Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model

It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy.     

Rapid auxin-mediated changes in the proteome of the epidermal cells in rye coleoptiles: implications for the initiation of growth

In axial organs of juvenile plants, the phytohormone auxin (indole-3-acetic acid, IAA) rapidly mediates cell wall loosening and hence promotes turgor-driven elongation. In this study, we used rye (Secale cereale) coleoptile sections to investigate possible effects of IAA on the proteome of the cells. In a first set of experiments, we document that IAA causes organ elongation via promotion of expansion of the rigid outer wall of the outer epidermis. A quantitative comparison of the proteome (membrane-associated proteins), using two-dimensional difference gel electrophoresis (2-D DIGE), revealed that, within 2 h of auxin treatment, at least 16 protein spots were up- or down-regulated by IAA. These proteins were identified using reverse-phase liquid chromatography electrospray tandem mass spectrometry. Four of these proteins were detected in the growth-controlling outer epidermis and were further analysed. One epidermal polypeptide, a small Ras-related GTP-binding protein, was rapidly down-regulated by IAA (after 0.5 h of incubation) by -35% compared to the control. Concomitantly, a subunit of the 26S proteasome was up-regulated by IAA (+30% within 1 h). In addition, this protein displayed IAA-mediated post-translational modification. The implications of these rapid auxin effects with respect to signal transduction and IAA-mediated secretion of glycoproteins (osmiophilic nano-particles) into the growth-controlling outer epidermal wall are discussed.     

Crystal structure of a ring-cleaving cyclohexane-1,2-dione hydrolase, a novel member of the thiamine diphosphate enzyme family

The thiamine diphosphate (ThDP) dependent flavoenzyme cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) catalyses a key step of a novel anaerobic degradation pathway for alicyclic alcohols by converting cyclohexane-1,2-dione (CDO) to 6-oxohexanoate and further to adipate using NAD(+) as electron acceptor. To gain insights into the molecular basis of these reactions CDH from denitrifying anaerobe Azoarcus sp. strain 22Lin was structurally characterized at 1.26 ? resolution. Notably, the active site funnel is rearranged in an unprecedented manner providing the structural basis for the specific binding and cleavage of an alicyclic compound. Crucial features include a decreased and displaced funnel entrance, a semi-circularly shaped loop segment preceding the C-terminal arm and the attachment of the C-terminal arm to other subunits of the CDH tetramer. Its structural scaffold and the ThDP activation is related to that observed for other members of the ThDP enzyme family. The selective binding of the competitive inhibitor 2-methyl-2,4-pentane-diol (MPD) to the open funnel of CDH reveals an asymmetry of the two active sites found also in the dimer of several other ThDP dependent enzymes. The substrate binding site is characterized by polar and non-polar moieties reflected in the structures of MPD and CDO and by three prominent histidine residues (His28, His31 and His76) that most probably play a crucial role in substrate activation. The NAD(+) dependent oxidation of 6-oxohexanoate remains enigmatic as the redox-active cofactor FAD seems not to participate in catalysis, and no obvious NAD(+) binding site is found. Based on the structural data both reactions are discussed.     

Can the Geranium Bronze, <Emphasis Type="Italic">Cacyreus marshalli</Emphasis>, become a threat for European biodiversity?

Cacyreus marshalli Butler is an invasive species in many parts of Europe and Mediterranean area. In Europe, its larvae normally feed on pelargoniums. We investigated its potential to spread to native Geranium spp. and evaluated the conservation risks that such a shift would pose for both native geraniums and cohabitant butterflies. The host plant preferences of the Geranium Bronze were investigated under controlled conditions. Studies included both no-choice and multi-choice tests, respectively using 9 and 6 Italian native Geranium spp. Host plant preferences were evaluated by counting the number of eggs laid on individual plants and following butterfly development until adult emergence. Under no-choice conditions, at least one egg was recorded on each tested plant, except for G. phaeum L. All the plants on which oviposition occurred were fully suitable for larval development. The butterfly, however, clearly preferred three species, i.e. G. pratense L., G. sanguineum L. and G. sylvaticum L. for oviposition. In multi-choice trials, females laid at least one egg on all the tested plants, with a preference for G. pratense and G. sylvaticum. In presence of Pelargonium spp. plants, however, no oviposition was observed on any Geranium spp. We assessed offspring fitness measuring their wingspan. No statistical differences were detected in the wingspan between adults emerged from Geranium and Pelargonium. Cacyreus marshalli represents a potential threat for both native geraniums and for Geranium-consuming lycaenids, such as Aricia nicias Meigen and Eumedonia eumedon Esper.     

Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.     

Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation

Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.     

