Aminoaciduria, but normal thyroid hormone levels and signalling, in mice lacking the amino acid and thyroid hormone transporter Slc7a8

LAT2 (system L amino acid transporter 2) is composed of the subunits Slc7a8/Lat2 and Slc3a2/4F2hc. This transporter is highly expressed along the basolateral membranes of absorptive epithelia in kidney and small intestine, but is also abundant in the brain. Lat2 is an energy-independent exchanger of neutral amino acids, and was shown to transport thyroid hormones. We report in the present paper that targeted inactivation of Slc7a8 leads to increased urinary loss of small neutral amino acids. Development and growth of Slc7a8(-/-) mice appears normal, suggesting functional compensation of neutral amino acid transport by alternative transporters in kidney, intestine and placenta. Movement co-ordination is slightly impaired in mutant mice, although cerebellar development and structure remained inconspicuous. Circulating thyroid hormones, thyrotropin and thyroid hormone-responsive genes remained unchanged in Slc7a8(-/-) mice, possibly because of functional compensation by the thyroid hormone transporter Mct8 (monocarboxylate transporter 8), which is co-expressed in many cell types. The reason for the mild neurological phenotype remains unresolved.     

Effective siRNA delivery and target mRNA degradation using an amphipathic peptide to facilitate pH-dependent endosomal escape

Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.     

Acid ceramidase expression modulates the sensitivity of A375 melanoma cells to dacarbazine

Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time- and dose-dependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that down-regulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma.     

Association between the chondrocyte phenotype and the expression of adipokines and their receptors: evidence for a role of leptin but not adiponectin in the expression of cartilage-specific markers

Although extensive evidence support the key role of adipokines in cartilage homeostasis, contradictory data have been found for their expression and their effects in chondrocytes. This study was then undertaken to determine whether a phenotypic modulation may affect the expression of adipokines and their receptors in human chondrocytes. The expression of leptin, adiponectin and their receptors, as well as cartilage-specific genes was examined in chondrocytes obtained from patients with osteoarthritis either directly after cells harvest or after culture in monolayer or in alginate beads. The results showed major changes in the gene expression pattern after culture in monolayer with a shift from the adipokines to their receptors. Interestingly, this downregulation of adipokines was associated with a loss of chondrocyte phenotype, and chondrocytes recovered a cartilage-like expression profile of leptin and adiponectin when cultured in a tridimensional chondrocyte phenotype-inducing system, but ceased expressing their receptors. Further experiments clearly showed that leptin but not adiponectin promoted the expression of cartilage-specific markers through mitogen-activated protein kinase, Janus kinase and phosphatidylinositol-3 kinase signaling pathways. In conclusion, our data indicate that any phenotypic modulation could affect chondrocyte responsiveness to leptin or adiponectin, and provide evidence for an important role for leptin in regulating the expression of cartilage-specific markers.     

The energetic costs of case construction in the caddisfly Limnephilus rhombicus: direct impacts on larvae and delayed impacts on adults

Caddisflies, whose aquatic larvae build a portable case with silk, are a suitable model organism to test the impacts of resource allocation trade-off during development and examine the evolution of life-history strategies. In the caddisfly Limnephilus rhombicus, adult feeding is minimal. Therefore, the whole resources are acquired during the larval phase and must be allocated to case construction, growth and reproduction. In this study, the larval energetic reserves of L. rhombicus were manipulated by forcing larvae to rebuild their cases in the final larval stage. This allowed us to measure the physiological cost of construction. First, we recorded oxygen consumption during case reconstruction. Second, we measured the sugar, protein and lipid contents of larvae forced to rebuild their case and of larvae required only to re-enter on their case. Larvae had their sugar, protein and lipid content measured after the rebuilding event and 72 h later. The same analyses were carried out with adults immediately after emergence. We found that larvae forced to rebuild a case consumed 1.5 times more oxygen than control larvae. This energy expenditure generated a cost that was estimated to be a loss of larval protein of approximately 35%. Insects were unable to compensate for this loss of proteins during the end of the larval stage, and their metamorphosis to adults was also impacted. Therefore, we suggest that loss of larval protein is linked to silk production and may alter fitness.     

