Biomedical subjects

A Chen

Publications and source records attributed to A Chen.

At least 19 recordsLinked to original sources

PubMed2000

Conjugation of Nedd8 to CUL1 enhances the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization.

The SCF-ROC1 ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase complex targets the ubiquitination and subsequent degradation of protein substrates required for the regulation of cell cycle progression and signal transduction pathways. We have previously shown that ROC1-CUL1 is a core subassembly within the SCF-ROC1 complex, capable of supporting the polymerization of ubiquitin. This report describes that the CUL1 subunit of the bacterially expressed, unmodified ROC1-CUL1 complex is conjugated with Nedd8 at Lys-720 by HeLa cell extracts or by a purified Nedd8 conjugation system (consisting of APP-BP1/Uba3, Ubc12, and Nedd8). This covalent linkage of Nedd8 to CUL1 is both necessary and sufficient to markedly enhance the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. A mutation of Lys-720 to arginine in CUL1 eliminates the Nedd8 modification, abolishes the activation of the ROC1-CUL1 ubiquitin ligase complex, and significantly reduces the ability of SCF(HOS/beta)(-TRCP)-ROC1 to support the ubiquitination of phosphorylated IkappaBalpha. Thus, although regulation of the SCF-ROC1 action has been previously shown to preside at the level of recognition of a phosphorylated substrate, we demonstrate that Nedd8 is a novel regulator of the efficiency of polyubiquitin chain synthesis and, hence, promotes rapid turnover of protein substrates.

Amino Acid Substitution↗

PubMed2000

Correction of glycogen storage disease type II by enzyme replacement with a recombinant human acid maltase produced by over-expression in a CHO-DHFR(neg) cell line.

Inherited genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in the autosomal recessive glycogen storage disease type II (GSD II). To investigate whether we could generate a functional recombinant human GAA (rhGAA) for enzyme replacement therapy, we subcloned the cDNAs for human GAA and mouse dihydrofolate reductase (DHFR) into DHFR(neg) Chinese hamster ovary cells and established a stable cotransformant that expressed rhGAA. We cultured the recombinant cells in media with progressively increasing concentrations of methotrexate and found that human GAA enzyme activity increased to over 2,000 IU per gram protein. Importantly, the human GAA enzyme activity correlated to equivalent amounts of human GAA protein by rocketimmunoelectrophoresis. We confirmed that the human GAA enzyme activity corresponded to an amplification in human GAA mRNA by Northern analysis and human GAA cDNA copy number by Southern analysis. Exposing the rhGAA to human GSDII fibroblast cells or patient's lymphocytes or monocytes resulted in uptake of the rhGAA and reversal of the enzymatic defect. Mannose-6-phosphate in the media blocked uptake. GAA -/- mice were treated with the rhGAA at 1 mg/kg, which resulted in heterozygous levels of GAA in tissues, most notably skeletal muscle, heart and diaphragm after two infusions. More importantly, after multiple infusions, hind, and fore-limb muscle weakness was reversed. This rhGAA would be ideal for enzyme replacement therapy in GSD II.

Animals↗

PubMed2000

Oligomerization of pro-opiomelanocortin is independent of pH, calcium and the sorting signal for the regulated secretory pathway.

Studies indicate that pro-opiomelanocortin (POMC) is sorted to the regulated secretory pathway by binding to a sorting receptor identified as membrane-bound carboxypeptidase E (CPE) [Cool et al. (1997) Cell 88, 73-83]. The efficiency of this sorting mechanism could be enhanced if POMC molecules were to self-associate to form oligomers, prior or subsequent to binding to CPE. Using cross-linking and gel filtration techniques, we demonstrated that POMC forms oligomers at both neutral and acidic pHs and calcium was not necessary. delta N-POMC, which lacks the N-terminal sorting signal for the regulated secretory pathway, also formed similar oligomers, indicating that the sorting and oligomerization domains are different.

Animals↗

PubMed2000

Isolation and characterization of two evolutionarily conserved murine kinases (Nek6 and nek7) related to the fungal mitotic regulator, NIMA.

