PAMAM dendrimeric conjugates with a Gd-DOTA phosphinate derivative and their adducts with polyaminoacids: the interplay of global motion, internal rotation, and fast water exchange

A series of dendrimeric conjugates based on a PAMAM (polyamidoamine) backbone with macrocyclic Gd-DO3A-P(ABn) complexes (monophosphinated analogue of DOTA) was prepared. The chelates were covalently attached to the G1-, G2-, and G4-PAMAM dendrimers through a thiourea linker in high loads (>90%). The prepared conjugates G1-(Gd-DO3A-P(BnN{CS}))(8), G2-(Gd-DO3A-P(BnN{CS}))(16), and G4-(Gd-DO3A-P(BnN{CS}))(59) showed relaxivities of 10.1, 14.1, and 18.6 s(-)(1) mM(-)(1) at 20 MHz and 37 degrees C and pH = 7.5, respectively. A variable-pH study (range 2-12) revealed up to 30% increase in the relaxivity at low pH for the G2-(Gd-DO3A-P(BnN{CS}))(16) conjugate. As confirmed by (1)H NMR titration of the unmodified G2 dendrimer, this is due to protonation of core tertiary amines leading to a more open and rigid structure. The variable-temperature (17)O NMR and (1)H NMRD relaxometric studies confirmed that the relaxivity is not controlled by water exchange but by rotational dynamics. A multiparametrical data evaluation using the Lipari-Szabo approach revealed that the water residence lifetime, (298)tau(M), for the conjugates studied was ca. 45-70 ns, which is longer than the value found for the monomeric model compound Gd-DO3A-P(ABn) (16 ns) but short enough so as not to limit the relaxivity. The global rotational correlation time, (298)tau(Rg), varied from 1.5 to 3.1 ns and seemed to indicate a sufficiently slow molecular tumbling to achieve the high relaxivities measured; however, the rigidity factor S(2) (approximately 0.26), describing the internal flexibility, was far from optimum. The overall relaxivity was significantly increased (e.g. by a factor of 1.8 for the G1-(Gd-DO3A-P(BnN{CS}))(8) conjugate) when a positively charged polyaminoacid like poly(Arg) or poly(Lys) was added to the conjugate solutions. The electrostatic interactions partially "freeze" the internal mobility of the conjugate and also slow down global motion. This assumption was confirmed by an evaluation of (1)H relaxometric data obtained for the G2-(Gd-DO3A-P(BnN{CS}))(16)-poly(Lys)(59) adduct. Importantly, it was proved that the adduct formation did not hamper the water exchange process.     

FRASS: the web-server for RNA structural comparison

Background  

The impressive increase of novel RNA structures, during the past few years, demands automated methods for structure comparison. While many algorithms handle only small motifs, few techniques, developed in recent years, (ARTS, DIAL, SARA, SARSA, and LaJolla) are available for the structural comparison of large and intact RNA molecules.     

Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices

The arbuscular mycorrhiza association results from a successful interaction between genomes of the plant and fungal symbiotic partners. In this study, we analyzed the effect of inactivation of late-stage symbiosis-related pea genes on symbiosis-associated fungal and plant molecular responses in order to gain insight into their role in the functional mycorrhizal association. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated wild-type and isogenic genotypes of pea mutated for the PsSym36, PsSym33, and PsSym40 genes where arbuscule formation is inhibited or fungal turnover modulated, respectively. Microdissection was used to corroborate arbuscule-related fungal gene expression. Molecular responses varied between pea genotypes and with fungal development. Most of the fungal genes were downregulated when arbuscule formation was defective, and several were upregulated with more rapid fungal development. Some of the plant genes were also affected by inactivation of the PsSym36, PsSym33, and PsSym40 loci, but in a more time-dependent way during root colonization by G. intraradices. Results indicate a role of the late-stage symbiosis-related pea genes not only in mycorrhiza development but also in the symbiotic functioning of arbuscule-containing cells.     

Genetic and physical mapping of new EST-derived SSRs on the A and B genome chromosomes of wheat

