Characterization of the interactions between Cucumber mosaic virus and Potato virus Y in mixed infections in tomato

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-FnyΔ2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVY-SON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.     

Individual and combined effects of Bacillus thuringiensis var. israelensis, temephos and Leptolegniachapmanii on the larval mortality of Aedes aegypti

Larvicidal effects of interaction between Bacillus thuringiensis var. israelensis (Bti), temephos and Leptolegnia chapmanii zoospores on larvae of Aedes aegypti were determined under laboratory and seminatural conditions. In laboratory bioassays, two concentrations of Bti (0.012, 0.027 ppm), two of temephos (0.00035, 0.001 ppm), and a single concentration of L. chapmanii zoospores (6.1 × 10⁴ zoospores ml⁻¹) were evaluated. Trials under field-like conditions were performed in a single container and then placed either in the shade or in direct exposure to sunlight. We evaluated concentrations of Bti and temephos at 3-fold those normally used in laboratory tests: 0.09 and 0.003 ppm, respectively, plus 1.8 × 10⁵ zoospores ml⁻¹ of L. chapmanii. The combined effect of sublethal concentrations of Bti, temephos, and L. chapmanii zoospores thus indicated that this fungus is not inhibited by the larvicides and also demonstrated the synergistic effect of the action of L. chapmanii when used together with Bti and temephos.     

Role of α7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP^C) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP^C extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrP^C-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP^C engagement induces an increase in intracellular Ca²⁺ levels. This effect was not detected in PrP^C-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP^C. Using a best candidate approach to test for potential channels involved in Ca²⁺ influx evoked by STI1-PrP^C, we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrP^C-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP^C and allowed reconstitution of signaling by PrP^C-STI1 interaction. These results indicate that STI1 can interact with the PrP^C·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.     

Tn502 and Tn512 Are res Site Hunters That Provide Evidence of Resolvase-Independent Transposition to Random Sites

