletion, mutants of annexin V were constructed using mutagenesis directed to Ca2+ site. We demonstrate that the Ca2+ site located in domain I is necessary for the inhibitory effect of annexin V on cPLA2 activity. The site in domain IV is also involved but with less efficiency. In contrast, mutations in site II and III do not modify this effect. Moreover, annexin V mutated on all sites does not inhibit cPLA2. Thus, we propose a predominant role of module (I/IV) in the biological action of annexin V, which, in physiological conditions, may control cPLA2 activity by depletion of the phospholipid substrate.

Annexins and protein kinases C.

Annexins and protein kinases C belong to two distinct families of ubiquitous cytoplasmic proteins involved in signal transduction. All annexins share the property of binding calcium and phospholipids in the presence of calcium. Protein kinases C belong to three distinct groups of kinases: cPKCs (conventional PKCs) depend on calcium, diacylglycerol and negatively charged phospholipids for their activity, nPKCs (novel PKCs) depend on diacylglycerol and negatively charged phospholipids and aPKCs (atypical PKCs) only require negatively charged phospholipids. Almost all annexins are both in vitro and in vivo substrates for PKCs except annexin V. All annexins have a putative binding site for PKCs but only annexin V would possess a potential pseudo-substrate site. We propose that annexin V modulates the activity of some cPKCs on their substrates which may be the other annexins.

Inhibitory effect of annexin V on protein kinase C activity in mesangial cell lysates.

Annexin V belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of the protein kinase C (PKC) activity. This study examines the effect of annexin V on the in vitro PKC activity in cultured mesangial cells using histone H1, the peptide [Ser25]PKC-(19-31), or endogenous proteins as substrates. The SDS/PAGE pattern of 32P-labeled mesangial proteins showed that the calcium-independent PKC [(n+a)PKC] phosphorylated several proteins from 70 kDa to 40 kDa and 22 kDa to 15 kDa. Three additional proteins from 34 kDa to 29 kDa, including annexin I and its proteolytic forms, were detected after activation of calcium-dependent PKC (cPKC). Increasing concentrations of annexin V did not alter the phosphorylation of (n+a)PKC substrates. By contrast, specific phosphorylation of proteins and annexin I by cPKC, was reduced in a dose-dependent manner. Addition of high concentration of calcium and phosphatidylserine did not reverse the inhibitory effect of annexin V. Annexin V also inhibited the phosphorylation of histone H1 or peptide [Ser25]PKC-(19-31) by cPKC. Moreover, removal of annexin V from cytosols increased the annexin I phosphorylation by these isoforms. From these results, we propose that annexin V may regulate the signal-transduction pathway involving the activation of cPKC, as they act in vitro as an inhibitor of these kinases.

In vivo and in vitro phosphorylation of annexin II in T cells: potential regulation by annexin V.

In order to understand how signal transduction occurs during T cell activation, it is necessary to identify the key regulatory molecules whose function is influenced by phosphorylation. Annexins II (A-II) and V (A-V) belong to a large family of Ca(2+)-dependent phospholipid-binding proteins. Among many putative functions, annexins may be involved in signal transduction during cellular proliferation and differentiation. In the present study we show that A-II is phosphorylated in vivo in the Jurkat human T cell line. Indeed, A-II is phosphorylated after stimulation by phorbol myristate acetate and on serine residues after T cell antigen receptor (TcR) stimulation. In cytosol from Jurkat cells, A-II is phosphorylated only by Ca2+/phospholipid-stimulated kinases such as Ca(2+)-dependent protein kinases C (cPKCs). A-V inhibits the phosphorylation of A-II and other substrates of cPKCs and has no effect on kinases activated only by phospholipids. In conclusion, A-II is phosphorylated both in vitro and in vivo in Jurkat cells, and may play a role as a substrate during signal transduction in lymphocytes via the TcR through the PKC pathway. On the other hand, A-V could act as a potent modulator of cPKCs in Jurkat cells.

High levels of antibodies to annexins V and VI in patients with rheumatoid arthritis.

