Studies have shown that peripheral levels of corticosterone correlate with the magnitudes of two well-described physiological models of memory, long-term potentiation (LTP) and primed burst (PB) potentiation. In the present experiments, the authors investigated the effects of experimenter-controlled manipulations of the levels of corticosterone on the magnitude of hippocampal PB potentiation in urethane-anesthetized rats. Primed burst potentiation is a long-lasting (at least 30 minutes) increase in the amplitude of the CA1 population spike and EPSP slope in response to physiologically patterned stimulation of the hippocampal commissure. The levels of serum corticosterone were controlled by implanting corticosterone pellets in adrenalectomized rats (ADX/PELLET). In the first experiment, a significant negative linear correlation between elevated (stress) levels of serum corticosterone (greater than 20 micrograms/dL) and the magnitude of PB potentiation in ADX/PELLET subjects (r = 0.60, P < .05) was found. In the second experiment, the shape of the corticosterone-PB potentiation function was different at low and intermediate levels of corticosterone than it was at high levels of corticosterone: There was a positive correlation at low levels (0-10 micrograms/dL), a peak response at intermediate levels (11-20 micrograms/dL), and a negative correlation at high levels (21-93 micrograms/dL) of corticosterone. Thus, the overall relationship between corticosterone and PB potentiation is an inverted-U function. These findings provide strong support for the hypothesis that corticosterone exerts a concentration-dependent biphasic influence on the expression of hippocampal plasticity.

Mutations in ␣-synuclein are known to be associated with Parkinson's disease (PD). The coexistence of this neuronal protein with ubiquitin and proteasome subunits in Lewy bodies in sporadic disease suggests that alterations of ␣-synuclein catabolism may contribute to the pathogenesis of PD. The degradation pathway of ␣-synuclein has not been identified nor has the kinetics of this process been described. We investigated the degradation kinetics of both wild-type and A53T mutant 6XHis-tagged ␣-synuclein in transiently transfected SH-SY5Y cells. Degradation of both isoforms followed firstorder kinetics over 24 h as monitored by the pulse-chase method. However, the t1 ⁄2 of mutant ␣-synuclein was 50% longer than that of the wild-type protein (p < 0.01). The degradation of both recombinant proteins and endogenous ␣-synuclein in these cells was blocked by the selective proteasome inhibitor ␤-lactone (40 M), indicating that both wild-type and A53T mutant ␣-synuclein are degraded by the ubiquitin-proteasome pathway. The slower degradation of mutant ␣-synuclein provides a kinetic basis for its intracellular accumulation, thus favoring its aggregation.

No abstract

IntroductionThe ability of neoplastic cells to evade the immune system remains a formidable barrier limiting the success of immunotherapy. Tumor cells can employ various mechanisms to escape detection by immune cells. These can include down-regulation of major histocompatibility complex (MHC) class I expression, 1-4 production of immunosuppressive cytokines such as transforming growth factor-␤, 1,5 up-regulation of Fas ligand, 6 and deregulation of zeta chain on T cells. 7 In both mouse and human, natural killer (NK) cells are composed of different subsets, which are characterized by the expression of inhibitory and/or activating receptors specific for MHC class I determinants. [8][9][10][11] In mice, these receptors belong to the family of Ly49 receptors, which are lectinlike molecules. 12 The human counterpart, killer immunoglobulin-like receptors, belongs to the immunoglobulin superfamily. 11 A small percentage of T cells in mice also express Ly49 receptors. 13 It has been shown that binding of the inhibitory receptors by the appropriate class I molecules results in generation of negative signals leading to inactivation of NK cell functions. [13][14][15] This inhibitory signal has been shown to dominate over activating stimuli. 15 Furthermore, the rapid rejection of tumors lacking the expression of MHC class I by NK cells demonstrates the pivotal role MHC plays in regulating NK function. [16][17][18] In spite of these studies demonstrating the functions of the inhibitory receptors in vitro, the in vivo functions of these receptors on either NK or T cells remain to be elucidated.One potential means for tumor escape may be by expressing MHC class I determinants at a level that allows sufficient binding of the Ly49 inhibitory receptors and thus escape from NKmediated killing. In To examine the effects of blockade of the inhibitory receptors on antitumor activity, we have used a C1498 mouse leukemia model and F(abЈ) 2 fragments of 5E6 monoclonal antibody (mAb), 21 which binds to Ly49C and I receptors, for in vitro as well as in vivo studies. The use of F(abЈ) 2 fragments allowed us to examine the responses that are due to blocking the Ly49 receptors without For personal use only. on May 12, 2018. by guest www.bloodjournal.org From depletion of the subset in vivo. The results from these studies demonstrate that blockade of Ly49 inhibitory receptors augments NK cell-mediated antitumor effects and that strategies to block NK inhibitory receptor interactions may be of potential use in cancer therapy. Materials and methods MiceC57BL/6 (B6, H2 b ) mice were obtained from the Animal Production Area (National Cancer Institute at Frederick [NCI-Frederick], MD), and B6 severe combined immunodeficient scid/scid (SCID) mice were generously provided by Dr Robert H. Wiltrout (NCI-Frederick). All mice were kept in a specific pathogen-free condition and used at 8 to 12 weeks of age. Antibodies and generation of F(ab) 2 fragmentsAntimouse Fc␥R (2.4G2, rat immunoglobulin [Ig]-G2a), fluorescein isothiocyanate (FITC)-conjugated anti...

