Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2). METHODS: In this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis. RESULTS: Elevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-ß upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion. CONCLUSIONS: These findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Protective effect of low-dose radiation on doxorubicin-induced brain injury in mice.

BACKGROUND: To investigate the protective effect of low-dose radiation (LDR) on brain injury in mice induced by doxorubicin (DOX). METHODS: Sixty female BALB/C mice were randomly divided into the control (CTR) group, low-dose radiation (LDR) group, doxorubicin treatment (DOX) group and low-dose radiation before doxorubicin treatment (COM) group. After 72 h of exposure to 75 mGy, the mice were intraperitoneally injected with 7.5 mg/kg of doxorubicin and sacrificed 5 days later. Neuron-specific enolase (NSE), lactate dehydrogenase (LDH), adenosine triphosphate (ATP), neurotransmitters, inflammatory mediators, apoptosis- and oxidative stress-related mediators as well as mitochondrial dysfunction were examined. RESULTS: Compared to the DOX group, the concentrations of DA, 5-HT, EPI and GABA in the COM group were significantly decreased, and the number of TUNEL-positive cells was decreased. In addition, the expression of proapoptotic proteins was downregulated in the COM group compared to the DOX group. Low-dose radiation in advance reduced reactive oxygen species and activated the SOD antioxidant defense system as indicated by significantly reduced GSH expression, increased GSSG expression, increased GPx expression and activation of the Nrf2 redox pathway. After low-dose radiation, the expression levels of ATP5f1, NDUFV1 and CYC1 were close to normal, and the mitochondrial respiratory control rate (RCR) and activity of respiratory chain complex enzymes also tended to be normal. Low-dose radiation upregulated the expression levels of IL-2 and IL-4 but downregulated the expression levels of IL-10 and TGF-ß. CONCLUSION: LDR has a protective effect on brain injury in mice treated with DOX. The mechanism is related to LDR alleviating mitochondrial dysfunction and oxidative stress, which promotes the production of antioxidant damage proteins, thus exerting an adaptive protective effect on cells.

Down-regulation of ABCB1 by collateral sensitivity drugs reverses multidrug resistance and up-regulates enolase I.

The emergence of drug resistance remains an obstacle in the clinical treatment of cancer. Recent developments in the studies of drug resistance have identified compounds such as verapamil and tamoxifen that specifically target ABCB1-expressing multidrug-resistant (MDR) cells, through an ATP-dependent ROS-generating mechanism. In this report, we demonstrate that treatment of ABCB1-expressing MDR cells (CHORC5 or MDA-Doxo400) or individual clones of the latter with sub-lethal concentrations of tamoxifen or verapamil down-regulates ABCB1 protein and mRNA expression in surviving clones. Consequently, tamoxifen- and verapamil-treated cells show increased sensitivity to chemotherapeutic drugs (e.g., colchicine and doxorubicin) and decreased sensitivity to collateral sensitivity drugs (e.g., verapamil and tamoxifen). Importantly, we show for the first time that down-regulation of ABCB1 expression resulting from tamoxifen treatment and CRISPR-knockout of ABCB1 expression up-regulate α-enolase (enolase I) protein levels and activity. These findings demonstrate a possible effect of ABCB1 expression on the metabolic homeostasis of MDR cells. Moreover, given the use of tamoxifen to prevent the recurrence of oestrogen receptor-positive breast cancer, the findings of this study may be clinically important in modulating activity of other drugs.

α-Enolase as a novel vaccine candidate against Streptococcus dysgalactiae infection in cobia (Rachycentron canadum L.).

Streptococcus dysgalactiae is an important pathogenic bacterium that has caused economic loss for the cobia industry in Taiwan, ROC. This study presents a highly effective subunit vaccine composed of a moonlight protein, α-enolase, for the prevention of S. dysgalactiae infection. First, α-enolase was cloned, transformed, and expressed in E. coli for production of recombinant protein. Then, the protective efficacies of α-enolase recombinant protein were evaluated in combination with either a pro-inflammatory cytokine, TNF-α, or an oil adjuvant, ISA 763 AVG. The results showed that the combination of α-enolase and ISA 763 AVG was highly protective (RPS = 88.89%), while a negative effect was found in the group immunised with α-enolase adjuvanted with TNF-α (RPS = 22.22%). A further study was conducted with double dose of ISA 763 AVG, which led to an increased RPS value of 97.37%. Moreover, immunised cobia exhibited significantly greater lysozyme activity, antibody responses, and expression of certain immune-related genes post-challenge. Altogether, our results demonstrated that a combination of α-enolase recombinant protein with ISA 763 AVG adjuvant is a promising vaccine that can be employed for protection of cobia against S. dysgalactiae infection.

