Cross-species amplification of Medicago truncatula microsatellites across three major pulse crops

Model plants are facilitating the genetic characterization and comparative mapping of a number of traditional crops. Medicago truncatula has been widely accepted as a model plant to this end as it provides the essential tools for multiple aspects of legume genetics and genomics. A large set of markers from highly conserved M. truncatula gene regions is being created and used to establish a worldwide framework for comparative genomic studies in legumes. We have investigated the potential for cross-species amplification of 209 expressed sequence tag (EST)-based and 33 bacterial artificial chromosome (BAC)-based microsatellites from M. truncatula in the three most important European legume pulses—pea, faba bean and chickpea—that might facilitate future comparative mapping. Our results revealed significant transferability of M. truncatula microsatellites to the three pulses (40% in faba bean, 36.3% in chickpea and 37.6% in pea). The percentage of M. truncatula EST-SSRs (simple sequence repeats) amplified in the three crops (39–43%) was twofold higher than that of the genomic SSRs (21–24%). Sequence analysis determined that the level of conservation in the microsatellite motif was very low, while the flanking regions were generally well conserved. The variations in the sequences were mainly due to changes in the number of repeat motifs in the microsatellite region combined with indel and base substitutions. None of the functional microsatellites showed direct polymorphism among the parental genotypes tested, consequently preventing their immediate use for mapping purposes.Electronic Supplementary Material Supplementary material is available for this article at     

Synthesis and characterisation of [[μ-(1,7,11,17-tetraaza-2,6,12,16-tetraoxacycloeicosane)][(μ-(aquo)]bis[(acac)(ethanol)nickel(II)]] [BPh4]2

The pale blue title compound, 4, was obtained from the reaction between 1,7,11,17-tetraaza-2,6,12,16-tetraoxacycloeicosane, Ni(acac)₂ and NaBPh₄ in aqueous acetone. X-ray structure determination at 120 K revealed that the dication of the ionic complex, 4, contains two independent octahedral Ni^II centres with trans-Ni₂N₂O₄ chromophores. The macrocyclic ligand and an aqua ligand act as bridges to the two nickel centres: the Ni-O(aquo)-N bond angle is 137.65(17)°. Each Ni centre is bonded to two nitrogens of the macrocycle, to a chelating acac unit, to an ethanol molecule as well as the bridging oxygen of the aqua group. The two nickel atoms sit outside the macrocycle cavity, such that the macrocyclic ligand acts as a canopy for the remainder of the dication. While none of the macrocycle oxygens are involved in the coordination to Ni, they are involved in internal hydrogen bonding with the aqua and ethanol ligands. Magnetic measurements show a paramagnetic behaviour down to 2 K, with an effective moment of 2.8 Bohr magnetons at room temperature.     

Peanut agglutinin high phenotype of activated CD8+ T cells results from de novo synthesis of CD45 glycans

Following activation in the periphery, murine CD8+ T cells exhibit a characteristic increased binding of peanut agglutinin (PNA), reflecting an increased expression of hyposialylated O-linked glycans (Galbeta1-3GalNAcalpha-O-Thr/Ser) on the cell surface. In this report, we show that the majority of the PNA receptors expressed on activated CD8+ T cells are carried by CD45. Other glycoproteins (e.g. CD8) and the glycolipid asialo-GM1 also carry PNA receptors, although to a much lesser extent. Analysis of enzymes involved in the sialylation/de-sialylation pathways showed that generation of PNA receptors in activated CD8+ T cells is not due to up-regulation of endogenous sialidases. Instead, our results indicate that the PNA(high) phenotype results from de novo synthesis of CD45 carrying reduced sialylated core 1 O-glycans.     

Differential inhibition of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3 rgulates pro-MMP-2 activation

