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J Wu

Publications and source records attributed to J Wu.

At least 1,135 records · Page 63Linked to original sources
PubMed1995

beta-Amyloid selectively augments NMDA receptor-mediated synaptic transmission in rat hippocampus.

The effect of beta-amyloid on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated glutamatergic excitatory postsynaptic currents measured with whole-cell voltage-clamp techniques, was investigated in the rat dentate gyrus in vitro. Application of beta-amyloid (1-40) by extracellular perfusion (200 nM) or intracellularly via the recording pipette (100 nM) resulted in a gradual enhancement of the NMDA receptor-mediated synaptic currents which did not reverse on washout. Basal AMPA receptor-mediated transmission, resting membrane potential or input resistance of the granule cells did not change. These results provide direct evidence that beta-amyloid selectively interacts with the potentially neurotoxic NMDA receptor via a postsynaptic site. It is proposed that this action may help explain the synaptic changes seen in Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed1995

Atrial natriuretic peptide suppresses the transcription of its guanylyl cyclase-linked receptor.

Atrial natriuretic peptide (ANP) treatment of rat aortic smooth muscle cells suppressed both 125I-ANP binding and ANP-dependent cGMP accumulation, suggesting reductions in the type C (NPR-C) and type A (NPR-A) natriuretic peptide receptor populations, respectively. NPR-A, but not NPR-C, mRNA levels were reduced in a dose-dependent fashion by ANP. The latter effect appeared to be due, at least in part, to suppression of NPR-A gene promoter activity. ANP effected a dose- and time-dependent reduction in a transiently transfected NPR-A luciferase reporter (-1575LUC). Analysis of 5' deletion mutants of the NPR-A promoter demonstrated that the ANP-dependent sequence lies between -1575 and -1290 relative to the transcription start site. Inhibition of the ANP promoter was also effected by brain natriuretic peptide, type C natriuretic peptide, and 8-bromo-cGMP, but not by the NPR-C-selective ligand cANF. In the case of 8-bromo-cGMP, the responsive element(s) was localized to the same 285-base pair region linked to the ANP effect above. These findings indicate that ANP autoregulates its own receptors in these cells and, at least in the case of NPR-A, it does so through suppression of receptor gene expression and receptor synthesis. This suppression may operate through a cGMP-dependent element located more than a kilobase upstream from the transcription start site.

Animals↗
PubMed1995

Axonal degeneration promotes abnormal accumulation of amyloid beta-protein in ascending gracile tract of gracile axonal dystrophy (GAD) mouse.

The GAD mouse is a spontaneous neurological mutant with axonal dystrophy in the gracile tract of the medulla oblongata and spinal cord. The immunoreactivity of amyloid precursor protein (APP-IR) and amyloid beta-protein (A beta P-IR) was examined in the gracile tract and the dorsal root ganglia of normal and GAD mice. The mice were studied at 4, 9, 18, and 32 weeks of age. These periods correspond clinically to the initial, progressive, critical, and terminal stages of the disease, respectively. The APP-IR in both axons and glial cells was already accentuated to a higher level as early as 4 weeks of age in the gracile nucleus of GAD mouse. Similarly there was increase in APP-IR of GAD mouse in the dorsal root ganglia. Almost all of the primary neurons in the dorsal root ganglia at the lumbar cord level of GAD mouse revealed stronger APP-IR than those of normal mouse throughout all stages. The cells showing immunoreactivity for amyloid beta-protein became positive in axons and glial cells in the gracile nucleus by approximately the 9th week, and followed by an increase of A beta P-IR in order of the cervical, thoracic and lumbar spinal cords. These results suggest that the initial feature in GAD mouse is an accumulation of amyloid precursor protein induced by axonal dystrophy which then leads to a deposition of amyloid beta-protein within the cytoplasm of both axons and glial cells in the gracile tract.

Amyloid beta-Protein Precursor↗
PubMed1995

Exact mass determination for elemental analysis of ions produced by matrix-assisted laser desorption.

