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H Wu

Publications and source records attributed to H Wu.

At least 235 records · Page 13Linked to original sources
PubMed2000

Thermodynamic characterization of the interaction between TRAF2 and tumor necrosis factor receptor peptides by isothermal titration calorimetry.

The tumor necrosis factor receptor (TNFR) superfamily can induce diverse biological effects, including cell survival, proliferation, differentiation, and apoptosis. The major signal transducers for TNFRs are the family of TNF receptor associated factors (TRAFs). The direct interaction between TRAFs and the intracellular tails of TNFRs is the first step of this signal relay process. Structural studies have revealed a trimeric nature of TRAF2 and a symmetrical mode of receptor binding, suggesting the involvement of trivalent TNFR2-receptor interaction in the signal transduction. In this study, using isothermal titration calorimetry (ITC), we report thermodynamic characterization of the interaction between TRAF2 and monomeric peptide sequences from TNFR members, including TNFR2, CD40, CD30, Ox40, and 4-1BB, and the Epstein-Barr virus (EBV)-transforming protein, latent infection membrane protein-1 (LMP1). The dissociation constants of the interaction were shown to range between 40 microM and 1.9 mM, which are substantially weaker than most protein-peptide interactions. The interaction is entirely driven by exothermic enthalpy, consistent with the abundance of polar contacts. The enthalpy of the interaction has a significant temperature dependence (DeltaCp = -245 cal/mol small middle dotK). The unfavorable entropy in the interaction and the comparison with structural energetics calculations suggest the involvement of conformational rearrangement in the interaction. The low affinity of TRAF2 to monomeric receptor peptides further supports the importance of avidity contribution in TRAF2 recruitment by these receptors upon ligand-induced trimerization or higher order oligomerization.

Amino Acid Sequence↗
PubMed2000

Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A. Determination of proteolytic cleavage sites in hormone storage granules.

The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A(332-364); identity of the peptide was confirmed by diagnostic Met(346) oxidation. Further analysis revealed two additional forms: bovine chromogranin A(333-364) and A(343-362). Synthetic longer (chromogranin A(332-364)) and shorter (chromogranin A(344-364)) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC(50) approximately 2.01 versus approximately 0.35 microm). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A(340-372), bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.

Adrenal Medulla↗
PubMed2000

Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation.

Fertilization in mammalian eggs is characterized by the presence of intracellular calcium ([Ca(2+)]i) oscillations. In mouse eggs, these oscillations cease after a variable period of time and this is accompanied by a decrease in inositol 1,4,5-trisphosphate receptor (IP3R) responsiveness and down-regulation of the IP3R type 1 (IP3R-1). To investigate the signaling pathway responsible for inducing IP3R-1 down-regulation during fertilization, mouse eggs were exposed to or injected with several Ca(2+)-releasing agonists and the amounts of IP3R-1 immunoreactivity evaluated by Western blotting. Exposure to ethanol or ionomycin, which induce a single [Ca(2+)]i rise, failed to signal down-regulation of IP3R-1. However, [Ca(2+)]i oscillations induced by injection of boar sperm fractions (SF), which presumably stimulate production of IP3, or adenophostin A, an IP3R agonist, both induced down-regulation of IP3R-1 of a magnitude similar to or greater than that observed after fertilization. Exposure to thimerosal, an oxidizing agent that modifies the IP3R without stimulating production of IP3, also initiated down-regulation of IP3R-1, although oscillations initiated by SrCl(2) failed to evoke down-regulation of IP3R-1. The degradation of IP3R-1 in mouse eggs appears to be mediated by the proteasome pathway because it was inhibited by preincubation with lactacystin, a very specific proteasome inhibitor. We therefore suggest that persistent stimulation of the phosphoinositide pathway in mouse eggs by the sperm during fertilization or by injection of SF leads to down-regulation of the IP3R-1.

Adenosine↗
PubMed2000

Fabrication of topologically complex three-dimensional microfluidic systems in PDMS by rapid prototyping.

