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At least 1,135 records · Page 63Linked to original sources
PubMed2005

TrkB has a cell-autonomous role in the establishment of hippocampal Schaffer collateral synapses.

Neurotrophin signaling has been implicated in the processes of synapse formation and plasticity. To gain additional insight into the mechanism of BDNF and TrkB influence on synapse formation and synaptic plasticity, we generated a conditional knock-out for TrkB using the cre/loxp system. Using three different cre-expressing transgenic mice, three unique spatial and temporal configurations of TrkB deletion were obtained with regard to the hippocampal Schaffer collateral synapse. We compare synapse formation in mutants in which TrkB is ablated either in presynaptic or in both presynaptic and postsynaptic cells at early developmental or postdevelopmental time points. Our results indicate a requirement for TrkB at both the presynaptic and postsynaptic sites during development. In the absence of TrkB, synapse numbers were significantly reduced. In vivo ablation of TrkB after synapse formation did not affect synapse numbers. In primary hippocampal cultures, deletion of TrkB in only the postsynaptic cell, before synapse formation, also resulted in deficits of synapse formation. We conclude that TrkB signaling has a cell-autonomous role required for normal development of both presynaptic and postsynaptic components of the Schaffer collateral synapse.

Animals↗
PubMed1997

Identification of an epitope of alpha-enolase (a candidate plasminogen receptor) by phage display.

Alpha-enolase is an ubiquitous cytoplasmic glycolytic enzyme which also exhibits cell surface mediated functions and a structural role in the lens of some species. An alpha-enolase related molecule (alpha-ERM) is present on the surfaces of neutrophils, monocytes and monocytoid cells and has the capacity to specifically bind plasminogen, suggesting that alpha-ERM may function as a plasminogen receptor. We have generated a monoclonal antibody (mAB), 9C12, against alpha-ERM. This mAB reacted with both alpha-ERM and purified human alpha-enolase in Western blotting and in enzyme linked immunosorbent assays (ELISA). mAB 9C12 detected a cell surface associated molecule on human peripheral blood neutrophils and on U937 human monocytoid cells as assessed by fluorescence activated cell sorting (FACS) analyses. In addition, mAB 9C12 recognized an intracellular pool of alpha-enolase/alpha-ERM in permeabilized U937 cells. A phage display approach was employed to identify the alpha-enolase epitope recognized by mAB 9C12. Random fragments of 100-300 base pairs (bp), obtained from the full length human alpha-enolase cDNA, were cloned into the filamentous phage vector pComb3B, to generate a phage-displayed peptide library. Recombinant phages binding to mAB 9C12 were selected and their DNA inserts characterized by direct sequencing. All of the fragments which bound to mAB 9C12 encoded the common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 of human alpha-enolase. This sequence is located within an external loop of the molecule. These data indicate that this sequence contains the epitope recognized by mAB 9C12 and is, therefore, exposed on the cell surface, further suggesting that alpha-enolase and alpha-ERM share common amino acid sequences.

Amino Acid Sequence↗
PubMed1975

Enolase catalyzed beta,gamma-alpha,beta isomerization of 2-phospho-3-butenoic acid to (Z)-phosphoenol-alpha-ketobutyrate.

2-Phospho-3-butenoic acid was synthesized and found to be a substrate for both yeast and rabbit muscle enolase (EC 4.2.1.11). Enolase catalyzes the isomerization of 2-phospho-3-butenoic acid to (Z)-phosphoenol-alpha-ketobutyrate, a beta,gamma-alpha,beta isomerization. Nuclear magnetic resonance studies on the product indicate only one isomer is formed. This reaction provides indirect evidence in further support of a carbanion intermediate for the enolase reaction. 2-Phospho-3-butenoic acid is also a good competitive inhibitor of both yeast and rabbit muscle pyruvate kinase (EC 2.7.1.40).

Animals↗
PubMed1993

Analysis of the tyrosine protein kinase p56lck expressed as a glutathione S-transferase fusion protein in Spodoptera frugiperda cells.

A baculovirus vector system that expresses cloned DNA sequences as glutathione S-transferase fusion proteins was developed. This system was used to express and purify the lymphocyte-specific tyrosine kinase p56lck. This recombinant p56lck was purified to homogeneity in a single chromatography step using glutathione resin. By SDS gel analysis recombinant p56lck was found to migrate as two species with molecular masses of approximately 56,000 Da. p56lck purified in this manner retained a high level of activity, phosphorylated an exogenous substrate on tyrosine residues, and underwent autophosphorylation on tyrosine residues. The Km (approximately 0.33 mmol) and Vmax (approximately 83 pmol min-1 mg-1) values were also determined by using enolase as a substrate.

