Corticotropin-releasing factor receptors couple to multiple g-proteins to activate diverse intracellular signaling pathways in mouse hippocampus: role in neuronal excitability and associative learning

Corticotropin-releasing factor (CRF) exerts a key neuroregulatory control on stress responses in various regions of the mammalian brain, including the hippocampus. Using hippocampal slices, extracts, and whole animals, we investigated the effects of human/rat CRF (h/rCRF) on hippocampal neuronal excitability and hippocampus-dependent learning in two mouse inbred strains, BALB/c and C57BL/6N. Intracellular recordings from slices revealed that application of h/rCRF increased the neuronal activity in both mouse inbred strains. Inhibition of protein kinase C (PKC) by bisindolylmaleimide I (BIS-I) prevented the h/rCRF effect only in hippocampal slices from BALB/c mice but not in slices from C57BL/6N mice. Inhibition of cAMP-dependent protein kinase (PKA) by H-89 abolished the h/rCRF effect in slices from C57BL/6N mice, with no effect in slices from BALB/c mice. Accordingly, h/rCRF elevated PKA activity in hippocampal slices from C57BL/6N mice but increased only PKC activity in the hippocampus of BALB/c mice. These differences in h/rCRF signal transduction were also observed in hippocampal membrane suspensions from both mouse strains. In BALB/c mice, hippocampal CRF receptors coupled to Gq/11 during stimulation by h/rCRF, whereas they coupled to Gs, Gq/11, and Gi in C57BL/6N mice. As expected on the basis of the slice experiments, h/rCRF improved context-dependent fear conditioning of BALB/c mice in behavioral experiments, and BIS-I prevented this effect. However, although h/rCRF increased neuronal spiking in slices from C57BL/6N mice, it did not enhance conditioned fear. These results indicate that the CRF system activates different intracellular signaling pathways in mouse hippocampus and may have distinct effects on associative learning depending on the mouse strain investigated

Rabaptin5 is recruited to endosomes by Rab4 and Rabex5 to regulate endosome maturation

Rab GTPases control membrane identity, fusion, and transport by interaction with effector proteins. Effectors that influence the activation/inactivation cycle of their own or other Rabs contribute to the timely conversion of Rab identities. Rab5 and its effector Rabaptin5 are generally considered the prime example for a positive feedback loop in which Rab5·GTP recruits Rabaptin5 complexed to Rabex5, the GDP/GTP exchange factor of Rab5, to early endosomes, thus maintaining the membrane's Rab5 identity. By deletion analysis, we found membrane recruitment of Rabaptin5 to require binding to Rab4 and Rabex5, but not Rab5. Deletion of either one of two Rab5 binding domains or silencing of Rab5 expression did not affect Rabaptin5 recruitment, but produced giant endosomes with early and late endosomal characteristics. The results contradict feedback activation of Rab5 and instead indicate that Rabaptin5 is recruited by Rabex5 recognizing ubiquitinated cargo and by Rab4 to activate Rab5 in a feed-forward manner

Asbestos modulates thioredoxin-thioredoxin interacting protein interaction to regulate inflammasome activation

BACKGROUND: Asbestos exposure is related to various diseases including asbestosis and malignant mesothelioma (MM). Among the pathogenic mechanisms proposed by which asbestos can cause diseases involving epithelial and mesothelial cells, the most widely accepted one is the generation of reactive oxygen species and/or depletion of antioxidants like glutathione. It has also been demonstrated that asbestos can induce inflammation, perhaps due to activation of inflammasomes. METHODS: The oxidation state of thioredoxin was analyzed by redox Western blot analysis and ROS generation was assessed spectrophotometrically as a read-out of solubilized formazan produced by the reduction of nitrotetrazolium blue (NTB) by superoxide. Quantitative real time PCR was used to assess changes in gene transcription. RESULTS: Here we demonstrate that crocidolite asbestos fibers oxidize the pool of the antioxidant, Thioredoxin-1 (Trx1), which results in release of Thioredoxin Interacting Protein (TXNIP) and subsequent activation of inflammasomes in human mesothelial cells. Exposure to crocidolite asbestos resulted in the depletion of reduced Trx1 in human peritoneal mesothelial (LP9/hTERT) cells. Pretreatment with the antioxidant dehydroascorbic acid (a reactive oxygen species (ROS) scavenger) reduced the level of crocidolite asbestos-induced Trx1 oxidation as well as the depletion of reduced Trx1. Increasing Trx1 expression levels using a Trx1 over-expression vector, reduced the extent of Trx1 oxidation and generation of ROS by crocidolite asbestos, and increased cell survival. In addition, knockdown of TXNIP expression by siRNA attenuated crocidolite asbestos-induced activation of the inflammasome. CONCLUSION: Our novel findings suggest that extensive Trx1 oxidation and TXNIP dissociation may be one of the mechanisms by which crocidolite asbestos activates the inflammasome and helps in development of MM

Transferrinfection. A Highly Efficient Way to Express Gene Constructs in Eukaryotic Cells

No abstract available

Fe-rich ferropericlase and magnesiow\ufcstite inclusions reflecting diamond formation rather than ambient mantle

At the core of many Earth-scale processes is the question of what the deep mantle is made of. The only direct samples from such extreme depths are diamonds and their inclusions. It is commonly assumed that these inclusions reflect ambient mantle or are syngenetic with diamond, but these assumptions are rarely tested. We have studied inclusion\u2013host growth relationships in two potentially superdeep diamonds from Juina (Brazil) containing nine inclusions of Fe-rich (XFe 480.33 to 650.64) ferropericlase-magnesiow\ufcstite (FM) by X-ray diffractometry, X-ray tomography, cathodoluminescence, electron backscatter diffraction, and electron microprobe analysis. The inclusions share a common [112] zone axis with their diamonds and have their major crystallographic axes within 3\ub0\u20138\ub0 of those of their hosts. This suggests a specific crystallographic orientation relationship (COR) resulting from interfacial energy minimization, disturbed by minor post-entrapment rotation around [112] due to plastic deformation. The observed COR and the relationships between inclusions and diamond growth zones imply that FM nucleated during the growth history of the diamond. Therefore, these inclusions may not provide direct information on the ambient mantle prior to diamond formation. Consequently, a \u201cnon-pyrolitic\u201d composition of the lower mantle is not required to explain the occurrence of Fe-rich FM inclusions in diamonds. By identifying examples of mineral inclusions that reflect the local environment of diamond formation and not ambient mantle, we provide both a cautionary tale and a means to test diamond-inclusion time relationships for proper application of inclusion studies to whole-mantle questions

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system