Abstract Background Mutations of MAGEL2 have been reported in patients presenting with autism, and loss of MAGEL2 is also associated with Prader-Willi syndrome, a neurodevelopmental genetic disorder. ...This study aimed to determine the behavioral phenotype of Magel2 -deficient adult mice, to characterize the central oxytocin (OT) system of these mutant mice, and to test the curative effect of a peripheral OT treatment just after birth. Methods We assessed the social and cognitive behavior of Magel2 -deficient mice, analyzed the OT system of mutant mice treated or not by a postnatal administration of OT, and determined the effect of this treatment on the brain. Results Magel2 inactivation induces a deficit in social recognition and social interaction and a reduced learning ability in adult male mice. In these mice, we reveal anatomical and functional modifications of the OT system and show that these defects change from birth to adulthood. Daily administration of OT in the first postnatal week was sufficient to prevent deficits in social behavior and learning abilities in adult mutant male mice. We show that this OT treatment partly restores a normal OT system. Thus, we report that an alteration of the OT system around birth has long-term consequences on behavior and on cognition. Importantly, an acute OT treatment of Magel2 -deficient pups has a curative effect. Conclusions Our study reveals that OT plays a crucial role in setting social behaviors during a period just after birth. An early OT treatment in this critical period could be a novel therapeutic approach for the treatment of neurodevelopmental disorders such as Prader-Willi syndrome and autism.
New designer drugs, as synthetic cannabinoids (SCs), continuously appear on the market and are booming due to their cannabis-like effect. New generation of smokable SCs, structurally dissimilar from ...Δ9-tetrahydrocannabinol (THC), have isomers with distinguishable pharmacokinetic parameters and therefore different in vivo effects. The isoforms are misidentified using conventional techniques such as gas or liquid chromatography coupled to mass spectrometry - or tandem mass - spectrometry. The aim of this study was to differentiate three positional isomers (JWH-007, JWH-019 and JWH-122) in single human hair samples, which store numerous substances revealing a way of life and consumption style.
Matrix-assisted laser desorption/ionization (MALDI) combined with imaging is an innovative and powerful tool used since few years, especially in forensic research. Herein, we propose an innovative method to monitor the drugs of abuse consumption through direct mapping of the compounds with a high spatial distribution in human hair samples, by state-of-art imaging MALDI-MSn. Three positional SC isomers (JWH-007, JWH-019 and JWH-122) were analysed using high and low fragmentation energy and the resulting MS/MS and even MS3 spectra differentiated the SCs. The MALDI-MS/MS and MS3 imaging was performed on hair soaked in a mixture of the three SCs as well as on hair from self-reported SC user, proving the potential of the technique for a forensic use. Keeping in mind that spatial distribution of organics from whole hair remains a challenge, the described methodology is a very promising analytical tool to probe the consumption of complex drugs and obtain correlation with its origin.
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•A high spatial resolution imaging method for synthetic cannabinoids is presented.•MSn permits more characterization and structural information of complex mixture.•MALDI-IMS lead to the drugs mapping in single hair.•High sensitivity was reached for the detection of JWH-122 from authentic hair.
Top-down mass spectrometry strategies allow identification and characterization of proteins and protein networks by direct fragmentation. These analytical processes involve a panel of fragmentation ...mechanisms, some of which preserve protein post-translational modifications. Thus top-down is of special interest in clinical biochemistry to probe modified proteins as potential disease biomarkers. This review describes separating methods, mass spectrometry instrumentation, bioinformatics, and theoretical aspects of fragmentation mechanisms used for top-down analysis. The biological interest of this strategy is extensively reported regarding the characterization of post-translational modifications in biochemical pathways and the discovery of biomarkers. One has to bear in mind that quantitative aspects that are beyond the focus of this review are also of critical important for biomarker discovery. The constant evolution of technologies makes top-down strategies crucial players in clinical and basic proteomics.