Amino Acid Sequence↗
PubMed2004

Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction.

The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in mediating the fusion of HIV with host cells. During the fusion process, three N-terminal helices and three C-terminal helices pack in an anti-parallel direction to form a six-helix bundle. X-ray crystallographic analysis of the gp41 core demonstrated that within each coiled-coil interface, there is a deep and large pocket, formed by a cluster of residues in the N-helix coiled-coil. In this report, we systematically analyzed the role of seven conserved residues that are either lining or packing this pocket on the infectivity and interhelical interaction using novel approaches. Our results show that residues L568, V570, W571, and K574 of the N-helix that are lining the side chain and right wall of the pocket are important for establishing a productive infection. Mutations V570A and W571A completely abolished replication, while replication of the L568A and K574A mutants was significantly attenuated relative to wild type. Similarly, residues W628, W631, and I635 of the C-helix that insert into the pocket are essential for infectivity. The impaired infectivity of these seven mutants is in part attributed to the loss in binding affinity of the interhelical interaction. Molecular modeling of the crystal structure of the coiled-coil further shows that alanine substitution of those residues disrupts the hydrophobic interaction between the N- and C-helix. These results suggest that the conserved residues in the coiled-coil domain play a key role in HIV infection and this coiled-coil pocket is a good target for development of inhibitors against HIV. In addition, our data indicate that the novel fluorescence polarization assay described in this study could be valuable in screening for inhibitors that block the interhelical interaction and HIV entry.

Amino Acid Sequence↗
PubMed1999

Structure and mechanism of glutamate racemase from Aquifex pyrophilus.

Glutamate racemase (MurI) is responsible for the synthesis of D-glutamate, an essential building block of the peptidoglycan layer in bacterial cell walls. The crystal structure of glutamate racemase from Aquifex pyrophilus, determined at 2.3 A resolution, reveals that the enzyme forms a dimer and each monomer consists of two alpha/beta fold domains, a unique structure that has not been observed in other racemases or members of an enolase superfamily. A substrate analog, D-glutamine, binds to the deep pocket formed by conserved residues from two monomers. The structural and mutational analyses allow us to propose a mechanism of metal cofactor-independent glutamate racemase in which two cysteine residues are involved in catalysis.

Amino Acid Isomerases↗
PubMed1999

Circulating antibodies against alpha-enolase in patients with primary membranous nephropathy (MN).

MN is characterized by the glomerular deposition of IgG4 immune complexes. This suggests that nephritogenic immune responses in MN are of the Th2 T helper cell type; however, the pathogenesis of MN is still unknown. In this study we examined sera from patients with primary MN for antibodies to renal proteins. A 47-kD protein in both human and porcine renal extracts was found by immunoblotting to react specifically with serum IgG from some patients. This protein was purified from porcine kidney and identified as alpha-enolase on the basis of its partial amino acid sequences. Sera from 87 patients with primary MN, 24 patients with secondary MN (15 rheumatoid arthritis patients, nine systemic lupus erythematosus patients), and 16 healthy subjects were examined by ELISA using purified alpha-enolase. In 60 (69%) patients with primary MN and 14 (58%) patients with secondary MN, the measured optical density values, and hence serum anti-alpha-enolase antibody levels, were greater than the mean + 2 s.d. of healthy subjects. Immunoblot analysis showed that IgG1 or IgG3 was the predominant subclass (Th1 T helper cell type subclass) of antibodies against alpha-enolase in patients with primary and secondary MN. Since circulating antibodies against alpha-enolase have recently been reported in patients with various autoimmune disorders, our results suggest that a number of patients with presumed primary MN may also have abnormalities in Th1 T helper cell-mediated immune responses.

Adolescent↗
PubMed1997

Intron variation in marbled murrelets detected using analyses of single-stranded conformational polymorphisms.

Combination of the targeted amplification of nuclear introns and the analysis of single-stranded conformational polymorphisms has the potential to provide an inexpensive, rapid, versatile and sensitive genetic assay for evolutionary studies and conservation. We are developing primers and protocols to analyse nuclear introns in vertebrates, and are testing them in a population genetic study of marbled murrelets Brachyramphus marmoratus. Here we present protocols and results for introns for aldolase B, alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase and lamin A. Results suggest that this approach presents a potentially powerful method for detecting genetic variation within and among local populations and species of animals: (i) a variety of genes can be surveyed, including genes of special interest such as those involved in disease resistance; (ii) assays are rapid and relatively inexpensive; (iii) large numbers of genes can be assayed, enabling accurate estimation of variation in the total genome; (iv) almost any mutation can be detected in the genes amplified; (v) the exact nature of variation can be investigated by sequence analysis if desired; (vi) statistical methods previously developed for proteins and/or sequence data can be used; (vii) protocols can be easily transferred to other species and other laboratories; and (viii) assays can be performed on old or degraded samples, blood or museum skins, so that animals need not be killed. Results of analyses for murrelets support earlier evidence that North American and Asiatic subspecies represent reproductively isolated species, and that genetic differences exist among murrelets from different sites within North America.