Targeting Enolase in Reducing Secondary Damage in Acute Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a complex debilitating condition leading to permanent life-long neurological deficits. The complexity of SCI suggests that a concerted multi-targeted therapeutic approach is warranted to optimally improve function. Damage to spinal cord is complicated by an increased detrimental response from secondary injury factors mediated by activated glial cells and infiltrating macrophages. While elevation of enolase especially neuron specific enolase (NSE) in glial and neuronal cells is believed to trigger inflammatory cascades in acute SCI, alteration of NSE and its subsequent effects in acute SCI remains unknown. This study measured NSE expression levels and key inflammatory mediators after acute SCI and investigated the role of ENOblock, a novel small molecule inhibitor of enolase, in a male Sprague-Dawley (SD) rat SCI model. Serum NSE levels as well as cytokines/chemokines and metabolic factors were evaluated in injured animals following treatment with vehicle alone or ENOblock using Discovery assay. Spinal cord samples were also analyzed for NSE and MMPs 2 and 9 as well as glial markers by Western blotting. The results indicated a significant decrease in serum inflammatory cytokines/chemokines and NSE, alterations of metabolic factors and expression of MMPs in spinal cord tissues after treatment with ENOblock (100 µg/kg, twice). These results support the hypothesis that activation of glial cells and inflammation status can be modulated by regulation of NSE expression and activity. Analysis of SCI tissue samples by immunohistochemistry confirmed that ENOblock decreased gliosis which may have occurred through reduction of elevated NSE in rats. Overall, elevation of NSE is deleterious as it promotes extracellular degradation and production of inflammatory cytokines/chemokines and metabolic factors which activates glia and damages neurons. Thus, reduction of NSE by ENOblock may have potential therapeutic implications in acute SCI.

Identification and characterization of an immunogenic antigen, enolase 2, among excretory/secretory antigens (ESA) of Toxoplasma gondii.

An immunogenic protein, enolase 2, was identified among the secreted excretory/secretory antigens (ESAs) from Toxoplasma gondii strain RH using immunoproteomics based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Enolase 2 was cloned, sequenced, and heterologously expressed. BLAST analysis revealed 75-96% similarity with enolases from other parasites. Immunoblotting demonstrated good immunoreactivity of recombinant T. gondii enolase (Tg-enolase 2) to T. gondii-infected animal serum. Purified Tg-enolase 2 was found to catalyze dehydration of 2-phospho-d-glycerate to phosphoenolpyruvate. In vitro studies revealed maximal activity at pH 7.5 and 37 °C, and activity was inhibited by K(+), Ni(2+), Al(3+), Na(+), Cu(2+) and Cr(3+). A monoclonal antibody against Tg-enolase 2 was prepared, 1D6, with the isotype IgG2a/κ. Western blotting revealed that 1D6 reacts with Tg-enolase 2 and native enolase 2, present among T. gondii ESAs. The indirect immunofluorescence assays showed that enolase 2 could be specifically detected on the growing T. gondii tachyzoites. Immunoelectron microscopy revealed the surface and intracellular locations of enolase 2 on T. gondii cells. In conclusion, our results clearly show that the enzymatic activity of T. gondii enolase 2 is ion dependent and that it could be influenced by environmental factors. We also provide evidence that enolase 2 is an important immunogenic protein of ESAs from T. gondii and that it is a surface-exposed protein with strong antigenicity and immunogenicity. Our findings indicate that enolase 2 could play important roles in metabolism, immunogenicity and pathogenicity and that it may serve as a novel drug target and candidate vaccine against T. gondii infection.

Enolase of Streptococcus Suis Serotype 2 Enhances Blood-Brain Barrier Permeability by Inducing IL-8 Release.