This paper describes a procedure for making topologically complex three-dimensional microfluidic channel systems in poly(dimethylsiloxane) (PDMS). This procedure is called the "membrane sandwich" method to suggest the structure of the final system: a thin membrane having channel structures molded on each face (and with connections between the faces) sandwiched between two thicker, flat slabs that provide structural support. Two "masters" are fabricated by rapid prototyping using two-level photolithography and replica molding. They are aligned face to face, under pressure, with PDMS prepolymer between them. The PDMS is cured thermally. The masters have complementary alignment tracks, so registration is straightforward. The resulting, thin PDMS membrane can be transferred and sealed to another membrane or slab of PDMS by a sequence of steps in which the two masters are removed one at a time; these steps take place without distortion of the features. This method can fabricate a membrane containing a channel that crosses over and under itself, but does not intersect itself and, therefore, can be fabricated in the form of any knot. It follows that this method can generate topologically complex microfluidic systems; this capability is demonstrated by the fabrication of a "basketweave" structure. By filling the channels and removing the membrane, complex microstructures can be made. Stacking and sealing more than one membrane allows even more complicated geometries than are possible in one membrane. A square coiled channel that surrounds, but does not connect to, a straight channel illustrates this type of complexity.

Dimethylpolysiloxanes↗
PubMed2000

Prototyping of masks, masters, and stamps/molds for soft lithography using an office printer and photographic reduction

This paper describes a practical method for the fabrication of photomasks, masters, and stamps/molds used in soft lithography that minimizes the need for specialized equipment. In this method, CAD files are first printed onto paper using an office printer with resolution of 600 dots/in. Photographic reduction of these printed patterns transfers the images onto 35-mm film or microfiche. These photographic films can be used, after development, as photomasks in 1:1 contact photolithography. With the resulting photoresist masters, it is straightforward to fabricate poly(dimethylsiloxane) (PDMS) stamps/molds for soft lithography. This process can generate microstructures as small as 15 microm; the overall time to go from CAD file to PDMS stamp is 4-24 h. Although access to equipment-spin coater and ultraviolet exposure tool-normally found in the clean room is still required, the cost of the photomask itself is small, and the time required to go from concept to device is short. A comparison between this method and all other methods that generate film-type photomasks has been performed using test patterns of lines, squares, and circles. Three microstructures have also been fabricated to demonstrate the utility of this method in practical applications.

Journal Article↗
PubMed2000

A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction.

TRAF proteins are major mediators for the cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily. They can be recruited to activated TNF receptors either by direct interactions with the receptors or indirectly via the adaptor protein TRADD. We now report the structure of the TRADD-TRAF2 complex, which is highly distinct from receptor-TRAF2 interactions. This interaction is significantly stronger and we show by an in vivo signaling assay that TRAF2 signaling is more readily initiated by TRADD than by direct receptor-TRAF2 interactions. TRADD is specific for TRAF1 and TRAF2, which ensures the recruitment of clAPs for the direct inhibition of caspase activation in the signaling complex. The stronger affinity and unique specificity of the TRADD-TRAF2 interaction are crucial for the suppression of apoptosis and provide a mechanistic basis for the perturbation of TRAF recruitment in sensitizing cell death induction.

Amino Acid Sequence↗
PubMed2000

Reduced anterior cingulate gyrus volume correlates with executive dysfunction in men with first-episode schizophrenia.

Although frontal lobe structural and functional abnormalities have been identified in schizophrenia, their relationship remains elusive. Because the frontal lobes are both structurally and functionally heterogeneous, it is possible that some measures of frontal lobe structure may not have accurately identified relevant frontal lobe subregions. The authors hypothesized that the volumes of two dorsal, 'archicortical' subregions (i.e. superior frontal gyrus and anterior cingulate gyrus), but not a ventral, 'paleocortical' subregion (i.e. orbital frontal region) would be significantly and selectively correlated with executive and motor dysfunction in patients with schizophrenia as previously reported for the anterior hippocampal region. Volumes of these frontal lobe subregions were measured from magnetic resonance images based on sulcal anatomy in 20 men and 15 women with first-episode schizophrenia. All patients completed a comprehensive neuropsychological test battery while clinically stabilized that encompassed six domains of functioning: attention, executive, motor, visuospatial, memory and language. Findings indicated that reduced anterior cingulate gyrus volume was significantly correlated with worse executive functioning in men; among women, there were no significant correlations. Among men, anterior cingulate gyrus volume was significantly more strongly correlated with executive functioning than with attention, visuospatial, memory, language and general intellectual functioning. Neither executive nor motor functioning was significantly more strongly correlated with the dorsal 'archicortical' volumes than with orbital frontal volume. These findings suggest a link between executive deficits and dysfunction of the dorsal 'archicortical' system and implicate sex differences in their relationship in first-episode schizophrenia.