The membrane type (MT)-matrix metalloproteinases (MMPs) constitute a subgroup of membrane-anchored MMPs that are major mediators of pericellular proteolysis and physiological activators of pro-MMP-2. The MT-MMPs also exhibit differential inhibition by members of the tissue inhibitor of metalloproteinase (TIMP) family. Here we investigated the processing, catalytic activity, and TIMP inhibition of MT3-MMP (MMP-16). Inhibitor profile and mutant enzyme studies indicated that MT3-MMP is regulated on the cell surface by autocatalytic processing and ectodomain shedding. Inhibition kinetic studies showed that TIMP-3 is a high affinity inhibitor of MT3-MMP when compared with MT1-MMP (K(i) = 0.008 nm for MT3-MMP versus K(i) = 0.16 nm for MT1-MMP). In contrast, TIMP-2 is a better inhibitor of MT1-MMP. MT3-MMP requires TIMP-2 to accomplish full pro-MMP-2 activation and this process is enhanced in marimastatpretreated cells, consistent with regulation of active enzyme turnover by synthetic MMP inhibitors. TIMP-3 also enhances the activation of pro-MMP-2 by MT3-MMP but not by MT1-MMP. TIMP-4, in contrast, cannot support pro-MMP-2 activation with either enzyme. Affinity chromatography experiments demonstrated that pro-MMP-2 can assemble trimolecular complexes with a catalytic domain of MT3-MMP and TIMP-2 or TIMP-3 suggesting that pro-MMP-2 activation by MT3-MMP involves ternary complex formation on the cell surface. These results demonstrate that TIMP-3 is a major regulator of MT3-MMP activity and further underscores the unique interactions of TIMPs with MT-MMPs in the control of pericellular proteolysis.     

Three‐dimensional cell culture microarray for high‐throughput studies of stem cell fate

We have developed a novel three‐dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high‐throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox‐1, whose levels were also measured in situ using a GFP reporter system. In addition, the high‐throughput capacity of the platform was tested using a dual‐slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor‐4 (FGF‐4) on the pluripotency of mouse ES cells. This high‐throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. Biotechnol. Bioeng. 2010; 106: 106–118. © 2010 Wiley Periodicals, Inc.     

An improved selection strategy and the use of acetosyringone in shoot induction medium increase almond transformation efficiency by 100-fold

An improved almond transformation system was developed with a 100× increase in efficiency (12.3%) as compared to the existing transformation method (0.1%) in spite of the lower regeneration ability of the explants used. Leaf transformation was performed with Agrobacterium EHA105/p35 SGUSINT. Main modifications introduced in the transformation protocol were the use of 150 μM acetosyringone (AS) during the 21-days induction period, and a different selection strategy. Transformed shoots were assayed using PCR, GUS analyses and Southern blotting. The improved methodology is being applied for transformation of in vitro propagated cultivars and opens the possibility of using almond as model for functional studies in Prunoideae.     

Arterial vascularization of the mandible and maxilla of neotropical primates

The objective of the present investigation was to conduct a comparative macroscopic study of the arterial vascularization of the mandible and maxilla of neotropical primates of the genera Cebus, Alouatta, Callithrix, and Leontopithecus. After vinyl was injected into the arterial system of the head of each specimen, the pieces were macerated and corroded. The level of the bifurcation of the common carotid artery into the internal and external carotids varied between the first and third cervical vertebrae. The external carotid artery accounts for most of the vascularization of the facial structures. The actual vessels responsible for the supply of this region are the sublingual, facial, angular, lingual, submandibular, submental, inferior and superior labial, maxillary, inferior alveolar, infraorbital, superior posterior alveolar, palatine major, and sphenopalatine arteries. We conclude that although the arterial vascular pattern was similar in all the genera studied, and resembles the human pattern, there are notable variations in the vasculature of the mandible and maxilla among these four neotropical genera.     

Arylfurans as potential trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 microM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 microg/ml (59 and 90 microM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 microg/ml (0.6-1.5 mM).     

Measurement of carnitine biosynthesis enzyme activities by tandem mass spectrometry: differences between the mouse and the rat

Although the mouse frequently is used to study metabolism and deficiencies therein, little is known about carnitine biosynthesis in this animal. To this point, only laborious procedures have been described to measure the activity of carnitine biosynthesis enzymes using subcellular fractions as the enzyme source. We developed two simple tandem mass spectrometry-based methods to determine the activity of three carnitine biosynthesis enzymes (6-N-trimethyllysine dioxygenase, 4-trimethylaminobutyraldehyde dehydrogenase, and 4-trimethylaminobutyric acid dioxygenase) in total homogenates that can be prepared from frozen tissue. The new assays were used to characterize these enzymes in mouse liver homogenate. Because carnitine biosynthesis has been studied extensively in the rat, we compared the mouse tissue distribution of carnitine biosynthesis enzyme activities and levels of the biosynthesis metabolites with those in the rat to determine which tissues contribute to carnitine biosynthesis in these species. Surprisingly, large differences in enzyme activities were found between the rat and the mouse, whereas carnitine biosynthesis metabolite levels were very similar in both species, possibly due to the different kinetic properties of the first enzyme of carnitine biosynthesis. Also, muscle carnitine levels were found to vary considerably between these two species, suggesting that there is a metabolic dissimilarity between the mouse and the rat.