The availability of genetic maps and phenotypic data of segregating populations allows to localize and map agronomically important genes, and to identify closely associated molecular markers to be used in marker-assisted selection and positional cloning. The objective of the present work was to develop a durum wheat intervarietal genetic and physical map based on genomic microsatellite or genomic simple sequence repeats (gSSR) markers and expressed sequence tag (EST)-derived microsatellite (EST-SSR) markers. A set of 122 new EST-SSR loci amplified by 100 primer pairs was genetically mapped on the wheat A and B genome chromosomes. The whole map also comprises 149 gSSR markers amplified by 120 primer pairs used as anchor chromosome loci, two morphological markers (Black colour, Bla1, and spike glaucousness, Ws) and two seed storage protein loci (Gli-A2 and Gli-B2). The majority of SSR markers tested (182) was chromosome-specific. Out of 275 loci 241 loci assembled in 25 linkage groups assigned to the chromosomes of the A and B genome and 34 remained unlinked. A higher percentage of markers (54.4%), localized on the B genome chromosomes, in comparison to 45.6% distributed on the A genome. The whole map covered 1,605 cM. The B genome accounted for 852.2 cM of genetic distance; the A genome basic map spanned 753.1 cM with a minimum length of 46.6 cM for chromosome 5A and a maximum of 156.2 cM for chromosome 3A and an average value of 114.5 cM. The primer sets that amplified two or more loci mapped to homoeologous as well as to non-homoeologous sites. Out of 241 genetically mapped loci 213 (88.4%) were physically mapped by using the nulli-tetrasomic, ditelosomic and a stock of 58 deletion lines dividing the A and B genome chromosomes in 94 bins. No discrepancies concerning marker order were observed but the cytogenetic maps revealed in some cases small genetic distance covered large physical regions. Putative function for mapped SSRs were assigned by searching against GenBank nonredundant database using TBLASTX algorithms.     

Towards improved boron neutron capture therapy agents: evaluation of in vitro cellular uptake of a glutamine-functionalized carborane

Abstract  Sodium borocaptate (BSH) is widely used for boron neutron capture therapy (BNCT) of brain tumors. One drawback is the large uptake by the liver causing a decrease of its availability at the tumor region as well as bringing about toxicity problems. A novel carborane-based compound containing a boron payload very similar to that of BSH has been synthesized and tested on rat glioma (C6) cells, hepatoma tissue culture (HTC) cells, and hepatocytes. The newly synthesized system consists of an o-carborane unit (C₂B₁₀H₁₁, o-CB) conjugated to a glutamine residue through a proper spacer, namely, o-CB-Gln. As compared with BSH, it showed the same uptake by C6 cells, but a 50% decrease in uptake by HTC cells and an 80% decrease in uptake by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to that of BSH accompanied by a much lower liver toxicity. Graphical Abstract  A novel carborane based compound, consisting in an o-carborane unit (C2B10H11, o-CB) conjugated to a glutamine residue through a proper spacer (namely o-CB-Gln) has been synthesized, characterized and tested on rat glioma (C6), hepatoma (HTC) and hepatocytes. As compared to sodium borocaptate (BSH), widely used for boron neutron capture therapy (BNCT) of brain tumors, the newly synthesized system showed the same uptake by C6 cells, but a 50% decrease by HTC and 80% decrease by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to BSH accompanied by a much lower liver toxicity.      

Semaphorin 3E Initiates Antiangiogenic Signaling through Plexin D1 by Regulating Arf6 and R-Ras

Recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, to provide a repulsive cue for plexin D1-expressing endothelial cells, thus providing a highly conserved and developmentally regulated signaling system guiding the growth of blood vessels. We show here that Sema3E acts as a potent inhibitor of adult and tumor-induced angiogenesis. Activation of plexin D1 by Sema3E causes the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix (ECM) and causing the retraction of filopodia in endothelial tip cells. Sema3E acts on plexin D1 to initiate a two-pronged mechanism involving R-Ras inactivation and Arf6 stimulation, which affect the status of activation of integrins and their intracellular trafficking, respectively. Ultimately, our present study provides a molecular framework for antiangiogenesis signaling, thus impinging on a myriad of pathological conditions that are characterized by aberrant increase in neovessel formation, including cancer.Pathological angiogenesis characterizes numerous human diseases, ranging from chronic inflammation, atherosclerosis, diabetic retinopathy, and age-related macular degeneration to cancer (5, 11, 30). Thus, elucidating the mechanisms underlying normal and aberrant blood vessel growth may provide new therapeutic options for many highly prevalent disease conditions. Ultimately, normal angiogenesis results from a precise balance between pro- and antiangiogenic mediators. Among the former, the family of vascular endothelial growth factors (VEGFs), basic fibroblastic growth factor (bFGF), sphingosine-1-phosphate (S1P), and the chemokines interleukin-8/CXCL8 and SDF-1/CXCL12 and their receptors are some of the most widely investigated (reviewed in references 3, 5, 8, and 17). The best-known angiogenesis inhibitors are proteolytic cleavage products of extracellular matrix (ECM) or serum components, such as endostatin, angiostatin, arresten, and tumstatin (reviewed in references 11 and 20). Antiangiogenic cytokines have also been described, including interferons and certain interleukins, which appear to act indirectly by limiting the expression of proangiogenic mediators or inducing antiangiogenic molecules (reviewed in references 11 and 20). In contrast, there are few known developmentally regulated, naturally occurring antiangiogenic molecules, which include platelet factor 4 (18), thrombospondin 1 (14), and pigment epithelium-derived factor (PEDF) (9). Their precise mechanism of action is not fully understood, thus limiting the ability to design new molecularly based antiangiogenic strategies.Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of the netrin, slit, eph, and semaphorin families, also play a critical role in blood vessel guidance during physiological and pathological blood vessel development (6). For example, multiple secreted class III semaphorins, which regulate developmental axonal growth (23, 27), are now known to act through their receptors, the A family plexins (plexins A1, A2, and A3), and their coreceptors, neuropilin 1 and neuropilin 2, to initiate pro- and antiangiogenic responses (reviewed in references 6 and 19). However, neuropilins also act as coreceptors for multiple angiogenic factors, such as VEGF, thus limiting our ability to distinguish the downstream signaling events initiated by semaphorins from those resulting from their interplay with endothelial growth and motility factors (19). In this regard, recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, independently of neuropilins to control endothelial cell (EC) positioning and patterning of the developing vasculature (13, 15). These findings prompted us to explore whether Sema3E acts as a natural antiangiogenic molecule and, if so, to investigate the underlying molecular mechanism. Indeed, we show here that Sema3E is a potent inhibitor of adult and tumor-induced angiogenesis. Sema3E causes filopodial retraction in endothelial tip cells and inhibits endothelial cell adhesion by disrupting integrin-mediated adhesive structures. At the molecular level, this process involves the stimulation of plexin D1 by Sema3E, which in turn interferes with R-Ras function and leads to the rapid activation of Arf6, thus revealing a novel physiological antiangiogenic signaling route.     

