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PubMedDate not supplied

Induced expression of alpha-enolase in differentiated diffuse large cell lymphoma.

A unique protein has been detected that is associated with the differentiation of diffuse large cell lymphoma (DLCL). The WSU-DLCL human cell line was cultured in the absence or presence of the biological agent, Bryostatin 1 (Bryo1). Cellular proteins of parent and differentiated WSU-DLCL cells were analyzed using one- and two-dimensional polyacrylamide gel electrophoresis (1D and 2D PAGE). In the 1D PAGE, a unique protein band of molecular mass approximately 47 kD was detected in the differentiated, but not the parent cells. Amino acid sequence of the band indicated the presence of more than one protein. The 2D PAGE analysis showed that one of the proteins of interest had an isoelectric point of 7.4. Partial amino acid sequencing of the spot by tryptic digest showed 100% homology with alpha-enolase. alpha-Enolase is a nonneuronal enzyme involved in the glycolytic pathway. This is the first report on the induction of alpha-enolase in human DLCL after treatment with the natural biological agent, Bryo1. We suggest that alpha-enolase may play a significant role in the differentiation of lymphoma in man.

Amino Acid Sequence↗
PubMed2005

The nine residue plasminogen-binding motif of the pneumococcal enolase is the major cofactor of plasmin-mediated degradation of extracellular matrix, dissolution of fibrin and transmigration.

The glycolytic enzyme alpha-enolase represents one of the nonclassical cell surface plasminogen-binding proteins of Streptococcus pneumoniae. In this study we investigated the impact of an internal plasminogen-binding motif of enolase on degradation of extracellular matrix and pneumococcal transmigration. In the presence of host-derived plasminogen activators (PA) tissue-type PA or urokinase PA and plasminogen S. pneumoniae expressing wild-type enolase efficiently degraded Matrigel or extracellular matrix (ECM). In contrast, amino acid substitutions in the nine residue plasminogen-binding motif of enolase significantly reduced degradation of ECM or Matrigel by mutated pneumococci. Similarly, recombinant wild-type enolase but not a mutated enolase derivative that lacks plasminogen-binding activity efficiently degraded ECM and Matrigel, respectively. In particular, bacterial cell enolase-bound plasmin potentiated dissolution of fibrin or laminin and transmigration of pneumococci through a fibrin matrix. In conclusion, these results provide evidence that the enolase is the major plasminogen-binding protein of pneumococci and that the nine residue plasminogen-binding motif of enolase is the key cofactor for plasmin-mediated pneumococcal degradation and transmigration through host ECM.

Amino Acid Motifs↗
PubMedDate not supplied

Neuronal and glial proteins in medulloblastomas. II. Heterotransplantation of human medulloblastoma.

Seven resected medulloblastomas were inoculated into hereditary athymic (nude) and asplenic athymic (Lasat) mice. From these, only one transplantable tumor line was obtained, in nude mice. We studied the transplanted medulloblastoma immunohistochemically and by electron microscope. In addition, using enzyme immunoassay, the gamma-enolase contents of the transplanted medulloblastomas were measured and the results were compared with those of transplanted glioblastomas. Neurofilament proteins (NF68Kd, NF160Kd) and gamma-enolase positive cells were maintained in the successful transplants of medulloblastomas, but the GFAP positive cells were lost. Under the electron microscope, a few cells with ten nanometer intermediate filaments and microtubules were observed in the transplanted tumor. The medulloblastoma possesses some of the neuronal characteristics which are maintained in the transplants in nude mice.

Animals↗
PubMed1993

Purification and characterization of an enzyme involved in oxidative carbon-carbon bond cleavage reactions in the methionine salvage pathway of Klebsiella pneumoniae.

The 5-methylthio-D-ribose moiety of 5'-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 2,3-Diketo-5-methylthio-1-phosphopentane is an advanced intermediate in the methionine recycling pathway present in the Gram-negative bacterium Klebsiella pneumoniae. This unusual metabolite is oxidatively cleaved to yield formate (from C-1), 2-keto-4-methylthiobutyrate (the transamination product of methionine), and 3-methylthiopropionate. To further characterize this oxidative conversion, the desthio analog of the naturally occurring diketone, namely 2,3-diketo-1-phosphohexane I, was synthesized. If the metabolism of I is analogous to that of 2,3-diketo-5-methylthio-1-phosphopentane it should be converted to formate, 2-ketopentanoate, and butyrate. An enzyme (E-1), which mediates the oxidative conversion of I to formate and 2-ketopentanoate, was isolated from extracts of K. pneumoniae. E-1 was purified 100-fold to homogeneity in 10% yield. The native enzyme is a monomeric protein of M(r) 27,000. The activity of E-1 requires magnesium ion as a cofactor. No other prosthetic groups were detected. Incubation of the enzyme with I, under anaerobic conditions, led to the discovery of two intermediates. These species have been identified by 1H and 13C NMR, UV-visible spectroscopy, and model chemistry studies as 2-hydroxy-3-keto-1-phospho-1-hexene II, generated by enolization of I; and 1,2-dihydroxy-3-keto-1-hexene III, generated by enzymatic dephosphorylation of II. Intermediates II and III are released from the active site of the enzyme; III accumulates under anaerobic conditions. Under aerobic conditions, III is non-enzymically oxidized to 2-ketopentanoate, formate, and other products. Compound II was also generated by heating I at pH 7.5 for 7 min. Action of alkaline phosphatase on II produces III.