In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502 and Tn512, which are members of the Tn5053 family. These transposons exhibit targeted and oriented insertion in the par region of plasmid RP1, since par-encoded components, namely, the ParA resolvase and its cognate res region, are essential for such transposition. Tn502 and, under some circumstances, Tn512 can transpose when par is absent, providing evidence for an alternative, par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of the res-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502 and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that the par-independent pathway enables the formation of deletions in the target molecule.It is widely recognized that mobile genetic elements contribute to genome plasticity and have been a driving force in the emergence and spread of resistance determinants within and between bacterial species; their impact is ongoing (10, 51). Significant among these elements are various classes of plasmids, transposons, and integrons which may lack resistance determinants or carry one or multiple determinants. Resistance determinants that have become globally dispersed in environmental and clinically significant bacteria include mercury(II) resistance (2, 17), evident even in ancient bacteria (27), and antibiotic resistance, which has increased in dominance since the advent of the antibiotic era (23, 40).This paper concerns the mercury resistance (mer) transposons Tn502 and Tn512, whose sequence organization and transpositional behavior show that they are new members of a family of elements exemplified by the mer transposon Tn5053 (22). These elements are closely related to those in the Tn402 family, which contain an integron (intI) recombination system (14, 36). Members of the two families differ in the positions of the mer or intI determinants (modules) near one end of the transposition (tni) module. The latter module contains four genes (tniABQR), and the entire transposon is bounded by 25-bp inverted-repeat termini (IRi and IRt). TniA, TniB, and TniQ are required to form the transpositional cointegrate, which is then resolved by the action of TniR (a serine resolvase) on a resolution (res) sequence located between tniR and tniQ (22). The transposon in its new location is flanked by 5-bp direct repeats (DRs) (20, 22). TniA, which contains a D,D(35)E transposase catalytic motif, is thought to function cooperatively with TniB, a putative nucleotide-binding protein, as the active TniAB transposase (21, 36). Studies of TniA conducted in vitro show binding to the IRs and to additional 19-bp repeat sequences that make up the complex termini of the transposon (21). The precise role of TniQ is unknown.An unexpected and unique feature of Tn5053 and Tn402 is that they depend on externally coded accessory functions for efficient transposition, namely, a res site served by a cognate resolvase (25). As a consequence, these transposons exhibit a strong transpositional bias for some target res sites (20, 25, 26) and have aptly been described as “res site hunters” (25). One such efficient interaction involves the res-ParA multimer resolution system of plasmid RP1 (IncPα); other plasmid- or transposon-encoded systems are less efficient or are refractory. Although the role of the external resolvase remains obscure, its capacity to bind to its cognate res is an essential requirement whereas its catalytic activity is not (20). For each interaction system, the target sites typically cluster in a single part of res but not necessarily within the same subregion and, on occasion, can lie in the vicinity of res. Typically, the transposon is in a single orientation with IRi closest to the resolvase gene. In one study, Tn402 clustered at two target sites, one within res and one nearby, and the orientations were different at the two sites (20).The experimentally observed target preference described above also occurs in natural associations of Tn5053/Tn402-like elements and became evident on sequencing class 1 integrons, which were often found positioned close to different res-resolvase gene regions (6, 20, 25). Most Tn402 family elements are comprised of an intI module that is flanked on the left by IRi and on the right by a 3′ conserved sequence (3′-CS) (13). In others, a remnant tni gene cluster may be present instead of the 3′-CS, and IRt occurs at the right flank. The structure of the latter category of integrons strongly indicated that they are defective transposons that were presumably capable of relocation provided that tni functions were supplied in trans (6, 32). The movement of In33 (Tn2521) from a chromosomal to a plasmid location appears to have been such an in trans event (30, 42), and others involving In0 and In2 are demonstrated in this study. In contrast, the integrons that lack the IRt end appear to be nonmobile remnants of Tn402-like transposons; they belong to several lineages, including those in which the incurred deletions are attributable to acquired insertion sequences (6). More recently, intact Tn5053/Tn402-like transposons and class 1 integrons have increasingly been detected in the res-parA region of IncP plasmids (39), which are arguably the most promiscuous of known plasmids (50). These various experimental and natural interactions provide insight into the dispersal pathways possible for Tn5053/Tn402-like elements.The res-hunting attribute is a striking feature that is experimentally supported by studies of four family members (namely, Tn5053 [22, 25], Tn402 [20, 26], and in this study, Tn502 [48] and Tn512). Another facet of the transposition of Tn502 is explored here. It concerns the observation that loss of the preferred par target region in RP1 does not abolish transposition of Tn502 (48), contrary to the finding with Tn5053 (25, 26) and, in this study, Tn512. The continued, low-frequency transposition of Tn502 involved at least three dispersed locations (48); however, nothing is known about the nature of these sites or about the features and requirements of the transposition process. Here we address these issues and uncover the existence of an alternative, par-independent pathway that is employed by Tn502 and is available to Tn512 under some circumstances. The study also provides information on the roles of the TniR and host (RecA) recombination systems in the resolution of transpositional cointegrates and on the ability of the par-independent transposition pathway to generate plasmid deletions.     

Trypanosoma cruzi: partial characterization of minor cruzipain isoforms non-adsorbed to Concanavalin A-Sepharose

The present paper reports the partial characterization of a subset of atypical cruzipain molecules which do not bind to Concanavalin A-Sepharose column. They are present in different strains of epimastigote forms of Trypanosoma cruzi and represent a 2-4% of total cruzipain. They were purified by affinity chromatography on Cystatin-Sepharose, recognized by the polyclonal anti-cruzipain serum, and their activity in gelatin-containing gels was completely abolished by E-64, TLCK, leupeptin, and aprotinin but not by PMSF, pepstatin A, EDTA or 1,10-phenantroline. These cysteine proteinases, as well as cruzipain showed to be endoproteinases able to hydrolize azocasein, hemoglobin, and bovine serum albumin at acidic pHs. However, evidences are presented indicating that this subset of cruzipain isoforms were also able to use the same blocked chromogenic peptidyl substrates than cruzipain at similar optimal alkaline pH values although with a different order of preference. Moreover, they showed a different oligosaccharide pattern after enzymatic treatment by high pH anion exchange chromatography, suggesting that this structural difference may account for the atypical behaviour in the lectin column.     