Amino Acid Sequence↗
PubMed2000

Identification of variants and dual promoters of murine serine/threonine kinase KKIAMRE.

KKIAMRE is a serine/threonine protein kinase whose transcripts increase in the deep cerebellar nuclei of the rabbit after eyeblink conditioning, a model of associative learning and memory. We here characterized the expression, isoforms, and promoters of murine KKIAMRE gene. The expression of KKIAMRE was detected, by in situ hybridization and immunohistochemistry, in neurons in various brain regions including deep cerebellar nuclei. The gene spans approximately 40 kb and consists of 15 exons. Analysis of cDNA clones revealed multiple variants, having diversity in the putative carboxy-terminal regulatory domain, generated by alternative splicing and intraexonal termination. Furthermore, they had alternative 5' noncoding sequences. Primer extension, RNase protection, and transient expression assays revealed that two alternative promoters linked to distinct noncoding exons direct the expression of KKIAMRE. The gene was mapped on chromosomes 5 and 4 in mouse and human, respectively.

Amino Acid Sequence↗
PubMed1973

Photosynthetic carbon metabolism in isolated maize bundle sheath strands.

Photosynthetically active bundle sheath strands capable of assimilating up to 8 micromoles CO(2) per milligram chlorophyll per hour have been isolated from fully expanded leaves of Zea mays L. Mesophyll cell contamination of the preparations was negligible, as evidenced by light and electron microscopy and by a high ratio of chlorophyll a to chlorophyll b in the strands. Ribose 5-phosphate markedly stimulated the rate of photosynthetic (14)CO(2) fixation by the isolated strands. In contrast, both pyruvate and phosphoenolpyruvate had a comparatively small stimulatory effect on bundle sheath (14)CO(2) fixation. After 5 minutes of photosynthesis in (14)C-bicarbonate, 95% of the incorporated (14)C was found in compounds other than C(4)-dicarboxylic acids, most notably in 3-phosphoglycerate and sugar phosphates. A similar distribution of (14)C was observed in the presence of exogenous ribose 5-phosphate. Extracts of bundle sheath strands contained high specific activities of "malic" enzyme, phosphoglycolate phosphatase, hydroxypyruvate reductase, and ribulose 1,5-diphosphate carboxylase, whereas the specific activities of NADP(+)-malate dehydrogenase and phosphopyruvate carboxylase were extremely low. These results indicate that the Calvin cycle occurs in the bundle sheath cells of maize.

Journal Article↗
PubMed2002

c-Src-mediated phosphorylation of hnRNP K drives translational activation of specifically silenced mRNAs.

hnRNPK and hnRNP E1/E2 mediate translational silencing of cellular and viral mRNAs in a differentiation-dependent way by binding to specific regulatory sequences. The translation of 15-lipoxygenase (LOX) mRNA in erythroid precursor cells and of the L2 mRNA of human papilloma virus type 16 (HPV-16) in squamous epithelial cells is silenced when either of these cells is immature and is activated in maturing cells by unknown mechanisms. Here we address the question of how the silenced mRNA can be translationally activated. We show that hnRNP K and the c-Src kinase specifically interact with each other, leading to c-Src activation and tyrosine phosphorylation of hnRNP K in vivo and in vitro. c-Src-mediated phosphorylation reversibly inhibits the binding of hnRNP K to the differentiation control element (DICE) of the LOX mRNA 3' untranslated region in vitro and specifically derepresses the translation of DICE-bearing mRNAs in vivo. Our results establish a novel role of c-Src kinase in translational gene regulation and reveal a mechanism by which silenced mRNAs can be translationally activated.

3' Untranslated Regions↗
PubMed1993

Transgenic mice expressing human beta-APP751, but not mice expressing beta-APP695, display early Alzheimer's disease-like histopathology.

Mice transgenic for the 751 amino acid isoform of the human beta-amyloid precursor protein (beta-APP) driven by the rat neuron specific enolase (NSE) promoter (NSE:beta-APP751) show features of early Alzheimer's disease (AD) pathology. These features, which were evident in multiple pedigrees, include: 1) preamyloid deposits which stain with antibodies that are specific for the beta-amyloid peptide and stain AD amyloid deposits and plaques, and 2) neuronal soma and processes which stain with an antibody (Alz50) that detects abnormal isoforms of tau which are characteristic of AD. The quality and distribution of both types of immunoreactivity revealed in the NSE:beta-APP751 mouse brains most closely resemble those seen in brains of young adults with Down's syndrome. Both structures are rarely, if ever, observed in brains from mice transgenic for the 695 amino acid isoform of beta-APP (NSE:beta-APP695) or in wild type mice.

