The AD3 locus of synaptotagmin-1 C2 domains modulates domain stability.

Synaptotagmin-1 (syt1) functions as the Ca-dependent sensor that triggers the rapid and synchronous release of neurotransmitters from neurotransmitter-containing vesicles during neuronal exocytosis. The syt1 protein has two homologous tandem C2 domains that interact with phospholipids in a Ca-dependent manner. Despite the crucial role of syt1 in exocytosis, the precise interactions between Ca, syt1, and phospholipids are not fully understood.

Structural Basis for the Distinct Membrane Binding Activity of the Homologous C2A Domains of Myoferlin and Dysferlin.

Dysferlin has been implicated in acute membrane repair processes, whereas myoferlin's activity is maximal during the myoblast fusion stage of early skeletal muscle cell development. Both proteins are similar in size and domain structure; however, despite the overall similarity, myoferlin's known physiological functions do not overlap with those of dysferlin. Here we present for the first time the X-ray crystal structure of human myoferlin C2A to 1.

FerA is a Membrane-Associating Four-Helix Bundle Domain in the Ferlin Family of Membrane-Fusion Proteins.

Ferlin proteins participate in such diverse biological events as vesicle fusion in C. elegans, fusion of myoblast membranes to form myotubes, Ca-sensing during exocytosis in the hair cells of the inner ear, and Ca-dependent membrane repair in skeletal muscle cells. Ferlins are Ca-dependent, phospholipid-binding, multi-C2 domain-containing proteins with a single transmembrane helix that spans a vesicle membrane.

Alternate splicing of dysferlin C2A confers Ca²⁺-dependent and Ca²⁺-independent binding for membrane repair.

Dysferlin plays a critical role in the Ca²⁺-dependent repair of microlesions that occur in the muscle sarcolemma. Of the seven C2 domains in dysferlin, only C2A is reported to bind both Ca²⁺ and phospholipid, thus acting as a key sensor in membrane repair. Dysferlin C2A exists as two isoforms, the "canonical" C2A and C2A variant 1 (C2Av1).

Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain.

Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation.

Structural mapping of post-translational modifications in human interleukin-24: role of N-linked glycosylation and disulfide bonds in secretion and activity.

Human interleukin-24 (IL-24) is unique among the IL-10 superfamily as there is considerable evidence that it possesses multiple anti-cancer properties, including direct tumor cell cytotoxicity, helper T cell (TH1) immune stimulation, and anti-angiogenic activities. The primary sequence of human IL-24 differs from homologous cytokines, because it possesses three consensus N-linked glycosylation sites and the potential for a single disulfide bond. To address the significance of these modifications in human IL-24, we analyzed the relationship between post-translational modifications and the cytokine activity of the human IL-24 protein.

The c2 domains of human synaptotagmin 1 have distinct mechanical properties.

Synaptotagmin 1 (Syt1) is the Ca(+2) receptor for fast, synchronous vesicle fusion in neurons. Because membrane fusion is an inherently mechanical, force-driven event, Syt1 must be able to adapt to the energetics of the fusion apparatus. Syt1 contains two C2 domains (C2A and C2B) that are homologous in sequence and three-dimensional in structure; yet, a number of observations have suggested that they have distinct biochemical and biological properties.

Structure of human synaptotagmin 1 C2AB in the absence of Ca2+ reveals a novel domain association.

Release of neurotransmitter from synaptic vesicles requires the Ca2+/phospholipid-binding protein synaptotagmin 1. There is considerable evidence that cooperation between the tandem C2 domains of synaptotagmin is a requirement of regulated exocytosis; however, high-resolution structural evidence for this interaction has been lacking. The 2.

Purification, crystallization and X-ray diffraction analysis of human synaptotagmin 1 C2A-C2B.

Synaptotagmin acts as the Ca(2+) sensor for neuronal exocytosis. The cytosolic domain of human synaptotagmin 1 is composed of tandem C2 domains: C2A and C2B. These C2 domains modulate the interaction of synaptotagmin with the phospholipid bilayer of the presynaptic terminus and effector proteins such as the SNARE complex.

MDA-7/IL-24 is a unique cytokine--tumor suppressor in the IL-10 family.

The melanoma differentiation associated gene-7 (mda-7) cDNA was isolated by virtue of being induced during melanoma differentiation. Initial gene transfer studies convincingly demonstrated potent antitumor effects of mda-7. Further studies showed that the mechanism of antitumor activity was due to induction of apoptosis.

Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP).

Fibroblast growth factor-8 protects cultured rat hippocampal neurons from oxidative insult.

Basic fibroblast growth factor (bFGF) has been reported to have neuroprotective properties following excitotoxic, metabolic, and oxidative insults. We report here that another FGF family member, FGF-8 is able to protect rat hippocampal cultures from oxidative stress. The b isoform of FGF-8 protected hippocampal cultures from hydrogen peroxide with an EC50 of approximately 25 ng/ml.

Comparison of the heme iron utilization systems of pathogenic Vibrios.

Vibrio alginolyticus, Vibrio fluvialis, and Vibrio parahaemolyticus utilized heme and hemoglobin as iron sources and contained chromosomal DNA similar to several Vibrio cholerae heme iron utilization genes. A V. parahaemolyticus gene that performed the function of V.

Stereoselective neuroprotection by novel 2,3-benzodiazepine non-competitive AMPA antagonist against non-NMDA receptor-mediated excitotoxicity in primary rat hippocampal cultures.