The exact masses of bastadins, cyclic peptides from marine sponges Ianthella basta, are determined using matrix-assisted laser desorption ionization (MALDI) coupled to a Fourier transform mass spectrometer. Two known compounds were mixed with the unknown to serve as internal calibrants. The mass of the calibrants bracketed the mass of the unknown compound. With this method, exact masses were obtained to within 5 ppm for single determinations, and less than 3 ppm for multiple determinations, allowing the derivation of elemental composition. This method is viable for routinely obtaining the exact masses of new compounds with MALDI.

Animals↗
PubMed1995

The glutamine hydrolysis function of human GMP synthetase. Identification of an essential active site cysteine.

GMP synthetase (EC 6.3.5.2) is an amidotransferase that catalyzes the amination of xanthosine 5'-monophosphate to form GMP in the presence of glutamine and ATP. Glutamine hydrolysis produces the necessary amino group while ATP hydrolysis drives the reaction. Ammonia can also serve as an amino group donor. GMP synthetase contains two functional domains, which are well coordinated. The "glutamine amide transfer" or glutaminase domain is responsible for glutamine hydrolysis. The synthetase domain is responsible for ATP hydrolysis and GMP formation. Inorganic pyrophosphate inhibits the synthetase and uncouples the two domain functions by allowing glutamine hydrolysis to take place in the absence of ATP hydrolysis or GMP formation. Acivicin, a glutamine analog, selectively abolishes the glutaminase activity. It inhibits the synthetase activity only when glutamine is the amino donor. When ammonia is used in place of glutamine, acivicin has no effect on the synthetase activity. Acivicin inhibits GMP synthetase irreversibly by covalent modification. Enzyme inactivation is greatly facilitated by the presence of substrates. Acivicin labels GMP synthetase at a single site, and a tryptic peptide containing the modified residue was isolated. Mass spectrometry and Edman sequence analysis show that Cys104 is the site of modification. This residue is conserved among GMP synthetases and is located within a predicted glutamine amide transfer domain. These data suggest that Cys104 is an essential residue involved in the hydrolysis of glutamine to produce an amino group and is not needed for the hydrolysis of ATP or amination of xanthosine 5'-monophosphate to produce GMP.

Adenosine Triphosphate↗
PubMed1995

Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.

Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and derivatized with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper [OP(Cu)]. Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease in dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximately 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody. Remarkably, the same cleavage products are observed with permease embedded in the native membrane. Comparison with the C-terminal half of the permease expressed independently as a standard indicates that the 19-kDa product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI. Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mutagenesis studies. Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximately 19 kDa and at 15-16 kDa. The larger fragment probably reflects cleavage at a site near the cytoplasmic end of helix VII, whereas the 15- to 16-kDa fragment is consistent with cleavage near the cytoplasmic end of helix VIII. When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys permease), a single product is observed at 19 kDa, suggesting fragmentation at the cytoplasmic end of helix VII. However, when the reagent is moved 100 degrees in the other direction to position 147 (Gly-147 --> Cys permease), cleavage is not observed. The results suggest that helix V is in close proximity to helices VII and VIII with position 148 in the interface between the helices, position 149 facing helix VII, and position 147 facing the lipid bilayer.

Amino Acid Sequence↗
PubMed1995

beta-Amyloid-(1-40) increases long-term potentiation in rat hippocampus in vitro.

The effect of beta-amyloid-(1-40) was investigated on long-term potentiation of glutamatergic excitatory postsynaptic field potentials recorded in the inner molecular layer in the rat dentate gyrus in vitro. In the presence of 200 nM beta-amyloid-(1-40) there was an increase in long-term potentiation of 51%. Basal synaptic transmission was not affected. These results provide direct evidence that a relatively low concentration of beta-amyloid-(1-40) increases synaptic plasticity.

Amyloid beta-Peptides↗
PubMed1995

Protein analysis by isoelectric focusing in a capillary array with an absorption imaging detector.