Structural characteristics of the plasmid-encoded toxin from enteroaggregative Escherichia coli

Intoxication by the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli requires toxin translocation from the endoplasmic reticulum (ER) to the cytosol. This event involves the quality control system of ER-associated degradation (ERAD), but the molecular details of the process are poorly characterized. For many structurally distinct AB-type toxins, ERAD-mediated translocation is triggered by the spontaneous unfolding of a thermally unstable A chain. Here we show that Pet, a non-AB toxin, engages ERAD by a different mechanism that does not involve thermal unfolding. Circular dichroism and fluorescence spectroscopy measurements demonstrated that Pet maintains most of its secondary and tertiary structural features at 37 degrees C, with significant thermal unfolding only occurring at temperatures >or=50 degrees C. Fluorescence quenching experiments detected the partial solvent exposure of Pet aromatic amino acid residues at 37 degrees C, and a cell-based assay suggested that these changes could activate an ERAD-related event known as the unfolded protein response. We also found that HEp-2 cells were resistant to Pet intoxication when incubated with glycerol, a protein stabilizer. Altogether, our data are consistent with a model in which ERAD activity is triggered by a subtle structural destabilization of Pet and the exposure of Pet hydrophobic residues at physiological temperature. This was further supported by computer modeling analysis, which identified a surface-exposed hydrophobic loop among other accessible nonpolar residues in Pet. From our data it appears that Pet can promote its ERAD-mediated translocation into the cytosol by a distinct mechanism involving partial exposure of hydrophobic residues rather than the substantial unfolding observed for certain AB toxins.     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Maternal Separation Induced Alterations of Neurogenesis in the Rat Rostral Migratory Stream

1. The aim of our study was to investigate the possibility that maternal separation, an experimental model for studies of early environmental influences, has an effect on postnatal neurogenesis in neurogenic pathway—the rostral migratory stream (RMS). 2. Rat pups were subjected to maternal separation daily for 3 h, starting from the first postnatal day (P1) till P14 or P21. In the first two groups, brains were analyzed at the age of P14 and P21, respectively. In the third group, after 3 weeks of maternal separation, 1 week of normal rearing was allowed, and the brains were analyzed at P28. The controls matched the age of maternally separated animals. Dividing cells were labeled by bromodeoxyuridine; dying cells were visualized by Fluoro-Jade C and nitric oxide (NO) producing cells by NADPH-diaphorase histochemistry. 3. Quantitative analysis of proliferating cells in the RMS showed that maternal separation decreased the number of dividing cells in all experimental groups. This decrease was most prominent in the caudal part of the RMS. The amount of dying cells was increased at the end of 3 weeks of maternal separation as well as 1 week later. The number of differentiated nitrergic cells in the RMS was increased at the end of 2 or 3 weeks of maternal separation, respectively. Besides quantitative changes, maternally separated animals showed an accelerated maturation of nitrergic cells. 4. Our results indicate that an exposure of rats to adverse environmental factors in early postnatal periods may induce acute site-specific changes in the RMS neurogenesis.     

Neutrophil adhesion to endothelial cells impairs the effects of catalase and glutathione in preventing endothelial injury

We studied the effects of the CuZn superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) on endothelial permeability to ¹²⁵I-albumin after activation of neutrophils (PMN) with phorbol 12-myristate-13-acetate (PMA; 10^?8M). PMN were either in direct contact with the endothelial cell monolayer grown on a porous gelatin-coated microporous 10-μm-thick polycarbonate filter (upright system) or separated from the endothelium by a similar filter (inverted system). Transendothelial ¹²⁵I-albumin clearance rates were measured as an index of endothelial permeability. In the absence of antioxidants, activation of PMN increased transendothelial ¹²⁵I-albumin clearnace rates in both systems from 0.041 ± 0.006 μl/min (baseline) to 0.262 ± 0.18 μl/min (upright system) and from 0.063 ± 0.02 μl/min to 0.244 ± 0.06 μl/min (inverted system). PMA induced 80–90% of PMN to adhere to either gelatin-coated filters or to endothelial cells, from the basal PMN adhesion value of 5.3 ± 2.2% and 4.3 ± 1.1%, respectively. SOD, which dismutates superoxide anion to hydrogen peroxide (H₂O₂), did not alter the transendothelial ¹²⁵I-albumin clearance rates in either systm at any concerntration from 10–300 U/ml. CAT (100–1,000 U/ml) and GSH (0.5–10 mM), which remove the H₂O₂ generated during PMN activation, did not alter the increase in transendothelial ¹²⁵I-clearance rates after PMN activation in the upright system, but both agents prvented the increase in transendothelial ¹²⁵I-clearance rates in the inverted system. We conclude that PMN activation with PMA causes endothelial injury irrespective of PMN contact to the endothelial monolayer. Moreover, H₂O₂, a release product of PMN activation, is a critical mediator of PMN-dependent endothelial injury. Finally, the results indicate that CAT and GSH prevent endothelial injury only in the absence of direct PMN contact with endothelial cells, suggesting that antioxidants such as GSH and CAT are excluded from sites of PMN-endothelial contact and thus are ineffective antioxidants. © 1993 Wiley-Liss, Inc.