Streptococcus suis serotype 2 (SS2) is an emerging zoonosis, and meningitis is the most frequent clinical manifestation, but mechanism of its virulent factor, enolase (Eno), is unknown in meningitis. In this study, Eno was inducibly expressed and added to an in vitro Transwell co-culture model of the blood-brain barrier (BBB) consisted of porcine brain microvascular endothelial cells (PBMECs) and astrocytes (ACs), the results showed that Eno induces a significant increase in BBB permeability and promotes the release of IL-8 et al. cytokines. Furthermore, IL-8 could significantly destroy the integrity of the BBB model in vitro. In mice models administered Eno for 24 h, Eno could significantly promote Evans blue (EB) moving from the blood to the brain and significantly increased the serum and brain levels of IL-8, as detected by ELISA. While G31P (IL-8 receptor antagonist) significantly decreased the concentration of EB in the brains of mice injected with Eno. The present study demonstrated that SS2 Eno may play an important role in disrupting BBB integrity by prompting IL-8 release.

Mitochondrial binding of α-enolase stabilizes mitochondrial membrane: its role in doxorubicin-induced cardiomyocyte apoptosis.

α-Enolase is a metabolic enzyme in the catabolic glycolytic pathway. In eukaryotic cells, the subcellular compartmentalization of α-enolase as well as its multifaceted functions has been identified. Here, we report that α-enolase is a regulator of cardiac mitochondria; it partially located in the mitochondria of rat cardiomyocytes. Doxorubicin treatment displaced α-enolase from mitochondria, accompanied by activation of mitochondrial cell death pathway. Furthermore, in isolated mitochondria, recombinant α-enolase significantly alleviated Ca(2+)-induced loss of membrane potential, swelling of matrix and permeabilization of membrane. In contrast, mitochondria from α-enolase knockdown H9c2 myoblasts underwent more severe membrane depolarization and swelling after Ca(2+) stimulation. In addition, α-enolase was further identified to interact with voltage dependent anion channel 1 in the outer membrane of mitochondria, which was weakened by doxorubicin. Collectively, the present study indicates that mitochondria-located α-enolase has a beneficial role in stabilizing mitochondrial membrane. In cardiomyocytes, the displacement of α-enolase from mitochondria by doxorubicin may involve in activation of the intrinsic cell death pathway.

C-terminal peptide of γ-enolase impairs amyloid-ß-induced apoptosis through p75(NTR) signaling.

γ-Enolase acts as a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. It is shown in this study to exert a protective effect against amyloid-ß-peptide (Aß)-induced neurotoxicity in rat pheochromocytoma PC12 cells. Aß-induced toxicity was abolished in the presence of the active C-terminal peptide of γ-enolase (γ-Eno) as measured by cell viability, lactate dehydrogenase release, sub-G1 cell population, intracellular reactive oxygen species, mitochondrial functions and apoptotic morphology. γ-Eno caused downregulation of the pro-apoptotic protein Bax and upregulation of the anti-apoptotic protein Bcl-2, as well as reduced caspase-3 activation. Exposure to Aß increased surface expression of p75 neurotrophin receptor (p75(NTR)), and the increase was abolished in the presence of γ-Eno peptide. Further, pretreatment with γ-Eno suppressed the activation of mitogen-activated protein kinases p38 and Jun-N-terminal kinase, which are p75(NTR) downstream effectors in apoptotic signaling. Moreover, Aß triggered γ-enolase co-immunoprecipitation with p75(NTR) as well as their strong association in the perimembrane region as shown by confocal microscopy, which further supports the interaction between these two proteins in cells insulted by Aß peptide. Our results indicate the possible use of γ-enolase C-terminal peptide for treating or preventing Alzheimer's disease.

Neuroprotective role of γ-enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X.

γ-Enolase is a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C-terminal end of the molecule. We have investigated the expression and colocalization of γ-enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ-enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C-terminally cleaved form of γ-enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ-enolase in microglial cells in response to amyloid-ß peptide (Aß) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ-enolase proved to be neuroprotective against Aß toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ-enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid-ß-related neurodegeneration.

γ-Enolase C-terminal peptide promotes cell survival and neurite outgrowth by activation of the PI3K/Akt and MAPK/ERK signalling pathways.

