A mouse model for monitoring calpain activity under physiological and pathological conditions

Calpains are Ca(2+)-dependent cysteine proteases known to be important for the regulation of cell functions and which aberrant activation causes cell death in a number of degenerative disorders. To provide a tool for monitoring the status of calpain activity in vivo under physiological and pathological conditions, we created a mouse model that expresses ubiquitously a fluorescent reporter consisting of eCFP and eYFP separated by a linker cleavable by the ubiquitous calpains. We named this mouse CAFI for calpain activity monitored by FRET imaging. Our validation studies demonstrated that the level of calpain activity correlates with a decrease in FRET (fluorescence resonance energy transfer) between the two fluorescent proteins. Using this model, we observed a small level of activity after denervation and fasting, a high level of activity during muscle regeneration and ischemia, and local activity in damaged myofibers after exercise. Finally, we crossed the CAFI mouse with the alpha-sarcoglycan-deficient model, demonstrating an increase of calpain activity at the steady state. Altogether, our results present evidence that CAFI mice could be a valuable tool in which to follow calpain activity at physiological levels and in disease states.     

A Nomadic Subtelomeric Disease Resistance Gene Cluster in Common Bean

The B4 resistance (R) gene cluster is one of the largest clusters known in common bean (Phaseolus vulgaris [Pv]). It is located in a peculiar genomic environment in the subtelomeric region of the short arm of chromosome 4, adjacent to two heterochromatic blocks (knobs). We sequenced 650 kb spanning this locus and annotated 97 genes, 26 of which correspond to Coiled-Coil-Nucleotide-Binding-Site-Leucine-Rich-Repeat (CNL). Conserved microsynteny was observed between the Pv B4 locus and corresponding regions of Medicago truncatula and Lotus japonicus in chromosomes Mt6 and Lj2, respectively. The notable exception was the CNL sequences, which were completely absent in these regions. The origin of the Pv B4-CNL sequences was investigated through phylogenetic analysis, which reveals that, in the Pv genome, paralogous CNL genes are shared among nonhomologous chromosomes (4 and 11). Together, our results suggest that Pv B4-CNL was derived from CNL sequences from another cluster, the Co-2 cluster, through an ectopic recombination event. Integration of the soybean (Glycine max) genome data enables us to date more precisely this event and also to infer that a single CNL moved from the Co-2 to the B4 cluster. Moreover, we identified a new 528-bp satellite repeat, referred to as khipu, specific to the Phaseolus genus, present both between B4-CNL sequences and in the two knobs identified at the B4 R gene cluster. The khipu repeat is present on most chromosomal termini, indicating the existence of frequent ectopic recombination events in Pv subtelomeric regions. Our results highlight the importance of ectopic recombination in R gene evolution.In the human genome, extensive cytogenetic and sequence analyses have revealed that subtelomeres are hot spots of interchromosomal recombination and segmental duplications (Linardopoulou et al., 2005). This peculiar dynamic activity of subtelomeres has been reported in such diverse organisms as yeast and the malaria parasite Plasmodium (Louis, 1995; Freitas-Junior et al., 2000, 2005). As expected for a plastic region of the genome subject to reshuffling through recombination events, subtelomeres exhibit unusually high levels of within-species structural and nucleotide polymorphism (Mefford and Trask, 2002). In plants, this plasticity of subtelomeres has not been identified in Arabidopsis (Arabidopsis thaliana; Heacock et al., 2004; Kuo et al., 2006) and, to our knowledge, has not yet been investigated at a large scale for other plant species with full genome sequences available. Regarding Arabidopsis, the apparent lack of high subtelomeric recombination may reflect its small and simple subtelomeres, mirroring its small genome size and relative paucity of repetitive sequences (Heacock et al., 2004; Kuo et al., 2006).Repetitive sequences, such as satellite DNA and retroelements, constitute an important fraction of every eukaryotic genome and therefore constitute the environment in which genes are expressed. Satellite DNA can be defined as highly reiterated noncoding DNA sequences, organized as long arrays of head-to-tail linked repeats of 150- to 180-bp or 300- to 360-bp monomers located in the constitutive heterochromatin (Plohl et al., 2008). Despite their ubiquity in eukaryotic genomes, little is known about the mechanisms that allow these elements to accumulate. Early hypotheses considered them to be nonfunctional “selfish” or “junk” DNA segments that increase or decrease their frequency without any advantage or disadvantage for an organism (Ohno, 1972; Orgel and Crick, 1980). However, identification of satellite DNA at structurally important parts of chromosomes, such as centromeres, has suggested functional roles of satellite DNA (Ma and Jackson, 2006; Kawabe and Charlesworth, 2007). Satellite DNA can also be localized in knobs, which are cytologically visible regions of highly condensed chromatin (heterochromatin) that are distinct from pericentromeric regions in pachytene chromosomes (Fransz et al., 2000; Gaut et al., 2007; Lamb et al., 2007).The survival of most organisms depends on the presence of specific genetic systems that maintain diversity in order to respond to changing environments. Plants, like animals, are continually challenged by a large array of pathogens. To perceive and counter pathogen attack, plants have evolved disease resistance (R) genes. The largest class of R genes encodes proteins containing a central Nucleotide-Binding Site (NBS) domain, a C-terminal Leucine-Rich Repeat (LRR) domain, and a variable N-terminal domain. These R proteins detect the presence of disease-causing bacteria, oomycetes, fungi, nematodes, insects, and viruses by sensing either specific pathogen effector molecules produced during the infection process or key molecules in the plant cell that may be attacked by pathogen effectors (Dangl and McDowell, 2006). The evolution of new R genes serves to counteract the