Entrance and exit from mitosis in Aspergillus nidulans require activation and proteolysis, respectively, of the NIMA (never in mitosis, gene A) serine/threonine kinase. Four different NIMA-related kinases were reported in mammals (Nek1-4), but none of them has been shown to perform mitotic functions related to those demonstrated for NIMA. We describe here the isolation of two novel murine protein kinase genes, designated nek6 and nek7, which are highly similar to each other (87% amino acid identity in the predicted kinase domain). Interestingly, Nek6 and Nek7 are also highly similar to the F19H6.1 protein kinase of Caenorhabditis elegans (76 and 73% amino acid identity in the kinase domain, respectively), and phylogenetic analysis suggests that these three proteins constitute a novel subfamily within the NIMA family of serine/threonine kinases. In contrast to the other documented NIMA-related kinases, Nek6/7 and F19H6.1 harbor their catalytic domain in the C-terminus of the protein. Immunofluorescence suggests that Nek6 and Nek7 are cytoplasmic. Linkage analysis, using the murine BXD recombinant inbred strain panel, localized nek6 to chromosome 2 at 28 cM. Using a mouse/hamster radiation hybrid panel, we assigned the nek7 gene to chromosome 1 at approximately 73 cM.

3T3 Cells↗

PubMed2000

Energy landscape of streptavidin-biotin complexes measured by atomic force microscopy.

The dissociation of ligand and receptor involves multiple transitions between intermediate states formed during the unbinding process. In this paper, we explored the energy landscape of the streptavidin-biotin interaction by using the atomic force microscope (AFM) to measure the unbinding dynamics of individual ligand-receptor complexes. The rupture force of the streptavidin-biotin bond increased more than 2-fold over a range of loading rates between 100 and 5000 pN/s. Moreover, the force measurements showed two regimes of loading in the streptavidin-biotin force spectrum, revealing the presence of two activation barriers in the unbinding process. Parallel experiments carried out with a streptavidin mutant (W120F) were used to investigate the molecular determinants of the activation barriers. From these experiments, we attributed the outer activation barrier in the energy landscape to the molecular interaction of the '3-4' loop of streptavidin that closes behind biotin.

Biotin↗

PubMed2000

Gene 33/Mig-6, a transcriptionally inducible adapter protein that binds GTP-Cdc42 and activates SAPK/JNK. A potential marker transcript for chronic pathologic conditions, such as diabetic nephropathy. Possible role in the response to persistent stress.

Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extracellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH(2)-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences.

3T3 Cells↗

PubMed2000

The conserved RING-H2 finger of ROC1 is required for ubiquitin ligation.

ROC1 is a common component of a large family of ubiquitin E3 ligases that regulate cell cycle progression and signal transduction pathways. Here we present evidence suggesting that a conserved RING-H2 structure within ROC1 is critical for its ubiquitin ligation function. Mercury-containing sulfhydryl modification agents (rho-hydroxymercuribenzoate and mercuric chloride) irreversibly inhibit the ROC1-CUL1 ubiquitin ligase activity without disrupting the complex. Consistent with this, these reagents also eliminate the ability of the Skp1-CUL1-HOS-ROC1 E3 ligase complex to support the ubiquitination of IkappaBalpha. Site-directed mutagenesis analysis identifies RING-H2 finger residues Cys(42), Cys(45), Cys(75), His(77), His(80), Cys(83), Cys(94), and Asp(97) as being essential for the ROC1-dependent ubiquitin ligase activity. Furthermore, C42S/C45S and H80A mutations reduce the ability of ROC1 to interact with CUL1 in transfected cells and diminish the capacity of ROC1-CUL1 to form a stable complex with Cdc34 in vitro. However, C75S, H77A, C94S, and D97A substitutions have no detectable effect on ROC1 binding activities. Thus, the ROC1 RING-H2 finger may possess multiple biochemical properties that include stabilizing an interaction with CUL1 and recruiting Cdc34. A possible role of the RING finger in facilitating the Ub transfer reaction is discussed.

Amino Acid Sequence↗

PubMed2000

The mechanical properties of compacts of microcrystalline cellulose and silicified microcrystalline cellulose.