γ-Enolase, a glycolytic enzyme, is expressed specifically in neurons. It exerts neurotrophic activity and has been suggested to regulate growth, differentiation, survival and regeneration of neurons. In the present study, we investigated the involvement of γ-enolase in PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) signalling, the two pathways triggered predominantly by neurotrophic factors. Whereas the PI3K/Akt pathway, rather than the MAPK/ERK pathway, is involved in γ-enolase-enhanced cell survival, γ-enolase-stimulated neurite outgrowth requires both pathways, i.e. the activation of both PI3K and ERK1/2, leading to subsequent expression of the growth-cone-specific protein GAP-43 (growth-associated protein of 43 kDa). MEK (MAPK/ERK kinase) and PI3K inhibition blocked or attenuated the neurite outgrowth associated with dynamic remodelling of the actin-based cytoskeleton. We show that γ-enolase-mediated PI3K activation regulates RhoA kinase, a key regulator of actin cytoskeleton organization. Moreover, the inhibition of RhoA downstream effector ROCK (Rho-associated kinase) results in enhanced γ-enolase-induced neurite outgrowth, accompanied by actin polymerization and its redistribution to growth cones. Our results show that γ-enolase controls neuronal survival, differentiation and neurite regeneration by activating the PI3K/Akt and MAPK/ERK signalling pathways, resulting in downstream regulation of the molecular and cellular processes of cytoskeleton reorganization and cell remodelling, activation of transcriptional factors and regulation of the cell cycle.

Immunization with Porphyromonas gingivalis enolase induces autoimmunity to mammalian α-enolase and arthritis in DR4-IE-transgenic mice.

OBJECTIVE: To examine the hypothesis that the subset of rheumatoid arthritis (RA) characterized by antibodies to citrullinated α-enolase is mediated by Porphyromonas gingivalis enolase in the context of DR4 alleles. METHODS: Recombinant human α-enolase and P gingivalis enolase, either citrullinated or uncitrullinated, were used to immunize DR4-IE-transgenic mice and control mice (class II major histocompatibility complex-deficient [class II MHC(-/-)] and C57BL/6 wild-type mice). Arthritis was quantified by measurement of ankle swelling in the hind paws and histologic examination. Serum IgG reactivity with α-enolase and citrullinated α-enolase was assayed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Antibodies to peptide 1 of citrullinated α-enolase (CEP-1) and its arginine-bearing control peptide, REP-1, were also assessed by ELISA. RESULTS: Significant hind-ankle swelling (≥0.3 mm) occurred in DR4-IE-transgenic mice immunized with citrullinated human α-enolase (9 of 12 mice), uncitrullinated human α-enolase (9 of 12 mice), citrullinated P gingivalis enolase (6 of 6 mice), and uncitrullinated P gingivalis enolase (6 of 6 mice). Swelling peaked on day 24. None of the control groups developed arthritis. The arthritic joints showed synovial hyperplasia and erosions, but there was a paucity of leukocyte infiltration. Antibodies to human α-enolase, both citrullinated and unmodified, and to CEP-1 and REP-1 were detectable in all immunized mice except the class II MHC(-/-) control mice. CONCLUSION: This is the first animal model that links an immune response to P gingivalis enolase to an important subset of RA, defined by antibodies to citrullinated α-enolase in the context of DR4. The fact that arthritis and anti-CEP-1 antibodies were induced independent of citrullination of the immunizing antigen suggests that the unmodified form of α-enolase may be important in initiating the corresponding subset of human RA.

Increased IgG antibody-induced cytotoxicity against airway epithelial cells in patients with nonallergic asthma.

BACKGROUND: IgG autoantibodies to airway epithelial cell proteins have been detected in patients with nonallergic asthma. OBJECTIVE AND METHODS: To evaluate the functional significance of these autoantibodies, we examined the presence of IgG antibody-induced cytotoxicity against airway epithelial cells (A549) by the microcytotoxicity assay using IgG antibodies purified from patients with nonallergic asthma. RESULTS: IgG antibody-induced cytotoxicity (expressed as percent cell lysis) was significantly increased in nine patients with nonallergic asthma (mean +/- standard deviation; 30.6 +/- 7.3%) as compared with eight healthy controls (13.9 +/- 5.1%) and nine patients with allergic asthma (20.3 +/- 10.4%; p < 0.05). In addition, IgG antibody-induced cytotoxicity was significantly inhibited when IgG antibodies from patients with nonallergic asthma were pre-incubated with recombinant human airway epithelial cell autoantigens (cytokeratin 18 or alpha-enolase proteins; p < 0.05). CONCLUSION: These results suggest a possible involvement of IgG autoantibody-induced cytotoxicity against airway epithelial cells in the pathogenesis of nonallergic asthma.