Isoelectric focusing (IEF) was successfully performed in capillary arrays with up to four capillaries. Separated proteins in the capillary array were detected by an UV absorption imaging detector. The whole analysis time for all samples in the capillary array was only 3 min due to the real-time imaging detector. The instrument was applied to analyse several protein samples including different human hemoglobin variants, myoglobin, transferrin, carbonic anhydrase and a monoclonal antibody to fluorescein. Because of good reproducibility of the focused pattern, unknown samples can be run simultaneously with a standard in the multichannel instrument and the components of unknown samples can be identified by comparing their zone positions to those of the standard. Minor components can be determined by the instrument in the presence of major components with 100 times higher concentrations in human hemoglobin samples. This instrument could be a powerful analytical tool for clinical analysis and for quality control in pharmaceutical companies.

Antibodies, Monoclonal↗
PubMed1995

Dynamics of lactose permease of Escherichia coli determined by site-directed chemical labeling and fluorescence spectroscopy.

Mutants with a single Cys residue in place of Phe27, Pro28, Phe29, Phe30, or Pro31 at the periplasmic end of putative transmembrane helix I were used to study the interaction of lactose permease with ligand by site-directed chemical modification or fluorescence spectroscopy. With permease embedded in the native membrane, mutant Phe27-->Cys or Phe28-->Cys is readily labeled with [14C]-N-ethylmaleimide (NEM), while mutant Phe29-->Cys, Phe30-->Cys, or Phe31-->Cys reacts less effectively. beta,D-Galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) has little or no effect on the reactivity of Phe27-->Cys, Phe29-->Cys, or Phe30-->Cys permease. Remarkably, however, Pro31-->Cys permease which is essentially unreactive in the absence of ligand becomes highly reactive in the presence of TDG. Ligand also enhances the NEM reactivity of the mutant with Cys in place of Pro28 which is presumably on the same face of helix I as position 31. The five single-Cys mutants which also contain a biotin acceptor domain in the middle cytoplasmic loop were purified by monomeric avidin-affinity chromatography in dodecyl beta,D-maltoside and subjected to site-directed fluorescence spectroscopy. Mutants Phe27-->Cys, Phe29-->Cys, and Phe30-->Cys react rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), and reactivity is not altered in the presence of TDG. In striking contrast, mutants Pro28-->Cys and Pro31-->Cys react extremely slowly with MIANS in the absent of ligand, and TDG dramatically enhances reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region.

The Ras-related nuclear protein, Ran, has been implicated in nuclear transport. By screening a HeLa cell lambda expression library with Ran-GTP and sequencing overlapping cDNA clones, we have obtained the derived primary structure of a protein with a calculated molecular mass of 358 kDa. Using antibodies raised against an expressed segment of this protein, we obtained punctate nuclear surface staining by immunofluorescence microscopy that is characteristic for nucleoporins. Electron microscopy of immunogold-decorated rat liver nuclear envelopes sublocalized the 358-kDa protein at (or near) the tip of the cytoplasmic fibers of the nuclear pore complex (NPC). In agreement with current convention, this protein was therefore termed Nup358 (for nucleoporin of 358 kDa). Nup358 contains a leucine-rich region, four potential Ran binding sites (i.e. Ran binding protein 1 homologous domains) flanked by nucleoporin-characteristic FXFG or FG repeats, eight zinc finger motifs, and a C-terminal cyclophilin A homologous domain. Consistent with the location of Nup358 at the cytoplasmic fibers of the NPC, we found decoration with Ran-gold at only the cytoplasmic side of the NPC. Thus, Nup358 is the first nucleoporin shown to contain binding sites for two of three soluble nuclear transport factors so far isolated, namely karyopherin and Ran-GTP.

Amino Acid Isomerases↗
PubMed1995

Sphingosine 1-phosphate rapidly activates the mitogen-activated protein kinase pathway by a G protein-dependent mechanism.