evolution of novel virulence factors from the pathogens (McDowell and Simon, 2008). Among this prevalent class of R gene, two subclasses, corresponding to two ancient lineages (Bai et al., 2002; Meyers et al., 2003; Ameline-Torregrosa et al., 2008), have been identified based on the N-terminal domain of the R protein: the Coiled-Coil (CC)-NBS-LRR (CNL) and the Toll-Interleukin receptor (TIR)-NBS-LRR (TNL). Genome studies have demonstrated that NBS-LRR (NL) sequences are abundant in any plant genome. For example, annotation of the Arabidopsis, rice (Oryza sativa), poplar (Populus trichocarpa), Medicago truncatula (Mt), grape (Vitis vinifera), Lotus japonicus (Lj), and papaya (Carica papaya) genomes identified at least 149, 480, 317, 333, 233, 229, and 55 genes encoding NL proteins, respectively (Bai et al., 2002; Meyers et al., 2003; Zhou et al., 2004; Tuskan et al., 2006; Velasco et al., 2007; Ameline-Torregrosa et al., 2008; Kohler et al., 2008; Ming et al., 2008; Sato et al., 2008). NL sequences are often located at complex loci (Smith et al., 2004), as exemplified by Arabidopsis, where two-thirds of them are organized in tightly linked clusters (Meyers et al., 2003; Leister, 2004; McDowell and Simon, 2006). Evolution of NL sequences in the Arabidopsis genome has been investigated according to their phylogenetic positions and physical locations. Although tandem duplications explain the origin of a large fraction of NLs, it seems that ectopic recombination has also played a role in Arabidopsis NL evolution, since mixed clusters comprising evolutionarily distant NL exist. Ectopic recombination is also evident when phylogenetically close R genes are physically dispersed on different chromosomes (Leister, 2004; McDowell and Simon, 2006). These results confirm pioneer macrosynteny studies between related monocot species suggesting the existence of NL movement in plant genomes. Indeed, extensive loss of collinearity between NL sequences between rice and barley (Hordeum vulgare), which diverged 50 million years ago (Mya), has suggested rapid reorganization of NL sequences (Leister et al., 1998; Leister, 2004). However, our knowledge of the molecular evolution of R genes remains limited due to the still small number of complete plant genome sequences available to date. Detailed comparative study across taxa at different evolutionary distances is needed to see how R gene clusters evolve at various time scales.Legumes (Fabaceae) constitute the third largest family of flowering plants and represent the second most important family of agronomically important plants after Poaceae (Graham and Vance, 2003). As a result of recent sequencing efforts, legumes are one of the few plant families with extensive genome sequences in different species, since the soybean (Glycine max [Gm]) genome sequence is complete (http://www.phytozome.net/soybean.php) and both Mt and Lj genome sequences are nearly complete (Young et al., 2005; Sato et al., 2008). Consequently, the legume family is extremely well adapted for comparative phylogenomic approaches, in which phylogenetic inference is combined with structural genomic analyses (Ammiraju et al., 2008). Common bean (Phaseolus vulgaris [Pv]) is the most important grain legume for direct human consumption (Broughton et al., 2003). Pv is a selfing species and has a small diploid genome (2n = 22) of 588 Mb (Bennett and Leitch, 1995). Conservation of genome macrostructure (macrosynteny) has been reported between several legumes, including common bean and the two model legume species Mt and Lj genomes (Zhu et al., 2005; Hougaard et al., 2008). However, the extent of gene order conservation at the DNA sequence level has not yet been evaluated within orthologous chromosome segments between Pv and the two model legume species.In the genome of common bean, many disease R genes are clustered at complex loci located at the ends (rather than the centers) of linkage groups (LGs; Vallejos et al., 2006; Geffroy et al., 2008). For example, Colletotrichum lindemuthianum Co-2 R specificity maps at one end of LG B11 (Adam-Blondon et al., 1994). Molecular analysis has revealed that this locus consists of a tandem array of CNL sequences (Geffroy et al., 1998; Creusot et al., 1999). Another CNL-rich region has been identified at the end of LG B4 in the vicinity of R specificities and R quantitative trait loci against a large selection of pathogens, including C. lindemuthianum, Uromyces appendiculatus, and the bacterium Pseudomonas syringae (Geffroy et al., 1998, 1999; Miklas et al., 2006). Recently, fluorescence in situ hybridization (FISH) analysis revealed that this complex R cluster is located in the subtelomeric region of the short arm of chromosome 4 and includes two knobs (Geffroy et al., 2009). In a sequencing effort focused on CNL sequences, we have previously identified 17 CNL sequences of the B4 locus (referred to as B4-CNL) from Pv genotype BAT93 (Ferrier Cana et al., 2003, 2005; Geffroy et al., 2009). In the BAT93 genotype, these B4-CNL sequences are located on both sides of the subterminal knob (Geffroy et al., 2009).To investigate the organization and the evolutionary origin of the subtelomeric B4 R gene cluster, we have sequenced approximately 650 kb of the Pv B4 R gene cluster, revealing that, in genotype BAT93, CNL are spread out in four subclusters, separated by non-CNL-encoding genes. This Pv sequence was then compared gene by gene with the sequenced portions of the three sequenced legume genomes, Mt, Lj, and Gm. Conserved microsynteny (conservation of local gene repertoire, order, and orientation) was observed, except for the CNL sequences, which appear to be completely absent in the corresponding regions of Mt and Lj. In this study, by combining genomics, phylogenetic, and cytogenetic approaches, we provide evidence that ectopic recombination in subtelomeric regions between nonhomologous chromosomes (4 and 11), involving a single CNL, gave rise to the Pv B4 R gene cluster. Chromosomal distribution of a new satellite DNA tandem repeat, referred to as khipu, suggests that ectopic recombination events in subtelomeric regions of bean nonhomologous chromosomes are frequent. Our results highlight the importance of ectopic recombination as an important evolutionary mechanism for the evolution of disease resistance genes.     

Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator

Transplantation of genetically manipulated cells to the central nervous system holds great promise for the treatment of several severe neurological disorders. The success of this strategy relies on sufficient levels of transgene expression after transplantation. This has been difficult to achieve, however, due to transgene silencing. In this study, we transduced the neural stem cell line RN33B with self-inactivating lentiviral vectors and analyzed transgenic expression of green fluorescent protein (GFP) in several different settings both in vitro and after transplantation to the brain. We found that the transgene was affected of silencing both when transduced cells were proliferating and after differentiation. To prevent silencing, the cHS4 insulator was incorporated into the lentiviral vector. We found that a vector carrying the cHS4 insulator was partially protected against differentiation-dependent downregulation in vitro and in vivo. However, in proliferating cells, we found evidence for variegation and positional effects that were not prevented by the cHS4 insulator, suggesting that the mechanism behind silencing in proliferating cells is not the same mechanism influencing differentiation-dependent silencing. Taken together, these findings favor vector optimization as a strategy for achieving efficient ex vivo gene transfer in the central nervous system.     

Effect of acetylcholine on the heart ventricle of the goldfish Carassius auratus, Teleosts, Cyprinidae. Modification of the response as a function of temperature]

Acetylcholine (Ach) is more efficient on the red fish heart at 14 degrees C than at 5 degrees C or 8 degrees C. It does not probably exist a cholinesterasic system and high concentrations of Ach are to be used to obtain significant variations of the heart frequency. Ach does not appear as a physiological mediator and temperature seems to be an essential factor in heart regulation and modulation of the pharmacological effects of eventual mediators. A thermal adaptation exists but the fast increase of the temperature, in the presence of Ach, results in heart blocage, whose contractions reappear spontanously during cooling. This is at the present time no valuable hypothesis could be made to explain this phenomenon.     

Parasitic hazard with sewage sludge applied to land.

A modification of the FAUST technique allowed a highly regular recovery of Taenia saginata eggs from sewage sludge, as well as their quantification. Despite the low viability (8%) noted, the viable T. saginata egg level remains high (20.10(6)/ha) and offers a serious risk for cattle even after a 3-week "no-grazing" period.     