Enolase and arrestin are novel nonmyelin autoantigens in multiple sclerosis.

INTRODUCTION: Although myelin autoimmunity is known to be a major factor in the pathogenesis of multiple sclerosis (MS), the role of nonmyelin antigens is less clear. Given the complexity of this disease, it is possible that autoimmunity against nonmyelin antigens also has a pathogenic role. Autoantibodies against enolase and arrestin have previously been reported in MS patients. The T-cell response to these antigens, however, has not been established. METHODS: Thirty-five patients with MS were recruited, along with thirty-five healthy controls. T-cell proliferative responses against non-neuronal enolase, neuron-specific enolase (NSE), retinal arrestin, beta-arrestin, and myelin basic protein were determined. RESULTS: MS patients had a greater prevalence of positive T-cell proliferative responses to NSE, retinal arrestin, and beta-arrestin than healthy controls (p<0.0001). The proliferative response against NSE, retinal arrestin, and beta-arrestin correlated with the response against myelin basic protein (p < or = 0.004). Furthermore, the proliferative response against retinal arrestin was correlated to beta-arrestin (p<0.0001), whereas there was no such correlation between non-neuronal enolase and NSE (p = 0.23). DISCUSSION: There is accumulating evidence to suggest that the pathogenesis of MS involves more than just myelin autoimmunity/destruction. Autoimmunity against nonmyelin antigens may be a component of this myriad of immunopathological events. NSE, retinal arrestin, and beta-arrestin are novel nonmyelin autoantigens that deserve further investigation in this respect. Autoimmunity against these antigens may be linked to neurodegeneration, defective remyelination, and predisposition to uveitis in multiple sclerosis. Further investigation of the role of these antigens in MS is warranted.

Interferon-gamma responses to Candida recover slowly or remain low in immunodeficient HIV patients responding to ART.

Extended assessments of memory T-cell responses in HIV patients who have a satisfactory virological response to combination antiretroviral therapy (CART) have been limited by availability of longitudinal samples and of antigens to which most individuals (including HIV-negative controls) have been exposed. Studies of cytomegalovirus (CMV) show that interferon-gamma (IFN-gamma) responses never recover completely, but this may be antigen-specific. Here we present responses to Candida and CMV antigens analyzed using a statistical approach that derives overall trends from samples collected at variable time points. Results were considered in relation to putative markers of T-regulatory cells. Blood mononuclear cells collected from seventeen HIV-1 patients (nadir <100 CD4 T cells/mL) 0-8 years after initiation of CART were stimulated with Candida spp lysate, Candida enolase protein or CMV lysate and production of IFN-gamma was assessed by ELISpot assay. CD4 T-cell counts increased fivefold and stabilized within 24 months on CART, following control of plasma viremia. IFN-gamma responses to Candida antigens began low and increased slowly, generating positive slope up to 60 months on CART (Candida enolase p=0.008; Candida lysate p=0.03; mixed-model Wald test). Only two patients displayed a CMV or Candida-specific IFN-gamma response above the median for seronegative controls. Proportions of T cells expressing CD25 or CD57 did not correlate with IFN-gamma responses. Slow reconstitution of IFN-gamma responses to CMV and Candida in previously immunodeficient patients with restored CD4+ T-cell counts on CART suggests a broad and nonresolving defect in memory T-cell responses.

The effect of enzyme treatment on the in vitro fermentation of lucerne incubated with equine faecal inocula.

