Drp1 mediates caspase-independent type III cell death in normal and leukemic cells

Ligation of CD47 triggers caspase-independent programmed cell death (PCD) in normal and leukemic cells. Here, we characterize the morphological and biochemical features of this type of death and show that it displays the hallmarks of type III PCD. A molecular and biochemical approach has led us to identify a key mediator of this type of death, dynamin-related protein 1 (Drp1). CD47 ligation induces Drp1 translocation from cytosol to mitochondria, a process controlled by chymotrypsin-like serine proteases. Once in mitochondria, Drp1 provokes an impairment of the mitochondrial electron transport chain, which results in dissipation of mitochondrial transmembrane potential, reactive oxygen species generation, and a drop in ATP levels. Surprisingly, neither the activation of the most representative proapoptotic members of the Bcl-2 family, such as Bax or Bak, nor the release of apoptogenic proteins AIF (apoptosis-inducing factor), cytochrome c, endonuclease G (EndoG), Omi/HtrA2, or Smac/DIABLO from mitochondria to cytosol is observed. Responsiveness of cells to CD47 ligation increases following Drp1 overexpression, while Drp1 downregulation confers resistance to CD47-mediated death. Importantly, in B-cell chronic lymphocytic leukemia cells, mRNA levels of Drp1 strongly correlate with death sensitivity. Thus, this previously unknown mechanism controlling caspase-independent type III PCD may provide the basis for novel therapeutic approaches to overcome apoptotic avoidance in malignant cells.     

Detection of Southern tomato virus by molecular hybridisation

Southern tomato virus (STV) is a double‐stranded RNA (dsRNA) virus belonging to the genus Amalgavirus from the family Amalgamaviridae. STV has been detected in tomato plants showing symptoms of stunting, fruit discoloration and size reduction, although its role on symptom development is unclear. Also, little is known about the incidence and epidemiology of this virus and how it spreads in tomato crops. In this work, we developed a molecular hybridisation method by using a digoxigenin‐labelled RNA probe based on the nucleotide sequence of the STV putative coat protein which was tested with different procedures for preparation of plant material. This technique was sensitive enough to detect STV from sap extracts (obtained just by grinding in buffer) from different plant tissues such as leaves, fruits, roots and seeds. This procedure is suitable for field surveys since it allows a cheap and quick processing of a high number of samples. Surveys performed in three important tomato production areas (Peninsular Spain, the Canary Islands and Sicily) showed that STV is widely spread, with incidences ranging from 18% to 74% in different local and commercial tomato varieties.     

Transmission of Tomato spotted wilt virus isolates able and unable to overcome tomato or pepper resistance by its vector Frankliniella occidentalis

Tomato spotted wilt virus (TSWV) causes serious diseases of many economically important crops. Disease control has been achieved by breeding tomato and pepper cultivars with the resistance genes Sw‐5 and Tsw, respectively. However, TSWV isolates overcoming these genetic resistances have appeared in several countries. To evaluate the risk of spread of these resistance‐breaking isolates, we tested their ability of transmission by the main vector of TSWV, the thrips Frankliniella occidentalis. We compared the transmission rate by thrips of six TSWV isolates of different biotype (able or unable to overcome this resistance in pepper and tomato), and with divergent genotype (A and B). Our results indicate that the transmission rate was related to the amount of virus accumulated in thrips but not to virus accumulation in the source plants on which thrips acquired the virus. No correlation was found between transmission efficiency by thrips and the genotype or between transmission efficiency and the ability of overcoming both resistances. This result suggests that resistance‐breaking isolates have the same potential to be transmitted as the isolates unable to infect resistant tomato and pepper cultivars.     

Donepezil-tacrine hybrid related derivatives as new dual binding site inhibitors of AChE

A new series of donepezil–tacrine hybrid related derivatives have been synthesised as dual acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. These new hybrids combined a tacrine, 6-chlorotacrine or acridine unit as catalytic binding site and indanone (the heterocycle present in donepezil) or phthalimide moiety as peripheral binding site of the enzyme, connected through a different linker tether length. One of the synthesised compounds emerged as a potent and selective AChE inhibitor, which is able to displace propidium in a competition assay. These results seem to confirm the ability of this inhibitor to bind simultaneously to both sites of the enzyme and make it a promising lead for developing disease-modifying drugs for the future treatment of Alzheimer’s disease. To gain insight into the molecular determinants that modulate the inhibitory activity of these compounds, a molecular modelling study was performed to explore their binding to the enzyme.     

High-affinity potassium and sodium transport systems in plants

All living cells have an absolute requirement for K+, which must be taken up from the external medium. In contrast to marine organisms, which live in a medium with an inexhaustible supply of K+, terrestrial life evolved in oligotrophic environments where the low supply of K+ limited the growth of colonizing plants. In these limiting conditions Na+ could substitute for K+ in some cellular functions, but in others it is toxic. In the vacuole, Na+ is not toxic and can undertake osmotic functions, reducing the total K+ requirements and improving growth when the lack of K+ is a limiting factor. Because of these physiological requirements, the terrestrial life of plants depends on high-affinity K+ uptake systems and benefits from high-affinity Na+ uptake systems. In plants, both systems have received extensive attention during recent years and a clear insight of their functions is emerging. Some plant HAK transporters mediate high-affinity K+ uptake in yeast, mimicking K+ uptake in roots, while other members of the same family may be K+ transporters in the tonoplast. In parallel with the HAK transporters, some HKT transporters mediate high-affinity Na+ uptake without cotransporting K+. HKT transporters have two functions: (i) to take up Na+ from the soil solution to reduce K+ requirements when K+ is a limiting factor, and (ii) to reduce Na+ accumulation in leaves by both removing Na+ from the xylem sap and loading Na+ into the phloem sap.     