In Parkinson's disease (PD), conformational changes in the alpha-synuclein monomer precede the formation of Lewy bodies. We examined postmortem PD and undiseased (control) substantia nigra for evidence of pathological crosslinking of alpha-synuclein by tissue transglutaminase (tTG) using immunohistochemistry, immunoprecipitation, and Western blot. Consistent with previous reports, we found that both tTG and its substrate-characteristic N(epsilon)-(gamma-glutamyl)-lysine crosslink are increased in PD nigral dopamine neurons. Furthermore, both the tTG protein and its substrate crosslink coprecipitated with alpha-synuclein in extracts of PD substantia nigra. Unexpectedly, the isodipeptide crosslink was detected in the alpha-synuclein monomer as well as in higher molecular mass oligomers of alpha-synuclein. Although the intramolecularly crosslinked alpha-synuclein monomer was present in control tissue, it was highly enriched in PD substantia nigra. Conversely, significantly less uncrosslinked alpha-synuclein remained in the postimmunoprecipitate lysate of PD tissue than in control. Crosslinked alpha-synuclein, formed at the expense of the total alpha-synuclein monomer, correlated with disease progression. These results demonstrate that much of the alpha-synuclein monomer in PD nigra is crosslinked by tTG and thus may be functionally impaired. This modification appears to be an early step in PD pathogenesis, preceding the aggregation of alpha-synuclein in Lewy bodies.

Helicobacter pylori causes a common chronic infection of humans that leads to epithelial cell damage. Studies have shown that apoptosis of the gastric epithelium is increased during infection and this response is associated with an expansion of gastric T-helper type 1 (Th1) cells. We report that gastric T cells contribute to apopto-Helicobacter pylori causes a lifelong infection of the gastric antrum that affects more than 50% of humanity. Infection is associated with chronic antral gastritis, characterized by a mucosal infiltration of polymorphonuclear and mononuclear leukocytes (10, 14). Evidence for a pathogenic role of H. pylori infection in peptic ulcer is derived from clinical investigations showing that cure of H. pylori infection accelerates ulcer healing and prevents ulcer relapse (20). Besides ulcer disease, H. pylori infection has also been implicated as a cause of gastric lymphoma or carcinoma in some patients (7). There are many reports that H. pylori infection interferes with the equilibrium between proliferation and apoptosis of the gastric epithelium (4,7,25,32,35,49). Most of the in situ studies have shown that the number of apoptotic epithelial cells increases during H. pylori infection (25,32,35,41). Numerous mechanisms could account for this, including the direct effects of the bacteria, as well as the inflammatory response elicited by the infection (13,24,41,49).Several independent approaches have suggested that Thelper type 1 (Th1) cells are selectively increased during infection (3,9,18,26,31). Th1 cytokines, such as gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣), can increase the release of proinflammatory cytokines, such as interleukin-8, from the epithelium (52), as well as Fas and Fas ligand (FasL) (6). Furthermore, these cytokines can also increase the expression of major histocompatibility complex class II molecules by gastric epithelial cells, thereby increasing the binding of H. pylori to the gastric epithelium (13). As Th1 cells are associated with cell-mediated immune responses, they may also play a role in damaging gastric tissues directly by triggering apoptosis in gastric epithelial cells.Th1 cells can also express higher levels of FasL than Th2 cells (40,46). This molecule belongs to the TNF family of proteins. The receptor of FasL, Fas (CD95), is a 45-kDa protein belonging to the TNF receptor family. Cross-linking of Fas with an agonistic immunoglobulin M (IgM) antibody or FasL will transduce the death signal to the cells, resulting in the induction of apoptosis. However, studies to date have not shown that gastric T cells express FasL and induce epithelial cell death by Fas/FasL interactions. The purpose of this study was to investigate the role of H. pylori in the modulation of lymphoepithelial interactions in the stomach that are mediated through Fas/FasL. MATERIALS AND METHODS Subjects.Material from human tissue was obtained from consenting adults aged 20 to 55 years as approved by the respective institutional review boards at the National University of...