A series of experiments was conducted to determine the effects of a fibrolytic enzyme preparation (enzyme 1; E1) on the in vitro fermentation of lucerne incubated with equine faecal inocula. In experiment 1, high-temperature-dried (HT) lucerne was treated with five levels of E1 (0 to 2.4 ml/g DM) and incubated at 50 degrees C for 20 h. Samples then received a simulated foregut digestion (SFD) treatment before DM and NSP analysis. In experiment 2, HT lucerne was treated with the same enzyme levels used in experiment 1. Samples were then split into two groups; plus or minus an SFD treatment before in vitro fermentation using an equine faecal inoculum. In experiment 3, fresh and wilted lucerne were treated with the same levels of E1 as experiments 1 and 2, incubated at 50 degrees C for 20 h, then fermented in vitro. For experiment 4, fresh and wilted lucerne were treated with low levels (0 to 0.008 ml/g DM) of E1 before fermentation. E1 significantly (P<0.05) enhanced DM and NSP losses from HT lucerne following SFD treatment compared with the control. High levels of E1 significantly (P<0.05) enhanced the rate, but not extent, of fermentation of HT, wilted and fresh lucerne; however, low levels of E1 were ineffective. At higher application levels, E1 appears to have considerable potential to enhance the nutritive value of lucerne for horses. Information on the fermentation kinetics of the substrates was valuable; all end-point measurements showed no effect of enzyme treatment.

Potent inhibitor of N-myristoylation: a novel molecular target for cancer.

N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational and/or posttranslational transfer of myristate to the NH(2) terminus of the glycine residue of a number of important proteins that have diverse biological functions and thus have been proposed as potential targets for chemotherapeutic drug design. Earlier, we demonstrated that NMT is more active in colonic epithelial neoplasms than in corresponding normal-appearing colonic tissue. Furthermore, an increased expression of NMT was also observed in gallbladder carcinoma. In the present study, we report a novel protein inhibitor of NMT. This protein caused a potent concentration-dependent inhibition of human NMT with half-maximal inhibition at 4.5 +/- 0.35 nM. This study will serve as a template for further investigations in the area of protein myristoylation.

Anti-inflammatory treatment influences neuronal apoptotic cell death in the dentate gyrus in experimental pneumococcal meningitis.

Apoptotic neuronal death and the increase of neuron-specific enolase (NSE) in cerebrospinal fluid (CSF) were studied in a rabbit model of experimental pneumococcal meningitis after treatment with antimicrobial (ceftriaxone) and antiinflammatory agents (dexamethasone, monoclonal antibodies against the beta-subunit of beta 2-integrins [anti-CD18 mAb]). Twenty-four hours after infection, apoptotic cell death was found solely in the granular cell layer of the dentate gyrus. Neurons with DNA fragmentation were quantified with the in situ tailing (IST) reaction. Dexamethasone and anti-CD18 mAb inhibited the NSE increase in CSF significantly (p = 0.003, p = 0.011). After administration of dexamethasone the density of apoptotic neurons was significantly higher than in control animals receiving only ceftriaxone (p = 0.044). The median of the density of apoptotic neurons was lower in the dentate gyrus in animals receiving anti-CD18 mAb and ceftriaxone vs those receiving only ceftriaxone, although the difference did not reach statistic significance (p = 0.058). In conclusion, apoptotic cell death occurs in the dentate gyrus during the early phase of bacterial meningitis. The extent was influenced by antiinflammatory therapy. The systemic administration of glucocorticoids increased the quantity of apoptotic neurons in the dentate gyrus but reduced overall neuronal damage as indicated by low levels of NSE concentration in CSF.

Expression of APP in transgenic mice: a comparison of neuron-specific promoters.

The beta-amyloid precursor protein (APP) carries mutations in codons 717 or 670/671, which cosegregate with familial forms of Alzheimer's disease (AD). As an initial step to study the related pathogenetic mechanisms in vivo we have generated transgenic mice expressing APP with these mutations. Several neuron-specific promoters were used to drive expression of human APP cDNAs. Only the Thy-1 promoter yielded transgene expression levels comparable to or above the endogenous mouse levels. Deletion of a 121 bp sequence from the 3' untranslated region of APP appeared to increase mRNA levels. Transgene mRNA was found throughout the brain with highest levels in hippocampus and cerebral cortex. Accordingly, human APP was detected in these regions by Western blotting. Protein levels paralleled mRNA levels reaching or exceeding the amount of endogenous APP. Variable reactivity of human APP in cell bodies was shown by immunocytochemistry. Although our initial histological examinations did not reveal any alterations characteristic of AD, further studied will be required.