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Exosomes are vesicles secreted by most hematopoietic cells on fusion of multivesicular endosomes with the plasma membrane. Many studies have reported that exosomes may also be released by ...tumor cells. Exosomes are believed to play an antitumor role through immune cells. We asked whether tumor exosomes have biological activities on tumor cells. We report that human pancreatic tumor nanoparticles, exosome‐like as characterized by proteomic analyses and rich in lipid rafts, decreased tumor cell proliferation. Nanoparticles increased Bax and decreased Bcl‐2 expressions. Caspase‐3 and −9 but not caspase‐8 inhibitors impaired apoptosis, which implicates the mitochondria apoptotic pathway. The ceramide‐sphingomyelin apoptotic pathway was inoperative. Moreover, nanoparticles induced phosphatase and tensin homolog (PTEN) and glycogen synthase kinase (GSK) −3β activation and decreased pyruvate dehydrogenase activity. In nanoparticle‐treated cells, PTEN formed complexes with actin, β‐catenin, and GSK‐3β. Thus, β‐catenin may no longer be available to activate the survival pathway. Nanoparticles triggered the down‐regulation of cyclin D1 and poly(ADP‐ribose) polymerase. Hence, nanoparticles counteracted the constitutively activated phosphatidylinositol 3‐kinase/Akt survival pathway to drive tumor cells toward apoptosis. Our study provides the first evidence of an apoptotic function of tumor‐derived nanoparticles on tumor cells. We propose a new role for nanoparticles, i.e., as signal carriers for interaction between cells, which may have implications in physiopathological situations.—Ristorcelli, E., Beraud, E., Verrando, P., Villard, C., Lafitte, D., Sbarra, V., Lombardo, D., Verine, A. Human tumor nanoparticles induce apoptosis of pancreatic cancer cells. FASEB J. 22, 3358–3369 (2008)
Glioblastoma multiforme is one of the most common intracranial tumors encountered in adults. This tumor of very poor prognosis is associated with a median survival rate of approximately 14 months. ...One of the major issues to better understand the biology of these tumors and to optimize the therapy is to obtain the molecular structure of glioblastoma. MALDI molecular imaging enables location of molecules in tissues without labeling. However, molecular identification in situ is not an easy task. In this paper, we used MALDI imaging coupled to in‐source decay to characterize markers of this pathology. We provided MALDI molecular images up to 30 μm spatial resolution of mouse brain tissue sections. MALDI images showed the heterogeneity of the glioblastoma. In the various zones and at various development stages of the tumor, using our top‐down strategy, we identified several proteins. These proteins play key roles in tumorigenesis. Particular attention was given to the necrotic area with characterization of hemorrhage, one of the most important poor prognosis factors in glioblastoma.
Gram-negative porins are the main entry for small hydrophilic molecules. We studied translocation of structurally related cephalosporins, ceftazidime (CAZ), cefotaxime (CTX) and cefepime (FEP). CAZ ...is highly active on E. coli producing OmpF (Outer membrane protein F) but less efficient on cells expressing OmpC (Outer membrane protein C), whereas FEP and CTX kill bacteria regardless of the porin expressed. This matches with the different capacity of CAZ and FEP to accumulate into bacterial cells as quantified by LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry). Furthermore, porin reconstitution into planar lipid bilayer and zero current assays suggest permeation of ≈1,000 molecules of CAZ per sec and per channel through OmpF versus ≈500 through OmpC. Here, the instant killing is directly correlated to internal drug concentration. We propose that the net negative charge of CAZ represents a key advantage for permeation through OmpF porins that are less cation-selective than OmpC. These data could explain the decreased susceptibility to some cephalosporins of enterobacteria that exclusively express OmpC porins.
This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin ...polymerization and microtubule targeting agent binding, we then highlight how the three-dimensional structures of drug-tubulin complexes obtained on stabilized tubulin are seeded by precise biological and biophysical data. New avenues opened by thermodynamics analysis, high throughput screening, and proteomics for the molecular pharmacology of these drugs are presented. The amount of data generated by biophysical, proteomic and cellular techniques shed more light onto the microtubule-tubulin equilibrium and tubulin-drug interaction. Combining these approaches provides new insight into the mechanism of action of known microtubule interacting agents and rapid in-depth characterization of next generation molecules targeting the interaction between microtubules and associated modulators of their dynamics. This will facilitate the design of improved and/or alternative chemotherapies targeting the microtubule cytoskeleton.
SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies ...demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a ΔsifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.
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