Protein synthesis has long been known to be required for associative learning to consolidate into long-term memory. Here we demonstrate that PKC isozyme activation on days before training can induce the synthesis of proteins necessary and sufficient for subsequent long-term memory consolidation. Bryostatin (Bryo), a macrolide lactone with efficacy in subnanomolar concentrations and a potential therapeutic for Alzheimer's disease, is a potent activator of PKC, some of whose isozymes undergo prolonged activation after associative learning. Under normal conditions, two training events with paired visual and vestibular stimuli cause short-term memory of the mollusc Hermissenda that lasts Ϸ7 min. However, after 4-h exposures to Bryo (0.25 ng͞ml) on two preceding days, the same two training events produced long-term conditioning that lasted >1 week and that was not blocked by anisomycin (1 g͞ml). Anisomycin, however, eliminated long-term memory lasting at least 1 week after nine training events. Both the nine training events alone and two Bryo exposures plus two training event regimens caused comparably increased levels of the PKC ␣-isozyme substrate calexcitin in identified type B neurons and enhanced PKC activity in the membrane fractions. Furthermore, Bryo increased overall protein synthesis in cultured mammalian neurons by up to 60% for >3 days. The specific PKC antagonist Ro-32-0432 blocked much of this Bryo-induced protein synthesis as well as the Bryo-induced enhancement of the behavioral conditioning. Thus, Bryo-induced PKC activation produces those proteins necessary and sufficient for long-term memory on days in advance of the training events themselves.bryostatin ͉ PKC isozymes T he requirement of protein synthesis for long-term memory has been demonstrated over several decades for a variety of memory paradigms (1-14). It was originally shown that druginduced inhibition of protein synthesis (e.g., with 5-propyluracil or anisomycin) blocked long-term memory when this inhibition occurred during a critical time interval after the training paradigm (9). It has remained a mystery as to what specific, critical proteins were so essential for memory consolidation and how their molecular regulation was so necessary for long-lasting memory storage.In many species the formation of long-term associative memory has also been shown to depend on translocation, and thus activation, of protein kinase C (PKC) isozymes to neuronal membranes. PKC activation has been shown to occur in single identified type B cells of the mollusc Hermissenda (15) with Pavlovian conditioning and a variety of mammalian associative learning protocols (16)(17)(18). Furthermore, a high-affinity substrate of the ␣-isozyme of PKC, calexcitin (CE) (19), was found within single identified type B cells to show Pavlovianconditioning-dependent increases of phosphorylation and absolute quantity (20).Consistent with these findings, administration of the potent PKC activator bryostatin (Bryo) enhanced rat spatial maze learning (21). Bryo, a macrolide lactone,...

The degeneration of nigral dopamine neurons in Parkinson's disease (PD) reportedly involves a defect in brain mitochondrial complex I in association with the activation of nuclear factor-jB (NF-jB) and caspase-3. To elucidate molecular mechanisms possibly linking these events, as well as to evaluate the neuroprotective potential of the cyclopentenone prostaglandin A 1 (PGA 1 ), an inducer of heat shock proteins (HSPs), we exposed human dopaminergic SH-SY5Y cells to the complex I inhibitor rotenone. Dose-dependent apoptosis was preceded by the nuclear translocation of NF-jB and then the activation of caspase-3 over the ensuing 24 h. PGA 1 increased the expression of HSP70 and HSP27 and protected against rotenone-induced apoptosis, without increasing necrotic death. PGA 1 blocked the rotenone-induced nuclear translocation of NF-jB and attenuated, but did not abolish, the caspase-3 elevation. Unexpectedly, the caspase-3 inhibitor, Ac-DEVD.CHO (DEVD), at a concentration that completely prevented the caspase-3 elevation produced by rotenone, failed to protect against apoptosis. These results suggest that complex I deficiency in dopamine cells can induce apoptosis by a process involving early NF-jB nuclear translocation and caspase-3 activation. PGA 1 appears to protect against rotenone-induced cell death by inducing HSPs and blocking nuclear translocation of NF-jB in a process that attenuates caspase-3 activation, but is not mediated by its inhibition.