OBJECTIVE: Glucocorticoids are powerful antiinflammatory agents widely used for the treatment of rheumatoid arthritis (RA). Synthesis and/or secretion of annexin I (A-I) is induced by these steroids. Annexins V and VI are also found extracellularly but are not induced by glucocorticoids. Annexins may be potent antagonists of phospholipase A2 (PLA2). Since autoantibodies to A-I have been reported in patients with RA, we studied the reactivity of sera from patients with RA to A-V and A-VI. METHODS: Sera from 26 patients with RA were assessed for anti-A-V and anti-A-VI antibodies and compared with sera from 26 sex/age matched healthy subjects. IgG and IgM antibodies were analyzed in an ELISA: A correlation study with disease activity and corticosteroid treatment schedule was performed. RESULTS: Sera from patients with RA contained significantly higher levels of IgG [anti-A-V and anti-A-VI] autoantibodies than control sera, both being correlated. This rise in antiannexin antibody titers was correlated with the RA activity score, and negatively correlated with the daily dose of corticosteroids. CONCLUSION: High levels of IgG (anti-A-V and anti-A-VI) antibodies were found in sera from patients with RA. We suggest that antiannexin autoantibodies may play a role in the clinical course of RA by impairing the anti-PLA2 effect of annexins.

A soluble protein negatively regulates phospholipase D activity. Partial purification and characterization.

Phosphatidylcholine-specific phospholipase D (PLD) is an important signalling phospholipase in mammalian cells. Recently, PLD activity has been shown to be positively regulated by the GTP-binding protein ARF (ADP-ribosylating factor). In the present work, we document the presence of a factor negatively regulating PLD activity in bovine brain cytosol. The inhibitory factor is characterized as a large protein or a complex of proteins with a molecular mass higher than 300 kDa. Using permeabilized and pre-permeabilized HL-60 cells depleted of their cytosol, we demonstrate that the inhibitor acts on GTP[S]-stimulated PLD activity. This effect is immediate, persistent and dose dependent for GTP[S]-stimulated PLD. Different possibilities for a mechanism of action of the inhibitory factor on the regulation of GTP binding of ARF were investigated. This inhibitory factor is not the guanine-dissociating inhibitor (GDI) for the small G-binding proteins Rho (Rho-GDI), reported to be a PLD inhibitor, since specific antibodies against this protein did not recognize a protein in the peak containing the inhibitory factor for PLD activity. Furthermore, the inhibitory factor does not prevent the binding of GTP[S] to ARF in the presence of HL-60 membranes. This excludes its possible role as an inhibitor of an ARF/guanine exchange factor. The inhibitory factor not only inhibits a pathway of PLD through GTP[S] activation in particular of the small GTP-binding protein, ARF, but it also inhibits PLD activated via either protein kinase C (PKC) or tyrosine kinase activation. The inhibitory factor also decreases PLC activity and this effect seems to be secondary to the inhibition of PLD activity. We discuss a mechanism of action of the inhibitor on PLD and the importance of this enzyme activity for membrane traffic.

Potential interaction between annexin VI and a 56-kDa protein kinase in T cells.

Annexins belong to a large family of calcium-dependent phospholipid binding proteins known to undergo post-translational modifications such as phosphorylation. Physiological function of each annexin is still unclear since they may participate in signal transduction. We have tested the presence of annexins in a T cell line (Jurkat) and studied their phosphorylation by protein tyrosine kinases of the src family. Among annexins I, II, V and VI found in Jurkat cells, annexin VI was shown to be phosphorylated in vitro by p56lck and annexins I and II by p60src. We could not detect the phosphorylation of A-VI in vivo, even after cell stimulation. However, a 56-kDa phosphoprotein was found to be associated with A-VI after T cell activation. This 56-kDa protein shares some characteristics with p56lck.

Annexin VI is secreted in human bile.

A calcium-binding protein of 68 kDa was isolated from human bile that precipitates upon addition of 5 mM CaCl2. This protein was recognized by an immunoaffinity purified anti-annexin VI antibody and it had a similar aminoacid composition as annexin VI. Phospholipase A2 activity was inhibited in vitro in a dose-dependent manner as reported for annexin VI. It is the first time that an annexin secretion is demonstrated in bile.

[Clinical value of interaural time discrimination]. / Intérêt clinique d'un test de discrimination interaurale de temps.

The interaural time discrimination (ITD) is one of the azimuthal sound location cues. In multiple sclerosis subjects, deficits in binaural timing discrimination are closely related to abnormalities in brainstem auditory evoked potentials (BAEPs). To determine if psychophysical performances could be related to the BAEPs in other otoneurological deficits, thresholds of ITD were measured in 168 patients and the results correlated to the electrophysiological responses to clicks. Both tests were normal in 29.8% and abnormal in 42.2% of cases. A discrepancy existed in 28.0%: ITD abnormal--BAEPs normal in 23.2%, ITD normal--BAEPs abnormal in 4.8%. In conclusion, in cases with normal ITD thresholds, the probability of a retrocochlear lesion is low. The test of ITD is helpful when the interpretation of the BAEPs is difficult because of desynchronized waves.