Degeneration of primary afferent terminals following brachial plexus extensive avulsion injury in rats

Important breakthroughs in the understanding regeneration failure in an injured CNS have been made by studies of primary afferent neurons. Dorsal rhizotomy has provided an experimental model of brachial plexus (BP) avulsion. This is an injury in which the central branches of primary afferents are disrupted at their point of entry into the spinal cord, bringing motor and sensory dysfunction to the upper limbs. In the present work, the central axonal organization of primary afferents was examined in control (without lesion) adult Wistar rats and in rats subjected to a C3-T3 rhizotomy. Specific sensory axon subtypes were recognized by application of antibodies to the calcitonin gene-related peptide (CGRP), the P2X3 purinoreceptor, the low-affinity p75-neurotrophin receptor and the retrograde tracer cholera toxin subunit beta (TCbeta). Other subtypes weres labeled with the lectin Griffonia simplicifolia 1B4. Using immunohistochemistry and high resolution light microscopy, brachial plexus rhizotomy in adult rats has proven a reliable model for several neural deficits in humans. This lesion produced different degrees of terminal degeneration in the several types of primary afferents which define sub-populations of sensitive neurons. Between the C6 and C8 levels of the spinal cord,, deafferentation was partial for peptidergic GCRP-positive fibers, in contrast with elimination of non peptidergic and myelinated fibers. Dorsal rhizotomy has provided an adequate experimental model to study sensory alterations such as acute pain and allodynia as well as factors that affect regeneration into the CNS., Therefore, the differential deafferentation response must be considered inr the evaluation of therapies for nociception (pain) and regeneration for brachial plexus avulsion. The anatomical diffierences among the primary afferent subtypes also affect their roles in normal and damaged conditions.     

Cellular prion protein: implications in seizures and epilepsy

1. Cellular prion (PrP^c) is a plasma membrane protein involved with copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling, and survival in the central nervous system2. Deletion of PrP^c gene (Prnp) in mice enhances sensitivity to seizures in vivo and neuronal excitability in vitro which can be related to: (i) disrupted Ca⁺²-activated K⁺ currents, with loss of I _HAP conductance in hippocampus; (ii) abnormal GABA-A inhibition in the hippocampus; (iii) mossy fiber reorganization in the hippocampus; (iv) changes in ectonucleotidases in both hippocampus and neocortex; and (v) higher levels of neocortical and subcortical oxidative stress. Moreover, postnatal Prnp knockout mice showed a significant reduction of after hyperpolarization potentials in hippocampal CA1 cells.3. Taken together, these findings suggest that loss of PrP^c function contributes to the hyperexcitable and synchronized activities underlying epileptic seizures generated in neocortex and hippocampus. Hence, the role of PrP^c on human symptomatic, cryptogenic or idiopathic epileptic syndromes deserves further investigation.     

Some factors affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells

Adhesion to the intestinal mucosa is generally considered an important property of probiotic microorganisms and has been related to many of their health benefits. This study investigated some factors that could affect or be involved in the adherence of Propionibacterium acidipropionici CRL 1198, a dairy strain with suggested probiotic effects and high adherence in vitro and in vivo to intestinal epithelial cells. In vitro adhesion of propionibacteria was decreased by gastric digestion but not affected by bile and pancreatic enzymes. Adherence was also decreased by pretreatment of bacterial cells with protease, sodium metaperiodate, and trichloroacetic acid, revealing that different features of the cell surface, like protein factors, carbohydrates, and teichoic acids, are involved in the process. Adherence to intestinal epithelial cells was enhanced by calcium and was dependent on other divalent cations. Adhesion to intestinal mucus was also demonstrated. The results should explain the metabolic effects in the host previously obtained with this strain and support the potential of Propionibacterium for development of new probiotics.