Amino Acid Sequence↗
PubMed2005

Medial preoptic area interactions with the nucleus accumbens-ventral pallidum circuit and maternal behavior in rats.

Several experiments explored the roles of nucleus accumbens (NA), ventral pallidum (VP) and medial preoptic area (MPOA) in the regulation of maternal behavior in rats. A preliminary experiment found that bilateral radiofrequency lesions of medial NA did not disrupt maternal behavior. Experiment 1 found that bilateral infusions of muscimol into VP, but not into medial NA, reversibly disrupted maternal behavior. Experiment 2 found that unilateral muscimol injections into VP disrupted maternal behavior to a greater extent when paired with a contralateral N-methyl-d-aspartic acid (NMDA) MPOA lesion than when paired with a sham MPOA lesion. Experiment 3 showed that a unilateral NMDA MPOA lesion paired with a contralateral NMDA VP lesion (Contra group) disrupted maternal behavior to a much greater extent than did sham NMLA lesions or NMDA lesions of MPOA and VP ipsilateral to one another. Experiment 3 focused on the specificity of the maternal behavior disruptions and found that the primary maternal deficit in the Contra females was a severe deficit in retrieval behavior. Importantly, these females showed normal hoarding behavior, home cage activity, and elevated plus maze activity. Experiment 3 used Neu N immunohistochemistry to define the extent of MPOA and VP excitotoxic lesions. It is hypothesized that MPOA acts to facilitate the active components of maternal behavior by inhibiting NA, which then releases VP from GABAergic inhibition, and such disinhibition of VP allows pup stimuli to trigger appropriate maternal responses.

Analysis of Variance↗
PubMed2006

In vivo transduction of murine cerebellar Purkinje cells by HIV-derived lentiviral vectors.

Cerebellar Purkinje cells are key elements in motor learning and motor coordination, and therefore, it is important to clarify the mechanisms by which Purkinje cells integrate information and control cerebellar function. Gene transfer into neurons, followed by the assessment of the effects on neural function, is an effective approach for examining gene function. However, this method has not been used fully in the study of the cerebellum because adenovirus vectors, the vectors most commonly used for in vivo gene transfer, have very low affinity for Purkinje cells. In this study, we used a human immunodeficiency virus (HIV)-derived lentiviral vector and examined the transduction profile of the vector in the cerebellum. A lentiviral vector carrying the GFP gene was injected into the cerebellar cortex. Seven days after the injection, Purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. GFP was also expressed, though less efficiently, in other cortical interneurons and Bergmann glias. In contrast to reported findings with other viral vectors, no transduced cells were observed outside of the cerebellar cortex. Thus, when HIV-derived lentiviral vectors were injected into the cerebellar cortex, transduction was limited to the cells in the cerebellar cortex, with the highest tropism for Purkinje cells. These results suggest that HIV-derived lentiviral vectors are useful for the study of gene function in Purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect Purkinje cells.

Animals↗
PubMed2006

Eimeria tenella enolase and pyruvate kinase: a likely role in glycolysis and in others functions.

Two cDNA codings for glycolytic enzymes were cloned from a cDNA library constructed from the schizont stage of the avian parasite Eimeria tenella. Enolase and pyruvate kinase cDNA were fully sequenced and compared with sequences of enzymes from other organisms. Although these enzymes were already detected in the sporozoite stage, their expression was enhanced during the first schizogony in accordance with the anaerobic conditions of this part of the life cycle of the parasite. Under activating conditions, microscopic observations suggest that these glycolytic enzymes were relocalised inside sporozoites and moreover were in part secreted. The enzymes were also localised at the apex of the first generation of merozoites. Enolase was partly observed inside the nucleus of sporozoites and schizonts. Taken together, these results suggest that glycolytic enzymes not only have a function in glycolysis during anaerobic intracellular stages but may also participate in the invasion process and, for enolase, in the control of gene regulation.