The mechanical properties of compacts of unlubricated microcrystalline cellulose and silicified microcrystalline cellulose were evaluated using the diametric tensile test. The results suggested that, under comparable testing conditions, compacts of silicified microcrystalline cellulose exhibited greater strength than those of microcrystalline cellulose. In addition to enhanced strength, silicified microcrystalline cellulose compacts exhibited greater stiffness and required considerably more energy for tensile failure to occur. Comparison of the data with that obtained for a dry blend of silicon dioxide/microcrystalline cellulose suggested that the functionality benefits of silicification were not due to a simple composite material model.

Cellulose↗

PubMed2000

The Akt proto-oncogene links Ras to Pak and cell survival signals.

The Ras oncogene regulates cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Ras signals through its effector phosphoinositide 3 (PI3) kinase to the Pak protein kinase (p65(pak)), but the steps from Ras to Pak remain to be elucidated. PI3 kinase can stimulate the small G protein, Rac, a direct activator of Pak, as well as the Akt proto-oncogene, a serine-threonine protein kinase. We found that activated Akt stimulated Pak, whereas a dominant negative Akt inhibited Ras activation of Pak in transfection assays. Akt stimulation of Pak was not inhibited by dominant negative mutants of either Rac or Cdc42 suggesting that Akt activated Pak through a GTPase-independent mechanism. We also developed a novel cell-free system to study Ras activation of Pak. In this system Ras activated Pak only in the presence of a crude cell extract but failed to activate Pak when Akt was immunodepleted from the extract. Akt protects cells from apoptosis through phosphorylation of downstream targets such as the Bcl-2 family member, Bad. We found that activated Pak decreased apoptosis and increased phosphorylation of Bad, whereas dominant negative Pak increased apoptosis and decreased phosphorylation of Bad. These studies define a new oncogene-mediated cell survival signal.

Animals↗

PubMed2000

Nitric oxide influences injury-induced microglial migration and accumulation in the leech CNS.

Damage to the leech or mammalian CNS increases nitric oxide (NO) production and causes accumulation of phagocytic microglial cells at the injury site. The aim of this study was to determine whether NO plays a role in microglial migration and accumulation at lesions in which NO is generated by a rapidly appearing endothelial nitric oxide synthase (eNOS) in leeches. Immunohistochemistry and cytochemistry demonstrated active eNOS before and throughout the period of microglial accumulation at the lesion. Decreasing NO synthesis by application of the NOS inhibitor N(w)-nitro-L-arginine methyl ester (1 mM) significantly reduced microglial accumulation, whereas its inactive enantiomer N(w)-nitro-D-arginine methyl ester (1 mM) resulted in microglial accumulation similar to that in crushed controls. Increasing NO with the donor spermine NONOate (SPNO) (1 mM) also inhibited accumulation, but not in the presence of the NO scavenger 2-(4-carboxyphenyl)-4,4,5, 5-teramethylimidazoline-oxyl-3-oxide (50 microM). The effect of SPNO was reversed by washout. SPNO application reduced average microglial migratory speeds and even reversibly arrested cell movement, as measured in living nerve cords. These results suggest that NO produced at a lesion may be a stop signal for microglia to accumulate there and that it can act on microglia early in their migration. Thus, NO may assume a larger role in nerve repair and recovery from injury by modulating accumulation of microglia, which appear to be important for axonal regeneration.

Animals↗

PubMed2000

Characterization of a novel human serine protease that has extensive homology to bacterial heat shock endoprotease HtrA and is regulated by kidney ischemia.

We report the isolation and characterization of a cDNA encoding the novel mammalian serine protease Omi. Omi protein consists of 458 amino acids and has homology to bacterial HtrA endoprotease, which acts as a chaperone at low temperatures and as a proteolytic enzyme that removes denatured or damaged substrates at elevated temperatures. The carboxyl terminus of Omi has extensive homology to a mammalian protein called L56 (human HtrA), but unlike L56, which is secreted, Omi is localized in the endoplasmic reticulum. Omi has several novel putative protein-protein interaction motifs, as well as a PDZ domain and a Src homology 3-binding domain. Omi mRNA is expressed ubiquitously, and the gene is localized on human chromosome 2p12. Omi interacts with Mxi2, an alternatively spliced form of the p38 stress-activated kinase. Omi protein, when made in a heterologous system, shows proteolytic activity against a nonspecific substrate beta-casein. The proteolytic activity of Omi is markedly up-regulated in the mouse kidney following ischemia/reperfusion.