Hypoxia impairs cell fusion and differentiation process in human cytotrophoblast,in vitro

During human pregnancy, the trophoblast develops from differentiation of cytotrophoblast cells into an endocrine active syncytiotrophoblast. In culture, isolated mononuclear cytotrophoblasts aggregate and then fuse to form a syncytium, reproducing the in vivo process. In this study, we examined the effect of low oxygen tension (approximately 9%, hypoxia) compared to standard conditions (approximately 19% oxygen, normoxia) on these cellular events. Under hypoxia, syncytial formation was less frequently observed, cell staining and electron microscopy revealed that cytotrophoblasts remain aggregated, with a positive proliferative cell nuclear antigen (PCNA) immunostaining. Desmoplakin and E-cadherin, both known to disappear with cytotrophoblast fusion, showed persistent expression in hypoxic cells after 3 days of culture. In contrast, the expression of actin and ezrin, two cytoskeletal proteins, was unchanged. hCG secretion and hPL expression were both decreased in hypoxic cells, reflecting a reduced syncytial formation. Thus, on day 3, the mean values for hCG secretion were 1,100 ± 155 and 289 ± 26 mlU/mL in normoxic and hypoxic conditions, respectively. The reduced cell fusion process as well as hCG secretion and hPL expression under hypoxia were reversed by reoxygenation of the cells. We conclude that under hypoxia, the formation of functional syncytiotrophoblast is impaired due to a defect in the cytotrophoblast fusion process. This may explain the observation of a higher number of cytotrophoblast cells and a reduced syncytial layer in placentas of some pathological pregnancies. © 1996 Wiley-Liss, Inc.     

Hypoxia-activated ligand HAL-1/13 is lupus autoantigen Ku80 and mediates lymphoid cell adhesion in vitro

Hypoxia is known toinduce extravasation of lymphocytes and leukocytes duringischemic injury and increase the metastatic potential ofmalignant lymphoid cells. We have recently identified a new adhesionmolecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates thehypoxia-induced increases in lymphocyte and neutrophil adhesion toendothelium and hypoxia-mediated invasion of endothelial cellmonolayers by tumor cells. In this report, we used expression cloningto identify this molecule as the lupus antigen and DNA-dependentprotein kinase-associated nuclear protein, Ku80. TheHAL-1/13-Ku80 antigen is present on the surface of leukemic and solidtumor cell lines, including T and B lymphomas, myeloid leukemias,neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells.Transfection and ectopic expression of HAL-1/13-Ku80 on (murine)NIH/3T3 fibroblasts confers the ability of these normally nonadhesivecells to bind to a variety of human lymphoid cell lines. This adhesioncan be specifically blocked by HAL-1/13 or Ku80-neutralizingantibodies. Loss of expression variants of these transfectantssimultaneously lost their adhesive properties toward human lymphoidcells. Hypoxic exposure of tumor cell lines resulted in upregulation ofHAL-1/13-Ku80 expression at the cell surface, mediated byredistribution of the antigen from the nucleus. These studies indicatethat the HAL-1/13-Ku80 molecule may mediate, in part, thehypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.

     

Hyperplasia of Interstitial Cells of Cajal in Sprouty Homolog 4 Deficient Mice

Gastrointestinal stromal tumors, which are thought to derive from interstitial cells of Cajal or their precursors, often harbor an oncogenic mutation of the KIT receptor tyrosine kinase. Sprouty homolog 4, a known negative regulator of ERK pathway, has been identified in the interstitial cells of Cajal in the Kit^K641E murine model of gastrointestinal stromal tumors. Sprouty homolog 4 was upregulated both at the mRNA and protein level in these cells, suggesting that Sprouty homolog 4 is downstream of oncogenic KIT activation and potentially engaged in the negative feedback loop of ERK activation in this model. Here, we used Kit^K641E heterozygous and Sprouty homolog 4 knock out animals to quantify interstitial cells of Cajal in situ, using quantitative immunofluorescence for the receptor tyrosine kinase Kit and for phosphodiesterase 3a (PDE3A). In the antrum of Sprouty homolog 4 knock out mice, hyperplasia of interstitial cells of Cajal was reminiscent of the Kit^K641E heterozygous mice antrum. Additionally, the density of interstitial cells of Cajal was higher in the colon of adult Sprouty homolog 4 knock out mice than in WT littermates, although hyperplasia seemed more severe in Kit^K641E heterozygous mice. Functional transit studies also show similarities between Sprouty homolog 4 knock out and Kit^K641E heterozygous mice, as the total transit time in 9 month old animals was significantly increased in both genotypes compared to WT littermates. We concluded that the lack of Sprouty homolog 4 expression leads to hyperplasia of the interstitial cells of Cajal and is functionally associated with a delayed transit time.     

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.