Amino Acid Sequence↗
PubMed2004

Continuous exposure to nitric oxide enhances diazepam binding inhibitor mRNA expression in mouse cerebral cortical neurons.

Effects of sustained exposure to nitric oxide (NO) formed by long-term activation of N-methyl-D-aspartate (NMDA) receptors and liberated from a long-lasting NO generator, DETA NONOate, on diazepam binding inhibitor (DBI) and its mRNA expressions were examined using mouse cerebral cortical neurons. Long-term exposure to NMDA increased DBI mRNA expression, and NO synthase inhibitors dose-dependently inhibited this increase. DETA NONOate dose-dependently increased DBI mRNA expression when exposing the neurons to this agent for 3 days and a maximal enhancement of the expression was found at 100 microM of the NO generator. In addition, a significant increase in DBI mRNA expression was observed 1 day after the exposure to 100 microM DETA NONOate, and the maximal expression was observed 2 days after the exposure, whereas transient exposure for less than 3 h to 100 microM DETA NONOate produced no changes in the expression. DETA NONOate (100 microM)-induced increase in DBI mRNA expression was completely abolished by concomitant exposure to hemoglobin. DBI content was also dose-dependently increased by DETA NONOate after the exposure for 3 days. The inhibition of cGMP formation by 1H-[1,2,4] oxadiazolo [4,3-alpha]quinoxalin-1-one (ODQ) showed no affects on the DETA NONOate-induced expression, suggesting that the increased expression of DBI mRNA is mediated via processes independent of cGMP. These results indicate that continuous exposure of the neurons to NO is an essential factor for increasing DBI mRNA expression in the neurons.

Analysis of Variance↗
PubMed2004

Enolase from Streptococcus sobrinus is an immunosuppressive protein.

A strategy of Streptococcus sobrinus, a major agent of dental caries, to survive and colonize the host consists of the production of a protein that suppresses the specific antibody responses. We have cloned the gene coding for a protein with immunosuppressive activity. It contains an open reading frame of 1302 base pairs encoding a polypeptide with 434 amino acid residues and a molecular mass of 46910 Da. The gene product is homologous to enolases from several organisms. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in a fluoride-sensitive enzymatically active form. Pretreatment of mice with the S. sobrinus recombinant enolase suppresses a primary immune response against T-cell dependent antigens. This immunosuppressive effect is specific to the antigen used in the immunization, as it is not observed when the immune response against other antigens is analysed. Furthermore, the S. sobrinus recombinant enolase stimulates an early production of interleukin-10, an anti-inflammatory cytokine, and not the pro-inflammatory cytokine IFN-gamma. These observations indicate that enolase acts in the suppression of the specific host immune response against S. sobrinus infection.

Amino Acid Sequence↗
PubMed1988

Tau-crystallin/alpha-enolase: one gene encodes both an enzyme and a lens structural protein.

tau-Crystallin has been a major component of the cellular lenses of species throughout vertebrate evolution, from lamprey to birds. Immunofluorescence analysis of the embryonic turtle lens, using antiserum to lamprey tau-crystallin showed that the protein is expressed throughout embryogenesis and is present at high concentrations in all parts of the lens. Partial peptide sequence for the isolated turtle protein and deduced sequences for several lamprey peptides all revealed a close similarity to the glycolytic enzyme enolase (E.C. 4.2.1.11). A full-sized cDNA for putative duck tau-crystallin was obtained and sequenced, confirming the close relationship with alpha-enolase. Southern blot analysis showed that the duck genome contains a single alpha-enolase gene, while Northern blot analysis showed that the message for tau-crystallin/alpha-enolase is present in embryonic duck lens at 25 times the abundance found in liver. tau-Crystallin possesses enolase activity, but the activity is greatly reduced, probably because of age-related posttranslational modification. It thus appears that a highly conserved, important glycolytic enzyme has been used as a structural component of lens since the start of vertebrate evolution. Apparently the enzyme has not been recruited for its catalytic activity but for some distinct structural property. tau-Crystallin/alpha-enolase is an example of a multifunctional protein playing two very different roles in evolution but encoded by a single gene.

Amino Acid Sequence↗
PubMed1996

Protein expression during exponential growth in 0.7 M NaCl medium of Saccharomyces cerevisiae.

