Extracellular levels of the excitatory neurotransmitter glutamate in the nervous system are maintained by transporters that actively remove glutamate from the extracellular space. Homozygous mice deficient in GLT-1, a widely distributed astrocytic glutamate transporter, show lethal spontaneous seizures and increased susceptibility to acute cortical injury. These effects can be attributed to elevated levels of residual glutamate in the brains of these mice.

To study the function of GLAST, a glutamate transporter highly expressed in the cerebellar Bergmann astrocytes, the mouse GLAST gene was inactivated. GLAST-deficient mice developed normally and could manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging task such as staying on a quickly rotating rod. Electrophysiological examination revealed that Purkinje cells in the mutant mice remained to be multiply innervated by climbing fibres even at the adult stage. We also found that oedema volumes in the mutant mice increased significantly after cerebellar injury. These results indicate that GLAST plays active roles both in the cerebellar climbing fibre synapse formation and in preventing excitotoxic cerebellar damage after acute brain injury.

Apoptotic cell death, characterized by chromatin condensation, nuclear fragmentation, cell membrane blebbing, and apoptotic body formation, is also accompanied by typical mitochondrial changes. The latter includes enhanced membrane permeability, fall in mitochondrial membrane potential (⌬ m ) and release of cytochrome c into the cytosol. Gelsolin, an actin regulatory protein, has been shown to inhibit apoptosis, but when cleaved by caspase-3, a fragment that is implicated as an effector of apoptosis is generated. The mechanism by which the full-length form of gelsolin inhibits apoptosis is unclear. Here we show that the overexpression of gelsolin inhibits the loss of ⌬ m and cytochrome c release from mitochondria resulting in the lack of activation of caspase-3, -8, and -9 in Jurkat cells treated with staurosporine, thapsigargin, and protoporphyrin IX. These effects were corroborated in vitro using recombinant gelsolin protein on isolated rat mitochondria stimulated with Ca 2؉ , atractyloside, or Bax. This protective function of gelsolin, which was not due to simple Ca 2؉ sequestration, was inhibited by polyphosphoinositide binding. In addition we confirmed that gelsolin, besides its localization in the cytosol, is also present in the mitochondrial fraction of cells. Gelsolin thus acts on an early step in the apoptotic signaling at the level of mitochondria.

Regulation of the human proto-oncogene MYC apparently plays an important role in cellular proliferation and the genesis of diverse tumors. Transcription from MYC is governed principally by two promoters known as P1 and P2. Previously we have detected a negative regulator of these promoters upstream of MYC. We now report that this regulator comprises no more than 26 bp of DNA, with sequence that resembles the regulators of at least two other genes, and we describe nuclear factors that interact with the regulator. Nuclear extracts from human cells form three distinctive complexes with the negative regulator. One of these complexes includes the product of the proto-oncogene FOS or an antigenically related protein, and the FOS protein may, in turn, be associated with the product of the proto-oncogene JUN. Similarly, FOS and JUN proteins produced by translation in vitro bind cooperatively to the negative regulator. These results raise the possibility that FOS and JUN participate in the regulation of MYC.