Amino Acid Sequence↗

PubMed2000

Hierarchical costimulator thresholds for distinct immune responses: application of a novel two-step Fc fusion protein transfer method.

Activation of T cells is dependent upon coordinate engagement of Ag and costimulator receptors on their surfaces. In the case of the Ag receptors (TCRs), activation thresholds have been defined, with the number of TCRs that must be triggered to stimulate cytokine secretion by individual activated T cells differing for the various cytokines. In the present study, we have determined whether comparable activation thresholds exist for the costimulator receptors on T cells. To facilitate this type of quantitative costimulator analysis, we developed a novel two-step protein transfer approach that permits delivery of graded amounts of proteins to APC surfaces. By adding a human B7-1. Fcgamma1 (Fc domain of human IgG1) fusion protein to cells precoated with palmitated protein A, fine titration of the B7-1 extracellular domain was achieved. The B7-1. Fcgamma1 reincorporated into cell membranes by this method retained costimulator function, as measured by an in vitro proliferation assay. The degree of proliferation was dependent on the surface density of B7-1. Fcgamma1. Significantly, the threshold B7-1. Fcgamma1 density required for cytokine production differed between IFN-gamma and IL-2 and mirrored the hierarchy (IFN-gamma < IL-2) described previously for the TCR activation threshold. Hence, this study invokes a novel protein transfer strategy to establish that the levels of surface costimulator on APCs can dictate both the magnitude and the quality of evoked T cell responses. The notion of costimulator receptor activation thresholds emerges.

B7-1 Antigen↗

PubMed2000

Platelet-derived growth factor is a principal inductive factormodulating mannose 6-phosphate/insulin-like growth factor-II receptorgene expression via a distal E-box in activated hepatic stellate cells.

Hepatic stellate cells (HSCs) become activated during the earlystages of hepatic injury associated with fibrogenesis. The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGFIIR) plays animportant role in early fibrogenesis by participating in the activationof latent transforming growth factor-beta, a potent inducer of thematrix proteins in activated stellate cells that produce the hepaticnodule. Platelet-derived growth factor (PDGF), a potent HSC mitogen, isreleased early in hepatic injury and activates several signallingpathways in HSCs. In this study we examined the role of PDGF-BB in HSCregulation of M6P/IGFIIR gene expression. Several promoter elementswere found and characterized that modulate M6P/IGFIIR expression inactivated stellate cells. The presence of a distal CACGTG E-box at-2695 was required for M6P/IGFIIR expression in transfectedstellate cells. When the distal E-box was removed there was no significant M6P/IGFIIR promoter activity. The distal E-box-binding protein responded specifically to PDGF-BB with increased binding. This coincided with PDGF-BB up-regulation of M6P/IGFIIR mRNA transcript levels. Downstream elements include two proximal (-2 to-48) CACGTG E-boxes that bind a different protein to the distal(-2695) E-box. The proximal E-boxes respond moderately to PDGF-BB. The promoter segment encompassing -144 to +109 is able to respond dramatically to serum but is refractory to PDGF-BB. However, a constitutively bound protein binding to the -611/-716 fragment appears to be a repressor that suppresses inductive changes in protein binding occurring downstream of -611. These results indicate that the M6P/IGFIIR promoter responds primarily and specifically to PDGF-BB through a distal E-box element and possibly through two proximal E-box elements.

Animals↗

PubMed2000

Microvascular surgery in a bloodless field.

Although tourniquets play an integral role in extremity surgery, no clear guidelines exist for the use of tourniquets in microsurgery. We undertook a study in 12 healthy volunteers to better understand the coagulation properties of blood distal to an inflated tourniquet. At a 15-min inflation time, blood distal to an inflated tourniquet clots faster than blood taken from the opposite arm after addition of exogenous thrombin (12.5 s vs 17.5 s, P < 0.0001). Neither fibrinopeptide A (FPA) levels nor tissue plasminogen activator (tPA) levels were different from those of controls. Tissue factor pathway inhibitor (TFPI), an endogenous local anticoagulant, was slightly but significantly elevated in tourniquet blood. Although much remains to be understood, we believe that microvascular surgery in a bloodless field is safe and efficacious. Nine patients are presented who successfully underwent microvascular surgery in a bloodless field, using various types of extremity tourniquets.