Saccharomyces cerevisiae exponentially growing in basic or 0.7 M NaCl medium were isotopically labelled with 35S-methionine, followed by protein separation and quantification by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with computerised image analysis. The electrophoretic separation resolved about 650 proteins of which 13 displayed significant and at least 2-fold changes in rate of synthesis during saline growth. By sequencing of 2D-PAGE resolved proteins, one of the 8 induced spot, p42.9/5.5, was shown to correspond to the full length (containing the N-terminal extension) product of the GPD1 gene encoding the cytoplasmic glycerol 3-phosphate dehydrogenase. The expression of the TDH3 gene, glyceraldehyde 3-phosphate dehydrogenase, and the ENO2 gene, enolase, decreased during growth in NaCl medium, declines hypothesised to have an impact on the flux to glycerol.

Amino Acid Sequence↗
PubMed2002

cDNA cloning and immunological characterization of a newly identified enolase allergen from Penicillium citrinum and Aspergillus fumigatus.

BACKGROUND: Penicillium citrinum and Aspergillus fumigatus are prevalent indoor airborne fungal species that have been implicated in human respiratory allergic disorders. It is important to understand the allergenic profile of these fungal species. The purpose of the present study is to characterize a newly identified enolase allergen from P. citrinum and A. fumigatus. METHODS: Fungal proteins were separated by two-dimensional (2D) gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Protein spots that reacted with IgE antibodies in serum samples from asthmatic patients were identified and the N-terminal amino acid sequences were determined by Edman degradation. The peptide sequences obtained were utilized in cloning the cDNA of the allergen genes by reverse transcriptase-polymerase chain reaction and the 5'- and 3'-rapid amplification cDNA end reactions. RESULTS: Our results from 2D immunoblotting identified a 47-kD IgE-reactive component in the extracts of P. citrinum and A. fumigatus. The N-terminal amino acid sequences of the 47-kD proteins are homologous to those of fungal enolases. The corresponding enolase cDNA from P. citrinum contains 1,552 bp and encodes a protein of 438 residues. In A. fumigatus, the isolated enolase cDNA has 1,649 bp and contains a 438-amino acid open reading frame. The deduced amino acid sequences of these two enolases have 94% identity. These enolases from P. citrinum and A. fumigatus were expressed in Escherichia coli as a His-tagged protein and designated as rPen c 22 and rAsp f 22, respectively. Sera from 7 (30%) of the 23 Penicillium-sensitized asthmatic patients showed IgE binding to the 47-kD P. citrinum component (Pen c 22) and rPen c 22. In addition, six of seven Pen c 22-positive serum samples have IgE immunoblot reactivity to the 47-kD A. fumigatus component (Asp f 22) and rAsp f 22. A polyclonal rabbit antiserum generated against the N-terminal peptide of Pen c 22 can react with Pen c 22, rPen c 22, Asp f 22 and rAsp f 22. In addition, the presence of IgE cross-reactivity between rPen c 22 and rAsp f 22 and between enolases from A. fumigatus and Alternaria alternata was also detected by immunoblot inhibition. CONCLUSIONS: These results demonstrated that a novel enolase allergen from P. citrinum (Pen c 22) and A. fumigatus (Asp f 22) was identified. In addition, IgE cross-reactivity between enolase allergens from A. fumigatus and P. citrinum and between enolases from A. fumigatus and A. alternata was also detected. Results obtained provide more information on fungal enolase allergens.

Allergens↗
PubMed1993

Cholesteryl ester loading of mouse peritoneal macrophages is associated with changes in the expression or modification of specific cellular proteins, including increase in an alpha-enolase isoform.

This report explores the hypothesis that massive cholesteryl ester (CE) accumulation in macrophages, such as that occurring in atheroma foam cells, results in changes in the expression or modification of specific cellular proteins. Two-dimensional (2-D) gel electrophoretic patterns of metabolically labeled cellular proteins from mouse peritoneal macrophages that were loaded with CE (through incubation with acetylated low density lipoprotein [acetyl-LDL] for 4 days) were compared with those of control macrophages. Densitometric analysis of 2-D gel autoradiograms from the cell lysates revealed statistically significant changes in seven cellular proteins (five decreases and two increases). The changes in protein expression (foam cell versus control) ranged from a 458 +/- 164% (p < 0.001) increase to a 35 +/- 34% (p < 0.001) decrease (n = 11). Incubation of macrophages with beta-very low density lipoprotein, which also increased the CE content of macrophages (albeit to a lesser extent than acetyl-LDL), resulted in changes in five of the seven proteins. In contrast, incubation of cells with LDL, fucoidan, or latex beads, none of which caused CE accumulation, did not lead to significant changes in four of these five proteins. One of these four proteins, which increased fourfold to fivefold in foam cells (M(r) = 49,000; isoelectric point of 6.8), was purified by preparative 2-D gel electrophoresis. Internal amino acid sequence of cyanogen bromide fragments of this protein as well as Western blot analysis identified this protein as an isoform of alpha-enolase. The increased expression of this alpha-enolase isoform, which was seen as early as day 2 of acetyl-LDL incubation of the macrophages, was diminished by including an inhibitor of cholesterol esterification during the acetyl-LDL incubation period. In conclusion, macrophage foam cell formation is associated with distinct changes in protein expression, including a marked increase in an isoform of alpha-enolase, suggesting a specific biological adaptation to CE loading.