Addition of sphingosine 1-phosphate induces proliferation of quiescent Swiss 3T3 fibroblasts by unknown mechanisms. To identify the pathways involved, the ability of sphingosine 1-phosphate to activate mitogen-activated protein (MAP) kinase was studied. Sphingosine 1-phosphate rapidly activated the Raf/MAP kinase kinase (MKK)/MAP kinase pathway, and the concentration dependence for MAP kinase activation correlated with that for induction of DNA synthesis. Both MKK1 and MKK2 were activated by sphingosine 1-phosphate, assessed by specific immune complex kinase assays. Prior treatment of the Swiss 3T3 cells with pertussis toxin inhibited 70-80% of the sphingosine 1-phosphate-stimulated MAP kinase activity. Thus, one of the direct or indirect targets of exogenous sphingosine 1-phosphate appears to be a G(i)/G(o) protein.

3T3 Cells↗
PubMed1995

Interleukin-1 beta converting enzyme requires oligomerization for activity of processed forms in vivo.

Interleukin-1 beta converting enzyme (ICE) is composed of 10' (p10) and 20 kDa (p20) subunits, which are derived from a common 45 kDa precursor. Recent crystallographic studies have shown that ICE exists as a tetramer (p20/p10)2 in the crystal lattice. We provide evidence that the p10 and p20 subunits of ICE associate as oligomers in transfected COS cells. Using intragenic complementation, we show that the activity of a p10/p10 interface mutant defective in autoprocessing can be restored by co-expression with active site ICE mutants. Different active site mutants can also complement each other by oligomerization to form active ICE. These studies indicate that ICE precursor polypeptides may associate in different quaternary structures and that oligomerization is required for autoprocessing. Furthermore, integenic complementation of active site mutants of ICE and an ICE homolog restores autoprocessing activity, suggesting that hetero-oligomerization occurs between ICE homologs.

Amino Acid Sequence↗
PubMed1995

Overproduction and physical characterization of SoxR, a [2Fe-2S] protein that governs an oxidative response regulon in Escherichia coli.

SoxR protein governs the soxRS (superoxide response) regulon of Escherichia coli by becoming a transcriptional activator when the cells are exposed to compounds that mediate univalent redox reactions, many of which produce superoxide as a by-product. SoxR was overproduced and purified to near homogeneity from a strain bearing an expression vector. It could bind specifically to the soxS operator even in the absence of RNA polymerase. The aerobically purified protein, which is readily autooxidized, could activate the transcription of soxS DNA even without exposure to known inducing agents. SoxR is a globular homodimer. It contains one [2Fe-2S] cluster per polypeptide chain, as demonstrated by optical and EPR spectroscopy combined with stoichiometric analysis of iron content, unpaired-electron-spin density, and reduction by dithionite. The protein is active in its oxidized ([2Fe-2S]2+) state. The presence of a prosthetic group capable of univalent redox reactions may help to explain the activation of the regulon in vivo by compounds that can mediate such reactions.

Bacterial Proteins↗
PubMed1995

Rapamycin graft pretreatment in small bowel and kidney transplantation in the rat.

The effect of rapamycin (RAPA) as graft pretreatment was evaluated in orthotopic small bowel and kidney allotransplantation (Tx) in the rat. In the small bowel Tx model, six groups were involved, each including three combinations for evaluation of host-versus-graft (HVG) [Lewis (LEW) x Brown Norway (BN) (LBN)-F1-->Lewis], graft-versus-host (GVH) (LEW-->F1), and combined HVG and GVH immune responses (BN-->LEW). RAPA graft pretreatment alone (16 micrograms/ml x 3 ml) was able to induce a modest but significant prolongation of survival in all three combination models compared with controls (P < 0.05). The same was observed for low dose CsA treatment (2 mg/kg/day x 14 days) of the recipient only (P < 0.05). Combination of graft pretreatment with RAPA and CsA recipient treatment produced a marked prolongation of survival especially in HVG response. Recipients treatment with one 48-microgram bolus of RAPA i.v. immediately after graft revascularization failed to achieve any prolongation of survival for the GVH or combined HVG and GVH responses. This seems to exclude a "carry-over" effect of RAPA from graft to recipient. RAPA efficacy was also clearly confirmed in the kidney graft pretreatment model as compared to recipient treatment with an equivalent RAPA dose. These results demonstrate that graft RAPA pretreatment prolongs SB survival after Tx in the rat for HVG, GVH, and bidirectional immune responses. Intragraft interaction with passenger leukocytes or APC function appears as one of the possible mechanisms. RAPA graft pretreatment potentiates low dose CsA recipient treatment suggesting a possible use in clinical organ Tx.

