Members only: induced systemic resistance to herbivory in a clonal plant network

The stoloniferous herb Trifolium repens was used to study the expression of induced systemic resistance (ISR) to the generalist caterpillar Spodoptera exigua in interconnected ramets of clonal fragments. The ISR was assessed as caterpillar preference in dual choice tests between control and systemically induced plants. The ISR was detected in young ramets, after inducing older sibling ramets on the same stolon by a controlled herbivore attack. However, older ramets did not receive a defense induction signal from younger ramets unless the predominant phloem flow was reversed by means of basal shading. This provides evidence for the notion that in T. repens the clone-internal expression of ISR is coupled to phloem transport and follows source–sink gradients. The inducibility of the genotypes was not linked to their constitutive ability to produce cyanide, implying the absence of a trade-off between these two defense traits. To our knowledge, this is the first study that explores ISR to herbivory in the context of physiological integration in potentially extensive clonal plant networks.     

Stability of <Emphasis Type="Italic">Potato virus X</Emphasis> expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert–deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.     

Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment.

The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimer's disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.     

Effects of centric fusions on chiasma frequency and position in Leptysma argentina (Acrididae: Orthoptera) I. Spontaneous and stable polymorphic centric fusions

Leptysma argentina is a highly polymorphic South-American grasshopper from the cytological point of view; all populations so far studied carry a polymorphic fusion between pairs 3 and 6. In heterozygotes, the trivalent 3-3/6-6 shows alternate orientation in 97.17% of the cells. Trivalent chiasma frequency is significantly lower than in the combined 3 and 6 bivalents of the standard homozygote; besides, there is a marked displacement of chiasmata to a distal position. In structural homozygotes the same effects, but not so marked, were observed.One individual was a double heterozygote for both the polymorphic centric fusion and a spontaneous one between pairs 5 and 7. The presence of a fragment, sometimes associated with the centromeric region of the nonfused 5 chromosome, was detected in more than 50% of the cells. The orientation of trivalent 5-5/7-7 in metaphase I was highly irregular (36% linear orientation). Neither frequency nor position of chiasmata were altered in trivalent 5-5/7-7 when compared with bivalents 5+7 of normal individuals.The results suggest that proximal and interstitial chiasma reduction observed in trivalent 3-3/6-6 of L. argentina is due to a later adaptation to the polymorphic condition or a fortuitous genetic condition present in the original mutant, rather than to a direct effect of the fusion itself on chiasma distribution.Fellow of the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET)     

Adaptor protein ARH is recruited to the plasma membrane by low density lipoprotein (LDL) binding and modulates endocytosis of the LDL/LDL receptor complex in hepatocytes

ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 degrees C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.