Hirschsprung's disease (HSCR) is a congenital intestinal disease, characterized by the absence of ganglion cells in the distal portion of the intestinal tract. Recently, three susceptibility genes have been identified in HSCR, namely the RET protooncogene, the endothelin B (ET B ) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3). To investigate whether mutations in EDNRB could be related with HSCR in non-inbred populations in Japan, we examined alterations of the gene in 31 isolated patients. Three novel mutations were detected as follows: two transversions, A to T and C to A at nucleotides 311 (N104I) and 1170 (S390R), respectively, and a transition, T to C at nucleotide 325 (C109R). To analyze functions of these mutant receptors, they were expressed in Chinese hamster ovary cells. S390R mutation did not change the binding affinities but caused the decreases in the ligand-induced increment of intracellular calcium and in the inhibition of adenylyl cyclase activity, showing the impairment of the intracellular signaling. C109R receptors were proved to be localized near the nuclei as an unusual 44-kDa protein with the extremely low affinity to endothelin-1 (ET-1) and not to be translocated into the plasma membrane. On the other hand, N104I receptors showed almost the same binding affinities and functional properties as those of the wild type. Therefore, we conclude that S390R and C109R mutations could cause HSCR but that N104I mutation might be polymorphous. Hirschsprung's disease (HSCR)1 is a congenital intestinal disorder, characterized by the absence of ganglion cells in the distal portion of the intestinal tract, as a consequence of premature arrest of cranio-caudal migration of neural crest cells. Recently, three susceptibility genes have been identified in HSCR, namely the RET protooncogene, the endothelin B (ET B ) receptor gene (EDNRB), and the endothelin-3 (ET-3) gene (EDN3).Mice with targeted null disruption of Ret exhibited an autosomal recessive phenotype comprising the total lack of the enteric nervous system and renal agenesis (1), that provided an excellent model of HSCR. Approximately 50 mutations in RET have so far been identified, and the RET mutations account for 50% of familial and 15-20% of sporadic cases of HSCR, over 75% of which are associated with long segment HSCR (2-6).Endothelins (ETs) are a family of 21 amino acid vasoactive peptides, consisting of three isopeptides, ET-1, ET-2 and ET-3, that act on G protein-coupled receptors, ET A and ET B (7-11). ET A exhibits different affinities to the three isopeptides in the order of ET-1 Ն ET-2 Ͼ Ͼ ET-3. ET B accepts all three isopeptides equally. These receptors regulate multiple effector pathways, for example, phospholipase C via G q , and adenylyl cyclase via G s in smooth muscle cells (ET A ), and via G i in endothelial cells (ET B ) (12, 13). Mice with targeted null disruption of either Ednrb or Edn3 exhibit an identical phenotype, coat color spotting, and aganglionic megacolon, similar to HSCR or Shah-Waardenburg syndrome in...

Epithelial-mesenchymal transition (EMT) describes a process occurring during development and oncogenesis by which epithelial cells obtain fibroblast-like properties and show reduced cell adhesion and increased motility. In this report, we demonstrated typical EMT in human mammary epithelial MCF10A small interfering (si)RNA gelsolin-knockdown cells. EMT was characterized by fibroblastic morphology, loss of contact inhibition and focus formation in monolayer growth, enhanced motility and invasiveness in vitro, increased actin filaments, overexpression of RAC, activation of both extracellular signal-regulated kinase and AKT, inactivation of glycogen synthase kinase-3, conversion of cadherin from the E-to N-type and induction of the transcription factor Snail. These results suggested that gelsolin functions as a switch that controls E-and N-cadherin conversion via Snail, and demonstrated that its knockdown leads to EMT in human mammary epithelial cells and possibly to the development of human mammary tumors. ' 2005 Wiley-Liss, Inc.

Amyloid-beta (Abeta) peptides play a central role in the development of Alzheimer's disease. They are known to induce mitochondrial dysfunction and caspase activation, resulting in apoptosis of neuronal cells. Here we show that human cytoplasmic gelsolin inhibits Abeta peptide-induced cell death of neuronally differentiated rat pheochromocytoma (PC-12) cells. We also show that the segment 5 but not 6 of human cytoplasmic gelsolin is the important region responsible for inhibition of Abeta-induced cytotoxicity. Mitochondrial dysfunction associated with cell death, membrane potential loss and the release of cytochrome c are all abrogated in the presence of human full-length or segment 5 cytoplasmic gelsolin. Furthermore, RNA interference to reduce expression of endogenous gelsolin in PC12 cells shows that rat gelsolin act as an inhibitor of Abeta cytotoxicity. These results demonstrate that cytoplasmic gelsolin plays a important role in inhibiting Abeta-induced cytotoxicity by inhibiting apoptotic mitochondrial changes. The segment 5 of human cytoplasmic gelsolin is sufficient for the function.

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