Rearrangements of large-insert T-DNAs in transgenic rice

Introduction of large-DNA fragments into cereals by Agrobacterium-mediated transformation is a useful technique for map-based cloning and molecular breeding. However, little is known about the organization and stability of large fragments of foreign DNA introduced into plant genomes. In this study, we produced transgenic rice plants by Agrobacterium-mediated transformation with a large-insert T-DNA containing a 92-kb region of the wheat genome. The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers (fiber FISH). By using this cytogenetic technique, we showed that rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. Deletion of long stretches of the large-insert DNA was also observed in Agrobacterium.     

Reduction mechanism of L-cysteine on keratin fibers using microspectrophotometry and Raman spectroscopy

In order to investigate the reduction mechanism of L-cysteine (Cys) on keratin fibers, cross-sectional samples of virgin white human hair treated with Cys were prepared. The heterogeneous reaction between Cys and keratin fibers involving the diffusion of Cys into human hair was analyzed at the molecular level using microspectrophotometry and Raman spectroscopy. The diffusion pattern of Cys into human hair showed non-Fickian type characteristics, thus indicating the free amino groups of electrostatically interacted with the anionic ions of the fiber surface. The disconnected relative concentration of -SS- groups at various depths of the hair samples with pH 9.0 was less than the Cys relative concentration, indicating that the reaction rate (the disconnection of -SS- groups) was slower than the diffusion rate of Cys into human hair. From these experiments, we concluded that the free amino groups of Cys electrostatically interacted with the anionic ions of the fiber surface, thereby decreasing the reaction rate (the disconnection of -SS- groups) of Cys at pH 9.0.     

RhoA inactivation is crucial to manganese-induced astrocyte stellation

Actin depolymerization through Rho GTPases or exogenous mechanical tension has been suggested as a key determinant for the formation of astrocyte stellation. Rho GTPases function as switching molecules to converge both extracellular and intracellular signals in regulation of cytoskeletal organization. Their involvement in manganese-induced astrocyte stellation was assessed. The disruption of cytoskeletal architecture by manganese indicated the decreased activity of RhoA. Pharmacological and biochemical approaches revealed the inactivation of RhoA by manganese. This inactivation was partly through the down-regulation of guanine nucleotide exchange factor phosphorylation. Furthermore, the dephosphorylation of myosin light chain and cofilin through the inactivated RhoA effectors synergistically destabilized actin stress fibers. We conclude that manganese regulates cytoskeletal organization in astrocytes by modulating the activity of p115RhoGEF and RhoA.     

DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA

In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.     

A test of adaptive hypotheses: Mandibular traits,nest construction materials,and feeding habits in neotropical social wasps (Vespidae,Polistinae)

Summary. The success of many behaviors in hymenoptera depends on the shape and structure of their mandibles. Neotropical social wasps exhibit variation in the shape of their mandibles; both the type of material used in the nest construction and the food sources have been proposed as selective forces that explain that variation. These hypotheses were studied using Independent Contrast analyses and combined tests of significance between: nine mandibles traits, the type of nest material and food source types. Necrophagy and short fiber use are derived conditions in the subfamily that have evolved five times each, while the use of long fibers and live prey are primitive conditions. The mandible structures appear related to the nest fiber type but not to necrophagy. Particularly, species that use long fibers have bigger internal dorsal tooth, longer internal ventral tooth, and shorter and more curved mandibles. Similar results were obtained by including plant hairs as a third nest material type. These findings open related questions for groups of social insects where studies on evolution of mandibles have emphasized food sources.Received 23 November 2003; revised 9 March 2004; accepted 21 April 2004.     

Plant fiber intrusive growth characterized by NMR method

Intrusively growing plant cells insert themselves between surrounding cells, thus increasing the number of membranes on the tissue cross-section. This parameter can be assessed by spin echo NMR method with a magnetic field pulse gradient. Diffusion echo decay was measured for stem regions of long-fiber flax (Linum usitatissimum L.) differing in the stages of primary fiber development, which elongate thousand-fold during intrusive growth. Additionally, the number of fibers on stem cross-sections was counted under microscope. An increase in the slow component of the echo diffusion decay was correlated with an increase in the number of fibers on the stem cross-section in the zone of intrusive growth, while other stem-structure characteristics remained unchanged. Thus, NMR method can be used for characterization of intrusive fiber growth in situ.     

Presence and co-localization of vasoactive intestinal polypeptide with neuronal nitric oxide synthase in cells and nerve fibers within guinea pig intrinsic cardiac ganglia and cardiac tissue

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VIP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia. This work was supported by NIH grant HL65481 (R.L.P.) and HL54633 (D.B.H.). Use of the DeltaVision Restoration microscope was provided through the Imaging/Physiology Core supported by NIH Grant P20 RR16435 from the COBRE program of the National Center for Research Resources     

GABAergic system of the pineal organ of an elasmobranch (Scyliorhinus canicula): a developmental immunocytochemical study

The present immunocytochemical study provides evidence of a previously unrecognized, rich, γ-aminobutyric acid (GABA)-ergic innervation of the pineal organ in the dogfish (Scyliorhinus canicula). In this elasmobranch, the pineal primordium is initially detected at embryonic stage 24 and grows to form a long pineal tube by stage 28. Glutamic acid decarboxylase (GAD)-immunoreactive (-ir) fibers were first observed at stage 26, and by stage 28, thin GAD-ir fibers were detectable at the base of the pineal neuroepithelium. In pre-hatchling embryos, most fibers gave rise to GAD-ir boutons that were localized in the basal region of the neuroepithelium, although a smaller number of labeled terminals ascended to the pineal lumen. A few pale GAD-ir perikarya were observed within the pineal organ of stage 29 embryos, but GAD-ir perikarya were not observed at other developing stages or in adults. In contrast, GABA immunocytochemistry revealed the presence of GABAergic perikarya and fibers in the pineal organ of late stage embryos and adults. Although high densities of GABAergic cells were observed in the paracommissural pretectum, posterior tubercle, and tegmentum of dogfish embryos (regions previously demonstrated to contain pinealopetal cells), the presence of GABA-ir perikarya in the pineal organ strongly suggests that the rich GABAergic innervation of the elasmobranch pineal organ is intrinsic. This contrasts with the central origin of GABAergic fibers in the pineal gland of some mammals. This work was supported by the Spanish Education and Science Ministry and FEDER (BXX2000-0453-C02 and BFU2004-03313/BF1), the Xunta de Galicia (PGIDT99BIO20002), and NIH/NIDCD awards R01 DC01705 and P01 DC01837 (to G.R.H.).     

Activation of latent efferent sympathetic fibers in a viscero-visceral reflex circle under conditions of visceral pain in rats

Activation of “silent” efferent fibers due to stimulation of the mesenteric nerve within a definite frequency range is described; the effect is supposed to result from sensitization in reflex circles related to visceral pain. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 368–369, July–August, 2006.     

Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers

An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.