Cytogenetic mapping of a major locus for resistance to Fusarium head blight and crown rot of wheat on <Emphasis Type="Italic">Thinopyrum elongatum</Emphasis> 7EL and its pyramiding with valuable genes from a <Emphasis Type="Italic">Th. ponticum</Emphasis> homoeologous arm onto bread wheat 7DL

Key message

A major locus for resistance to different Fusarium diseases was mapped to the most distal end of Th. elongatum 7EL and pyramided with Th. ponticum beneficial genes onto wheat 7DL.

Abstract

Perennial Triticeae species of the Thinopyrum genus are among the richest sources of valuable genes/QTL for wheat improvement. One notable and yet unexploited attribute is the exceptionally effective resistance to a major wheat disease worldwide, Fusarium head blight, associated with the long arm of Thinopyrum elongatum chromosome 7E (7EL). We targeted the transfer of the temporarily designated Fhb-7EL locus into bread wheat, pyramiding it with a Th. ponticum 7el₁L segment stably inserted into the 7DL arm of wheat line T4. Desirable genes/QTL mapped along the T4 7el₁L segment determine resistance to wheat rusts (Lr19, Sr25) and enhancement of yield-related traits. Mapping of the Fhb-7EL QTL, prerequisite for successful pyramiding, was established here on the basis of a bioassay with Fusarium graminearum of different 7EL-7el₁L bread wheat recombinant lines. These were obtained without resorting to any genetic pairing promotion, but relying on the close 7EL-7el₁L homoeology, resulting in 20% pairing frequency between the two arms. Fhb-7EL resided in the telomeric portion and resistant recombinants could be isolated with useful combinations of more proximally located 7el₁L genes/QTL. The transferred Fhb-7EL locus was shown to reduce disease severity and fungal biomass in grains of infected recombinants by over 95%. The same Fhb-7EL was, for the first time, proved to be effective also against F. culmorum and F. pseudograminearum, predominant agents of crown rot. Prebreeding lines possessing a suitable 7EL-7el₁L gene/QTL assembly showed very promising yield performance in preliminary field tests.

     

Memory topology of neural nets

Aiming at a topology of memory-growth, a computer-simulated mechanism is developed here, based on the probabilistic neural model previously developed by Anninos, Csermeley & Harth.The essential topology of this mechanism reflects the generally accepted idea that memory growth is achieved by means of association processes, i.e. through the cross-referencing of new inputs with already acquired information. In terms of computer-simulation such mechanism is developed by following the formalism of Set Theory. So the memory units, i.e. the neural netlets are equated to subsets of a set which represents a larger memory-system. Consistently with this formalism, association is a necessary, although non-sufficient condition for the growth of memory and the Boolean overlap of two subsets, i.e. of two netlets, is identified with the cross-reference mechanism which we assume to be the basis of memory growth.     

The role of chemokines in Schistosoma mansoni infection: insights from human disease and murine models

Chemokines are a superfamily of low-molecular-weight cytokines that were initially described for their chemoattractant activity. It is now clear chemokines have several other activities that modulate immune processes. More than 50 chemokines ligands and at least 19 receptors have been described to date. Depending on the number of N-terminal cysteine residues, chemokines are grouped in the subfamilies CXC, CC, C or CX3C. A growing body of evidence suggests a role for chemokines in the pathogenesis of several inflammatory diseases. Our studies involving mice and humans infected with Schistosoma mansoni suggest an important role of the chemokine CCL3 and its receptors (CCR1 and CCR5) in the pathogenesis of severe schistosomiasis. We suggest that the differential activation of CCR1 or CCR5 during the course of schistosomiasis may dictate the outcome of the disease.