Targeted surveillance of raccoon rabies in Québec,Canada

Data from wildlife disease surveillance programs are used to inform implementation of disease control (e.g., vaccination, population reduction) in space and time. We developed an approach to increase detection of raccoon rabies in raccoons (Procyon lotor) and skunks (Mephitis mephitis) of Québec, Canada, and we examined the implications of using this approach for targeted surveillance. First we modeled the probability of a rabid animal relative to environmental characteristics of sampling locations. Rabid animals were more likely to be found in low-lying flat landscapes that had higher proportions of corn-forest edge habitat and hay agriculture, and that were within 20 km of one or more known rabies cases. From the model, we created 2 complementary risk maps to identify areas where rabid animals were most likely to be sampled. One map accounted for habitat and known rabies case locations, and can be used to define an infection zone from which surveillance can be targeted along the periphery to determine if disease is continuing to spread. The other map only accounted for habitat and can be used to locate areas most likely to contain rabid animals when the disease is present. In a further analysis we compared the 2 most successful methods for detecting raccoon rabies in Québec, given the disease was present. Government trapping operations (active surveillance) detected more cases in the short-term, but citizen notification (passive and enhanced) was more effective after 12 trapping days from which the initial rabies case was found. Our approach can benefit wildlife and public health agencies wanting to assess the disease status of regions by targeting surveillance to habitats most likely to contain infected animals and by defining the duration over which sampling methods are effective. © 2011 The Wildlife Society.     

True stress and Poisson's ratio of tendons during loading

Excessive axial tension is very likely involved in the aetiology of tendon lesions, and the most appropriate indicator of tendon stress state is the true stress, the ratio of instantaneous load to instantaneous cross-sectional area (CSA). Difficulties to measure tendon CSA during tension often led to approximate true stress by assuming that CSA is constant during loading (i.e. by the engineering stress) or that tendon is incompressible, implying a Poisson's ratio of 0.5, although these hypotheses have never been tested. The objective of this study was to measure tendon CSA variation during quasi-static tensile loading, in order to assess the true stress to which the tendon is subjected and its Poisson's ratio. Eight equine superficial digital flexor tendons (SDFT, about 30cm long) were tested in tension until failure while the CSA of each tendon was measured in its metacarpal part by means of a linear laser scanner. Axial elongation and load were synchronously recorded during the test. CSA was found to linearly decrease with strain, with a mean decrease at failure of -10.7±2.8% (mean±standard deviation). True stress at failure was 7.1-13.6% higher than engineering stress, while stress estimation under the hypothesis of incompressibility differed from true stress of -6.6 to 2.3%. Average Poisson's ratio was 0.55±0.12 and did not significantly vary with load. From these results on equine SDFT it was demonstrated that tendon in axial quasi-static tension can be considered, at first approximation, as an incompressible material.     

Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants

Resistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum?×?almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed.     

Drosophila Perilipin/ADRP homologue Lsd2 regulates lipid metabolism

Many cells store neutral lipids, as triacylglycerol and sterol esters, in droplets. PAT-domain proteins form a conserved family of proteins that are localized at the surface of neutral lipid droplets. Two mammalian members of this family, Perilipin and adipose differentiation-related protein, are involved in lipid storage and regulate lipolysis. Here, we describe the Drosophila PAT-family member Lsd2. We showed that Lsd2 is predominantly expressed in tissues engaged in high levels of lipid metabolism, the fat body and the germ line of females. Ultrastructural analysis in the germ line showed that Lsd2 localizes to the surface of lipid droplets. We have generated an Lsd2 mutant and described its phenotype. Mutant adults have a reduced level of neutral lipid content compared to wild type, showing that Lsd2 is required for normal lipid storage. In addition, ovaries from Lsd2 mutant females exhibit an abnormal pattern of accumulation of neutral lipids from mid-oogenesis, which results in reduced deposition of lipids in the egg. Consistent with its expression in the female germ line, we showed that Lsd2 is a maternal effect gene that is required for normal embryogenesis. This work demonstrates that Lsd2 has an evolutionarily conserved function in lipid metabolism and establishes Drosophila melanogaster as a new in vivo model for studies on the PAT-family of proteins.     

Quinoline-carboxylic acids are potent inhibitors that inhibit the binding of insulin-like growth factor (IGF) to IGF-binding proteins

4-benzylquinolines 5, based on a series of isoquinolines 1, were prepared and tested as inhibitors of the IGF/IGFBP-3 complex based on their ability to displace IGF-I from its binding to IGF-binding protein-3. SAR studies on the 6,7-dihydroxy moiety of the quinoline 5a showed that the catecol moiety could be replaced with other functional groups. Computational modeling of the 5a/mini-IGFBP-5 complex revealed the possible binding site of 5a on IGFBP-5.