Adult↗
PubMed2000

A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase.

Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk(lo)) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk(-/-) mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk(lo) phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk(lo) phenotype in vitro but not in vivo, whereas CD19 and the p85alpha form of phosphoinositide 3-kinase behaved as Btk(lo) enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85alpha haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C beta, Fyn, CD22, Galphaq, or Galpha11 had no detectable effect on the function of Btk(lo) B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2000

Carbocyclic influenza neuraminidase inhibitors possessing a C3-cyclic amine side chain: synthesis and inhibitory activity.

As part of our continuing work in the area of influenza neuraminidase inhibitors, a series of C3-aza inhibitors possessing a cyclic amine side chain was synthesized and evaluated for influenza neuraminidase inhibitory activity. Analogues possessing a six-, seven- and eight-membered ring, 4c-e, respectively, at the C3 position exhibited excellent influenza B neuraminidase inhibition.

Amines↗
PubMed2000

Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase.

Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis. The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor. While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible. We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes. Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases. While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence seems to be more important for proper positioning of the ligand than for catalysis. Also, Ser49 and/or Cys147 were found to possibly act as an activator for water in the cyclohydrolase step. These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases. On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase.

Amino Acid Sequence↗
PubMed2000

MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type.

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Recently, we proposed that the influence of motor nerve activity on skeletal muscle fiber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Here we demonstrate that calcineurin-dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium-regulated signaling inputs, specifically calmodulin-dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow fiber-specific enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up-regulates a MEF2-dependent reporter gene. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myofiber subtypes are established and maintained.

Animals↗
PubMed2000

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases.

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.

Animals↗
PubMed2000

A protein encoded within the Down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling.

Here we describe a small family of proteins, termed MCIP1 and MCIP2 (for myocyte-enriched calcineurin interacting protein), that are expressed most abundantly in striated muscles and that form a physical complex with calcineurin A. MCIP1 is encoded by DSCR1, a gene located in the Down syndrome critical region. Expression of the MCIP family of proteins is up-regulated during muscle differentiation, and their forced overexpression inhibits calcineurin signaling to a muscle-specific target gene in a myocyte cell background. Binding of MCIP1 to calcineurin A requires sequence motifs that resemble calcineurin interacting domains found in NFAT proteins. The inhibitory action of MCIP1 involves a direct association with the catalytic domain of calcineurin, rather than interference with the function of downstream components of the calcineurin signaling pathway. The interaction between MCIP proteins and calcineurin may modulate calcineurin-dependent pathways that control hypertrophic growth and selective programs of gene expression in striated muscles.

Alternative Splicing↗
PubMed2000

Transforming activity of receptor tyrosine kinase tyro3 is mediated, at least in part, by the PI3 kinase-signaling pathway.

Protein tyrosine phosphorylation is an integral part of cytokine-induced proliferation and differentiation of hematopoietic cells. The authors previously reported cloning and characterization of the receptor tyrosine kinase Tif, also termed Tyro3. Using the yeast 2-hybrid technology, they recently identified that the p85 subunit of phosphatidylinositol 3-kinase (PI3 kinase) interacted with the cytoplasmic domain of Tyro3. On treatment with epidermal growth factor (EGF), NIH3T3 cells expressed EGFR/Tyro3 (a fusion receptor with the extracellular domain from epidermal growth factor receptor and the transmembrane and cytoplasmic domains from Tyro3), and EGFR/Tyro3 was rapidly phosphorylated on tyrosine residues. The interaction between Tyro3 and p85 was also confirmed by glutathione S-transferase (GST) pull-down experiments. Co-immunoprecipitation followed by Western blot analysis revealed that PI3 kinase was associated with and phosphorylated by the activated Tyro3. Tyro3-associated PI3 kinase exhibited an enhanced kinase activity. In addition, EGF treatment of EGFR/Tyro3-expressing cells led to enhanced phosphorylation of Akt, a downstream component of PI3 kinase. Treatment of NIH3T3 cells expressing a full length of rat Tyro-3, but not NIH3T3 cells, with protein S also resulted in phosphorylation of Akt. Soft agar colony assays showed that the addition of EGF to EGFR/Tyro3-transfected cells, but not to the parental NIH3T3 cells, resulted in a concentration-dependent increase in the formation of anchorage-independent colonies. Tyro3-mediated transformation of NIH3T3 cells was significantly blocked by wortmannin, a PI3 kinase-specific inhibitor. Results of these combined studies strongly suggested that the oncogenic transforming ability of Tyro3 was mediated at least in part by the PI3 kinase pathway. (Blood. 2000;95:633-638)