Aging↗
PubMed1995

A novel strategy to identify maternal and paternal inheritance in the mouse.

A novel strategy for identifying proteins which reveal maternal or paternal inheritance in the mouse is presented. Using two-dimensional electrophoresis we investigated protein expression patterns of adult liver and different embryonic and extraembryonic tissue in C57BL/6Crl and in DBA/2Crl mice, as well as in their reciprocal hybrids. We found three groups of protein spots which showed maternal or paternal inheritance of quantitative variations. These proteins were characterized by N-terminal or internal amino acid sequencing, by determination of the amino acid composition, by glycoprotein staining and RNA expression analysis. The three proteins identified were: alpha-enolase, cyclophilin and beta-group hemoglobins. The parental effects observed for alpha-enolase and cyclophilin were found to be due to parent-specific post-translational modifications of these proteins. For the beta-group hemoglobins our results suggested parental effects on the transcriptional level.

Amino Acid Isomerases↗
PubMed1990

A helix-turn-strand structural motif common in alpha-beta proteins.

By exhaustive structural comparisons, we have found that about one-third of the alpha-helix-turn-beta-strand polypeptides in alpha-beta barrel domains share a common structural motif. The chief characteristics of this motif are that first, the geometry of the turn between the alpha-helix and the beta-strand is somewhat constrained, and second, the beta-strand contains a hydrophobic patch that fits into a hydrophobic pocket on the alpha-helix. The geometry of the turn does not seem to be a major determinant of the alpha-beta unit, because the turns vary in length from four to six residues. However, the motif does not occur when there are few constraints on the geometry of the turn-for instance, when the turns between the alpha-helix and the beta-strands are very long. It also occurs much less frequently in flat-sheet alpha-beta proteins, where the topology is much less regular and the amount of twist on the sheet varies considerably more than in the barrel proteins. The motif may be one of the basic building blocks from which alpha-beta barrels are constructed.

Alcohol Dehydrogenase↗
PubMed2006

Regulation of thalamocortical patterning and synaptic maturation by NeuroD2.

During cortical development, both activity-dependent and genetically determined mechanisms are required to establish proper neuronal connectivity. While activity-dependent transcription may link the two processes, specific transcription factors that mediate such a process have not been identified. We identified the basic helix-loop-helix (bHLH) transcription factor Neurogenic Differentiation 2 (NeuroD2) in a screen for calcium-regulated transcription factors and report that it is required for the proper development of thalamocortical connections. In neuroD2 null mice, thalamocortical axon terminals fail to segregate in the somatosensory cortex, and the postsynaptic barrel organization is disrupted. Additionally, synaptic transmission is defective at thalamocortical synapses in neuroD2 null mice. Total excitatory synaptic currents are reduced in layer IV in the knockouts, and the relative contribution of AMPA and NMDA receptor-mediated currents to evoked responses is decreased. These observations indicate that NeuroD2 plays a critical role in regulating synaptic maturation and the patterning of thalamocortical connections.

2-Amino-5-phosphonovalerate↗
PubMed2002

Direct phosphorylation of focal adhesion kinase by c-Src: evidence using a modified nucleotide pocket kinase and ATP analog.

A single mutation in the nucleotide binding pocket of select protein kinases allows for use of a bulky, substituted-ATP analog not used by the wild-type kinase [1]. Using this approach with the protein tyrosine kinase c-Src, we have generated a mutant T338G and expressed it in Src/Yes/Fyn null fibroblasts (SYF1) at near endogenous levels. T338G Src exhibits high specificity for a substituted ATP analog N(6)-2-phenyl ethyl ATP (peATP), which is not used by wild-type c-Src in autophosphorylation nor substrate phosphorylation assays. By employing the T338G Src mutant and [gamma-(32)P]peATP analog, we demonstrate that c-Src can directly phosphorylate focal adhesion kinase (Fak) in vitro. We also show that incubation of permeabilized, T338 Src-expressing cells with peATP causes an increase in Fak tyrosine phosphorylation not observed in wild-type Src cells. Taken together, these data provide evidence that Src directly phosphorylates Fak and demonstrates the limitations of using this modified ATP strategy for analysis of direct substrates of protein kinases in permeabilized cells.

Adenosine Triphosphate↗