Animals↗
PubMed1995

Regulation of the Caulobacter crescentus rpoN gene and function of the purified sigma 54 in flagellar gene transcription.

The sequential transcription of flagellar (fla) genes in the Caulobacter crescentus cell cycle is controlled by the organization of these genes in a regulatory hierarchy of four levels (I-IV). Level III and level IV genes at the bottom of the hierarchy are dependent on level II genes and are transcribed late in the cell cycle from sigma 54-dependent promoters. To study the regulation of genes at levels III and IV, we have isolated and sequenced the rpoN gene in order to analyze its expression, purified the rpoN gene product, and examined the role of the RpoN protein in initiation of transcription from sigma 54-dependent promoters. We report here epistasis experiments that show rpoN is required for transcription of level III genes, but that the expression of the rpoN gene itself is not dependent on any of the fla genes examined; these results place rpoN at level II near the top of the hierarchy. Consistent with this conclusion were nuclease S1 assays that mapped the rpoN transcription start site and identified a sequence centered at -24, GTTA/TACCA/TT, which is similar to the core consensus sequence of the level IIB fliF, fliL, and fliQ promoters. We purified the full-length rpoN gene product to near homogeneity and demonstrated that the RpoN protein is required for transcription from the well-characterized sigma 54-dependent glnAp2 promoter of Escherichia coli and specifically recognizes the level III flbG gene promoter of C. crescentus. These last results confirm that rpoN encodes the C. crescentus sigma 54 factor and opens the way for the biochemical analysis of transcriptional regulation of level III and IV fla genes.

Amino Acid Sequence↗
PubMed1995

Reversible dissociation and unfolding of the Escherichia coli aspartate receptor cytoplasmic fragment.

The thermal denaturation of a 31-kDa soluble fragment derived from the Escherichia coli aspartate receptor cytoplasmic region (c-fragment) was found to be reversible. Denaturation monitored by differential scanning calorimetry (DSC) and circular dichroism (CD) was typically over 90% reversible in pH 7.0 buffer. The wild-type c-fragment exhibited one transition (Tm = 51 degrees C), which was taken as the main denaturation transition. c-Fragments derived from signaling mutants, shown to form oligomers by gel filtration chromatography (GFC), displayed a second low-temperature transition that correlated with the disappearance of the oligomeric form in the GFC traces over the same temperature range. The CD and DSC experiments also indicated that oligomers were more folded than monomers, observations that may provide an explanation for the structural basis of the smooth-swimming signaling state of the receptor. Octyl glucoside (OG), phospholipid (PL), and glycerol were added to characterize factors that contribute to c-fragment stability. At 10 mg/mL OG, the van't Hoff enthalpy of unfolding was reduced ca. 10-fold, although at room temperature the CD spectrum indicated little change in the secondary structure. The van't Hoff enthalpy was not affected by 35% (w/v) glycerol, but the Tm increased by ca. 18 degrees C. Cooperative transitions were detected in buffer containing OG, PL, and glycerol (10 mg/mL, 2 mg/mL, 35%, respectively). The correlation between conditions where cooperative transitions are observed, and where aspartate-modulated receptor signaling has been previously observed, provides an explanation for the inhibition of signaling in OG-containing buffers without glycerol and PL.

Aspartic Acid↗