3T3 Cells↗
PubMed2000

Identification of unexpected modifications of fluorescein-labeled oligodeoxynucleotides by nuclease P1 digestion and mass spectrometric techniques.

Fluorescein-labeled oligodeoxynucleotides (ODNs) from automated synthesis commonly produce multiple peaks in high performance liquid chromatography (HPLC) chromatograms. We found that these peaks are due to chemical modifications of the ODNs instead of the common perception of isomers. To identify the modifications, a model ODN, fluorescein-T(25), was synthesized and five compounds were isolated. Nuclease P1 (NP1) digestion was employed to cleave these compounds into nucleotides and fluorescein-nucleotides in order that the modifications be determined by mass spectrometry (MS). Analyses of NP1 digestion products containing fluorescein by MS revealed the expected product F1-T (M) and four unexpected compounds with MWs at M-1, M-17, M-16 and M + 16, respectively. Collision-induced dissociation (CID) spectra of these digestion products indicate that all modifications occur on the thiourea linkage [-NH-C( = S)-NH-] to the fluorescein moiety and the adjacent phosphate group, and the modifications were determined. The modifications were also confirmed by accurate mass measurement with Fourier transform mass spectrometry (FT-MS), by the synthesis of a reference compound, and by a mechanistic study using model compounds. These results demonstrate the power of the mass spectrometric techniques by determining the structures of two pairs of ODNs with MW difference of 1 Da. The results also suggest that fluorescein phosphoramidite with a thiourea linkage is not appropriate for the automated synthesis of fluorescein-labeled ODNs of high purity.

Chromatography, High Pressure Liquid↗
PubMed2000

Fabrication of microfluidic systems in poly(dimethylsiloxane).

Microfluidic devices are finding increasing application as analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems for fundamental research. Conventional methods of fabricating microfluidic devices have centered on etching in glass and silicon. Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes than these conventional methods to devices that handle aqueous solutions. These soft-lithographic methods are based on rapid prototyping and replica molding and are more accessible to chemists and biologists working under benchtop conditions than are the microelectronics-derived methods because, in soft lithography, devices do not need to be fabricated in a cleanroom. This paper describes devices fabricated in PDMS for separations, patterning of biological and nonbiological material, and components for integrated systems.

Animals↗
PubMed2000

Desferrioxamine (DFO) conjugated with starch decreases NAD redox potential of intact red blood cells (RBC): evidence for DFO as an extracellular inducer of oxidant stress in RBC.

Desferrioxamine (DFO) is an important iron-chelating agent. It has also been thought of as an agent with anti-oxidant potential as it chelates ferric iron in various parts of the body. However, there is evidence suggesting that it may paradoxically affect red blood cells (RBCs) by inducing intracellular oxidant stress. Recently we observed that incubation of RBCs with DFO decreases NAD redox potential in normal RBC. To further understand the mechanism of DFO's interaction with RBC, we conducted a study to determine the effect of extracellular DFO upon RBC's redox status. We examined NAD redox potential in intact RBC (N = 7) incubated with DFO conjugated to starch. RBCs were incubated with 4 mM DFO for 3(1/2) hr and with 6 mM DFO for 2 and 3(1/2) hr. Significant decreases in NAD redox potential were observed after the incubations. With 4 mM DFO at the 3 (1/2) hr time point the mean decrease was 12.37% +/- 9.96% (P < 0.0085). With 6 mM DFO, the mean decreases were 18.54% +/- 9.79% (P < 0.0013) and 19.16% +/- 8.78% (P < 0.0006) for the 2 and 3 (1/2) hr incubations, respectively. DFO by itself is very poorly permeable to RBC. Conjugation with starch further ensured impermeability of DFO. The data presented here confirm the oxidant effect of DFO on RBC. The data also demonstrate that the effect of DFO on RBC's NAD redox potential originates extracellularly.

Adult↗