Adult↗

PubMed2000

Cross-linking of cell surface receptors enhances cooperativity of molecular adhesion.

Cooperativity of molecular adhesion has been proposed as a mechanism for enhanced binding strength of adhesion molecules on the cell surface. Direct evidence for its mechanism, however, has been lacking until now. Atomic force microscopy (AFM) was used to measure the adhesive strength between concanavalin A (Con A) coupled to an AFM tip and Con A receptors on the surface of NIH3T3 fibroblast cells. Cross-linking of receptors with either glutaraldehyde or 3, 3'-dithio-bis(sulfosuccinimidylproprionate) (DTSSP) led to an increase in adhesion that could be attributed to enhanced cooperativity among adhesion complexes. An increase in loading rate due to greater stiffness of fixed cells also contributed to the twofold increase in binding strength. These results show that receptor cross-linking can greatly contribute to a total increase in cell adhesion by creating a shift toward cooperative binding of receptors.

3T3 Cells↗

PubMed2000

In vitro IgE inhibition in B cells by anti-CD23 monoclonal antibodies is functionally dependent on the immunoglobulin Fc domain.

CD23, the low affinity receptor for IgE (FcvarepsilonRII), is involved in regulation of IgE synthesis by B-lymphocytes. Five monoclonal antibodies to human CD23 were generated from cynomolgus macaques immunized with purified soluble CD23 (sCD23). Four of the five primate antibodies blocked the binding of IgE complexes to CD23 positive cells and also inhibited the production of IgE in vitro by IL-4 induced human peripheral blood mononuclear cells (PBMC). The variable domains of several primate antibodies were utilized to construct chimeric macaque/human (PRIMATIZED((R))) monoclonal antibodies. PRIMATIZED((R)) p5E8G1, containing human gamma 1 constant region, inhibited IgE production in vitro as efficiently as the parent primate antibody, but the human gamma 4 constant version, PRIMATIZED((R)) p5E8G4, was not as effective in IgE inhibition. An F(ab')(2) of p5E8G1 did not inhibit IgE production but did interfere with IgE inhibition by the intact anti-CD23 antibody in a dose dependent fashion. The murine monoclonal antibody MHM6 recognizes human CD23 at a different epitope than primate antibody 5E8, and inhibits IgE production by IL-4 induced PBMC. As with the F(ab')(2) of p5E8G1, the F(ab')(2) of MHM6 also failed to inhibit IgE production. These data imply that the mechanism by which anti-CD23 antibodies inhibit IgE production requires cross-linking of CD23 to an IgG receptor. These data also imply that neither bivalent cross-linking of CD23 alone or inhibition of CD23 binding to its natural ligands is sufficient to inhibit IgE production.

Animals↗

PubMed2000

SPEEDY: spin-echo enhanced diffusion filtered spectroscopy. A new tool for high resolution MAS NMR

The pulsed field gradient diffusion edited experiment, bipolar LED, has been combined with the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo sequence for the analysis of solid phase resin samples. Spin-echo enhanced diffusion filtered spectroscopy (SPEEDY), when optimized, filters both the compounds that demonstrate fast diffusion rates as well as the compounds that demonstrate fast T(2) relaxation rates. Using this technique, compounds that are not covalently attached to the resin are not observed and contributions from the resin matrix are greatly attenuated. The interpretation of the resulting spectrum is more readily accessible. This technique lessens the importance of completely removing reaction residues or the wash solvents simply for analytical evaluation. The utility of the combined filtering scheme was demonstrated by the implementation into a NOESY sequence.

Journal Article↗

PubMed2000

Identification of two subtypes of infantile acid maltase deficiency.

Infantile patients with acid maltase deficiency have severe hypertrophic cardiomyopathy, left ventricular outflow obstruction, and generalized muscle weakness and die before 1 year of age. We identified 12 infants with acid maltase deficiency who had a similar clinical presentation but less severe cardiomyopathy and absence of left ventricular outflow obstruction, and 9 of 12 had longer survival with assisted ventilation and supplemental intubation.

Age of Onset↗