Glutamate excitotoxicity has been implicated in a variety of acute and chronic neurodegenerative diseases but early phase clinical trials with competitive antagonists at both N-methyl-D-aspartate (NMDA)-receptors and alpha-amino-3-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptors have been disappointing. A family of atypical 2,3 benzodiazepines, exemplified by GYKI 52466, have been described recently which function as non-competitive AMPA-receptor antagonists. We have investigated the neuroprotective efficacy of LY303070 and LY300164, two analogs of GYKI-52466, in an embryonic rat hippocampal culture model of non-NMDA receptor-mediated excitotoxicity using kainic acid (KA) as an agonist at the AMPA/KA receptor.

Characterization of LY231617 protection against hydrogen peroxide toxicity.

The compound LY231617 [2,6-bis(1,1-dimethylethyl)-4-[[(1-ethyl)amino]methyl]phenol hydrochloride] has been reported to afford significant neuroprotection against hydrogen peroxide (H2O2)-induced toxicity in vitro and global ischemia in vivo. We now report on further mechanistic studies of H2O2 toxicity and protection by LY231617. Brief exposure to H2O2 (15 min) elicited an oxidative insult comparable with that generated by overnight treatment.

Characterization of 8-epiprostaglandin F2alpha as a marker of amyloid beta-peptide-induced oxidative damage.

The amyloid beta-peptide (A beta) is a major component of the neuritic plaques that are a defining histological characteristic of Alzheimer's disease. A beta can be directly toxic and pro-inflammatory to cells in vitro. Numerous reports have shown that oxidative damage and reactive oxygen species play a role in A beta-mediated neurotoxicity.

Induction of sulfated glycoprotein-2 (clusterin) and glial fibrillary acidic protein (GFAP) RNA expression following transient global ischemia is differentially attenuated by LY231617.

Sulfated glycoprotein-2 (SGP-2) is a secreted glycoprotein that along with GFAP has emerged as a prominent molecular marker of neurodegeneration. In the present study, we have evaluated further the relationship between SGP-2, GFAP and neurodegeneration, by examining the effects of LY231617, a potent antioxidant, on expression of SGP-2 and GFAP following four vessel occlusion (4VO). GFAP and SGP-2 RNA levels increased several fold in hippocampus and caudate nucleus in response to 30 min of 4VO.

Amyloid beta-mediated oxidative and metabolic stress in rat cortical neurons: no direct evidence for a role for H2O2 generation.

H2O2 and free radical-mediated oxidative stresses have been implicated in mediating amyloid beta (1-40) [A beta (1-40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H2O2-scavenging enzyme catalase protects neurons in culture against A beta-mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H2O2. A beta-mediated elevation in intracellular H2O2 production is suppressed by addition of a potent H2O2 scavenger without any significant neuroprotection.

Clusterin (Apo J) protects against in vitro amyloid-beta (1-40) neurotoxicity.

Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimer's disease (AD), where it is found associated with amyloid-beta (A beta) plaques. Clusterin also coprecipitates with A beta from CSF, suggesting a physiological interaction with A beta.

Selective protection against AMPA- and kainate-evoked neurotoxicity by (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahyd roisoquinoline- 3-carboxylic acid (LY293558) and its racemate (LY215490).

Glutamate receptor-mediated excitotoxicity is linked to the activation of multiple receptors including those activated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate. In this study, the novel glutamate receptor antagonist, as its active isomer (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-decahyd roisoquinoline-3- carboxylic acid ((-)LY293558) and it's +/- racemate (LY215490), was examined for neuroprotectant effects against excitotoxic injury in vitro and in vivo. This agent selectively protected against AMPA and kainate injury in cultured primary rat hippocampal neurons, an in vivo rat striatal neurotoxicity model, and against agonist-evoked seizures in mice.

Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro.

Amyloid beta peptide (A beta), the major protein constituent of senile plaques in patients with Alzheimer's disease, is believed to facilitate the progressive neurodegeneration that occurs in the latter stages of this disease. Early attempts to characterize the structure-activity relationship of A beta toxicity in vitro were compromised by the inability to reproducibly elicit A beta-dependent toxicity across different lots of chemically equivalent peptides. In this study we used CD spectroscopy to demonstrate that A beta secondary structure is an important determinant of A beta toxicity.

Neurotoxicity of human amylin in rat primary hippocampal cultures: similarity to Alzheimer's disease amyloid-beta neurotoxicity.

Amylin, a 37-amino-acid amyloidogenic peptide, bears biophysical similarities to the amyloid-beta peptide (A beta) deposited in Alzheimer's disease. Using embryonic rat hippocampal cultures we tested whether amylin induces neurotoxicity similar to that previously observed with A beta(1-40). Treatment with human amylin(1-37) resulted in prominent toxicity as assessed by phase-contrast microscopy and quantification of lactate dehydrogenase in the medium.

Thyrotropin-releasing hormone gene expression and receptors are differentially modified in limbic foci by seizures.

Previous studies using two seizure paradigms, electroconvulsive shock and kindling, suggested potential sites of endogenous thyrotropin-releasing hormone (TRH) action in specific epileptogenic areas. We studied TRH gene expression and TRH receptors in rat limbic areas using the kindling model of epilepsy. Immunoassayable TRH increased 4- to 20-fold over control levels in specific subregions of the hippocampus 24 hours after a single stage 5 seizure.

Sulfated glycoprotein-2 expression increases in rodent brain after transient global ischemia.

Sulfated glycoprotein-2 (SGP-2) is emerging as a prominent marker of neurodegeneration in mammalian brain. Regulation of brain SGP-2 was studied in adult male Wistar rats subjected to 30 min of forebrain ischemia by four vessel occlusion. By 3 days after the ischemic insult, SGP-2 RNA levels were increased two fold in caudate nucleus and hippocampus.