A method for the quantitation of intestinal metaplasia of the stomach by morphometry

A novel method to quantitate the extent of intestinal metaplasia in gastrectomy specimens is presented. Morphometric measurements of histochemically labeled mucin-producing goblet cells were done in three selected gastrectomies (one having a peptic ulcer, one with adenocarcinoma of the intestinal type, and one with adenocarcinoma of the diffuse type). The sectioning of the gastrectomy specimens was standardized. The results indicated that the intestinal metaplasia was significantly higher in the specimen with adenocarcinoma of the intestinal type (as compared to the other two specimens), both along the lesser and greater curvatures as well as in the fundic area. These quantitative results confirm nonquantitative reports based on subjective visual impressions. This quantitative histochemical method for measuring the actual length as well as the topographical distribution of intestinal metaplasia in resected stomachs will be used in future studies of intestinal metaplasia with the aim of disclosing possible differences among populations with disparate incidences of gastric carcinoma.     

Modulation of K+ translocation by AKT1 and AtHAK5 in Arabidopsis plants

Root cells take up K⁺ from the soil solution, and a fraction of the absorbed K⁺ is translocated to the shoot after being loaded into xylem vessels. K⁺ uptake and translocation are spatially separated processes. K⁺ uptake occurs in the cortex and epidermis whereas K⁺ translocation starts at the stele. Both uptake and translocation processes are expected to be linked, but the connection between them is not well characterized. Here, we studied K⁺ uptake and translocation using Rb⁺ as a tracer in wild‐type Arabidopsis thaliana and in T‐DNA insertion mutants in the K⁺ uptake or translocation systems. The relative amount of translocated Rb⁺ to the shoot was positively correlated with net Rb⁺ uptake rates, and the akt1 athak5 T‐DNA mutant plants were more efficient in their allocation of Rb⁺ to shoots. Moreover, a mutation of SKOR and a reduced plant transpiration prevented the full upregulation of AtHAK5 gene expression and Rb⁺ uptake in K⁺‐starved plants. Lastly, Rb⁺ was found to be retrieved from root xylem vessels, with AKT1 playing a significant role in K⁺‐sufficient plants. Overall, our results suggest that K⁺ uptake and translocation are tightly coordinated via signals that regulate the expression of K⁺ transport systems.     

Phylogenetic analysis in the genus Cicer and cultivated chickpea using RAPD and ISSR markers

Seventy five accessions belonging to 14 species of the genus Cicer were analysed with PCR-based molecular markers to determine their phylogenetic relationships. Eight of the species were annuals and included the Section Monocicer which contains cultivated chickpea (Cicer arietinum L.). The remaining six species were perennials (five from Section Polycicer and one from Section Acanthocicer). More than one accession per species was analysed in most of the wild species; within C. arietinum, 26 accessions including Kabuli and Desi types, were studied. RAPD analyses using 12 primers gave 234 polymorphic fragments. Variability within species was detected. A dendrogram based on the Jaccard similarity index showed that the distribution pattern of variability between species was related to both growth habit and geographical origin. An accession of Cicer reticulatum was closer to accessions of Cicer echinospermum than to the four remaining of C. reticulatum, suggesting the possibility of gene flow between species. Cluster analysis for cultivated chickpea differentiated Kabuli and Desi types but we did not detect a clear relationship between groups and the geographical origin of the accessions. Received: 5 April 2001 / Accepted: 13 July 2001     

Assessment of Environmental Hazards to Public Health in Temperate Urban Argentina

EcoHealth - Human health risk in urban areas depends on multiple environmental features. We performed a year-round survey in a highly urbanized district located in temperate Argentina (General San...     

CD47 engagement inhibits cytokine production and maturation of human dendritic cells

Upon encounter with bacterial products, immature dendritic cells (iDCs) release proinflammatory cytokines and develop into highly stimulatory mature DCs. In the present study, we show that human monocyte-derived DCs functionally express the CD47 Ag, a thrombospondin receptor. Intact or F(ab')2 of CD47 mAb suppress bacteria-induced production of IL-12, TNF-alpha, GM-CSF, and IL-6 by iDCs. 4N1K, a peptide derived from the CD47-binding site of thrombospondin, also inhibits cytokine release. The inhibition of IL-12 and TNF-alpha is IL-10-independent inasmuch as IL-10 production is down-modulated by CD47 mAb and blocking IL-10 mAb fails to restore cytokine levels. CD47 ligation counteracts the phenotypic and functional maturation of iDCs in that it prevents the up-regulation of costimulatory molecules, the loss of endocytic activity, and the acquisition of an increased capacity to stimulate T cell proliferation and IFN-gamma production. Interestingly, regardless of CD47 mAb treatment during DC maturation, mature DC restimulated by soluble CD40 ligand and IFN-gamma, to mimic DC/T interaction, produce less IL-12 and more IL-18 than iDCs. Finally, CD47 ligation on iDCs does not impair their capacity to phagocytose apoptotic cells. We conclude that following exposure to microorganisms, CD47 ligation may limit the intensity and duration of the inflammatory response by preventing inflammatory cytokine production by iDCs and favoring their maintenance in an immature state.