Amino Acid Sequence↗
PubMed1970

Gluconeogenesis in the cow. The effects of a glucocorticoid on hepatic intermediary metabolism.

1. The hepatic concentrations of the ketone bodies and of metabolites and activities of enzymes involved in gluconeogenesis were measured in healthy lactating and non-lactating cows 48h after administration of Voren, an ester of dexamethasone, and compared with those found in control animals given saline. Parallel measurements were also made of the blood concentrations of several of the metabolites. 2. Blood glucose concentrations were raised in the Voren-treated animals, whereas blood ketone body and free fatty acid concentrations were unaltered. Similarly there was no change in the hepatic concentrations of the ketone bodies. 3. Significant increases were found in the hepatic concentrations of citrate, 2-oxo-glutarate and malate in both groups of animals given Voren. 4. The hepatic concentrations of those glycolytic intermediates that were measured either decreased or did not change after Voren treatment. 5. The enzymes aspartate transaminase and fructose 1,6-diphosphatase were unchanged in activity after Voren administration, whereas phosphopyruvate carboxylase (EC 4.1.1.32) activity was depressed in the lactating group. However, glucose 6-phosphatase, tryptophan oxygenase and tyrosine aminotransferase increased in activity. 6. In several cases those hepatic metabolites that increased in concentration after Voren administration were present in lower concentration in normal lactating cows than in normal non-lactating cows. The same applied mutatis mutandis to those metabolites that were decreased by Voren. 7. These findings are discussed in relation to the use of glucocorticoids in the treatment of bovine ketosis.

Amino Acids↗
PubMed2006

Fluorescence resonance energy transfer-based assay for DNA-binding protein tagged by green fluorescent protein.

Specific interaction between green fluorescent protein (GFP)-tagged human alpha- or gamma-enolase(97-242) (alpha or gammaENO(97-242)) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a "real-time FRET assay." The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO(97-242) and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R(as)) of ENO(97-242) mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.

DNA-Binding Proteins↗
PubMed2004

Nestin expression is lost in a neural stem cell line through a mechanism involving the proteasome and Notch signalling.

Neural stem cells (NSCs) are believed to repair brain damage primarily through cell replacement: i.e., the ability to regenerate lost neurons and glia in a site-specific fashion. The neural stem cell line, MHP36, has been shown to have this capacity, but we have little idea of the molecular mechanisms that control the differentiation of such cells during brain repair. In this study we show that an early event in the differentiation of MHP36 cells, both in vivo and in vitro, is the loss of expression of the intermediate filament protein, nestin. We use a co-culture assay to show that loss of nestin is fast, being detectable after just 1 h and complete in 4 h, and is controlled by proteasome degradation rather than down-regulation of de novo nestin synthesis. We also show that nestin loss is regulated by Notch, and mediated by cell contact.

Acetylcysteine↗
PubMed2005

FAS deficiency reduces apoptosis, spares axons and improves function after spinal cord injury.

After spinal cord injury (SCI), apoptosis of neurons and oligodendrocytes is associated with axonal degeneration and loss of neurological function. Recent data have suggested a potential role for FAS death receptor-mediated apoptosis in the pathophysiology of SCI. In this study, we examined the effect of FAS deficiency on SCI in vitro and in vivo. FAS(Lpr/lpr) mutant mice and wildtype background-matched mice were subjected to a T5-6 clip compression SCI, and complementary studies were done in an organotypic slice culture model of SCI. Post-traumatic apoptosis in the spinal cord, which was seen in neurons and oligodendrocytes, was decreased in the FAS-deficient mice both in vivo and in vitro particularly in oligodendrocytes. FAS deficiency was also associated with improved locomotor recovery, axonal sparing and preservation of oligodendrocytes and myelin. However, FAS deficiency did not result in a significant increase in surviving neurons in the spinal cord at 6 weeks after injury, likely reflecting the importance of other cell death mechanisms for neurons. We conclude that inhibition of the FAS pathway may be a clinically attractive neuroprotective strategy directed towards oligodendroglial and axonal preservation in the treatment of SCI and neurotrauma.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