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C H Chung

Publications and source records attributed to C H Chung.

At least 37 records · Page 2Linked to original sources
PubMed2001

The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease.

HslVU is a two-component ATP-dependent protease, consisting of HslV peptidase and HslU ATPase. CodW and CodX, encoded by the cod operon in Bacillus subtilis, display 52% identity in their amino acid sequences to HslV and HslU in Escherichia coli, respectively. Here we show that CodW and CodX can function together as a new type of two-component ATP-dependent protease. Remarkably, CodW uses its N-terminal serine hydroxyl group as the catalytic nucleophile, unlike HslV and certain beta-type subunits of the proteasomes, which have N-terminal threonine functioning as an active site residue. The ATP-dependent proteolytic activity of CodWX is strongly inhibited by serine protease inhibitors, unlike that of HslVU. Replacement of the N-terminal serine of CodW by alanine or even threonine completely abolishes the enzyme activity. These results indicate that CodWX in B.subtilis represents the first N-terminal serine protease among all known proteolytic enzymes.

Adenosine Triphosphate↗
PubMed2001

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.

BACKGROUND: The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS: We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS: Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

ATP-Dependent Proteases↗
PubMed2001

Evidence for a short form of RGS3 preferentially expressed in the human heart.

RGS proteins (regulators of G protein signalling) negatively regulate G protein function as GTPase-activating proteins (GAP) for G protein alpha-subunits. The existence of mRNAs of different size for some of the RGS proteins, e.g. RGS3, suggests that these proteins may exist in isoforms due to alternative splicing. We therefore investigated RGS3 mRNA and protein expression in different human tissues. Ribonuclease protection assays and Northern blot analysis showed two specific mRNAs for RGS3 (RGS3L, RGS3S) in human myocardium, suggesting an additional, N-terminally truncated form of approximately 168 aa. When expressed as a recombinant protein RGS3S was recognized at approximately 23 kDa by an antipeptide antiserum originally raised against an RGS2 sequence. In membranes of human tissues this antiserum detected specific signals for RGS3L (approximately 70 kDa), RGS2 (approximately 30 kDa) and a 25-kDa protein, most likely RGS3S. Both RGS3S mRNA and the 25 kDa protein were abundant in human heart, whereas expression in liver, brain and myometrium was much weaker. To characterize RGS3S functionally, single turnover GTPase, adenylyl cyclase (AC) and phospholipase C (PLC) activities were determined. Both recombinant RGS3S and RGS16 increased Pi release from Galphai1 by about 150% and increased GTP- and GTP plus isoprenaline-stimulated AC activity by 20-30% in human left ventricular myocardial membranes. Additionally, both RGS proteins reduced basal and endothelin-stimulated PLC activity in these membranes by about 40%. We conclude that an additional truncated form of RGS3 is expressed in the human heart. As described for the full-length protein, RGS3S negatively regulates the activity of Gi/o- and Gq-, but not Gs-subfamily members.

Brain↗
PubMed2001

Leaching characteristics of paraffin waste forms generated from Korean nuclear power plants.

Leaching tests of paraffin waste forms including boric acid, cobalt, strontium and cesium were performed to investigate the leaching characteristics of paraffin waste forms which had been generated in Korean nuclear power plants. The leaching tests were conducted according to ANSI/ANS-16.1 test procedure and the cumulative fractions leached (CFLs) of boric acid, cobalt, strontium and cesium were obtained. The compressive strength before and after the leaching test was measured for various waste forms with different mixing ratios of boric acid to paraffin. It was observed that boric acid and other nuclides immobilized within paraffin waste forms were congruently released and the leaching rates were influenced by reacted layer depth as the dissolution reaction progressed. A shrinking core model based on the diffusion-controlled dissolution kinetics was developed in order to simulate the test results. The CFLs and the leaching rates were well expressed by the shrinking core model and the cross-sectional view of specimen after the test demonstrated the applicability of this model with the shrinking dissolution front to the leaching analysis of paraffin waste forms.

Boric Acids↗
PubMed2001

Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU.

BACKGROUND: The bacterial heat shock locus ATPase HslU is an AAA(+) protein that has structures known in many nucleotide-free and -bound states. Nucleotide is required for the formation of the biologically active HslU hexameric assembly. The hexameric HslU ATPase binds the dodecameric HslV peptidase and forms an ATP-dependent HslVU protease. RESULTS: We have characterized four distinct HslU conformational states, going sequentially from open to closed: the empty, SO(4), ATP, and ADP states. The nucleotide binds at a cleft formed by an alpha/beta domain and an alpha-helical domain in HslU. The four HslU states differ by a rotation of the alpha-helical domain. This classification leads to a correction of nucleotide identity in one structure and reveals the ATP hydrolysis-dependent structural changes in the HslVU complex, including a ring rotation and a conformational change of the HslU C terminus. This leads to an amended protein unfolding-coupled translocation mechanism. CONCLUSIONS: The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.

ATP-Dependent Proteases↗
PubMed2001

A deubiquitinating enzyme, UCH/CeUBP130, has an essential role in the formation of a functional microtubule-organizing centre (MTOC) during early cleavage in C. elegans.

BACKGROUND: Deubiquitinating enzymes generate monomeric ubiquitin in protein degradation pathways and are known to be important for the early development in many organisms. RESULTS: RNA interference experiments targeted for a UBP homologue, UCH/CeUBP130, in C. elegans resulted in cell division defective embryos. Immunostaining localized UCH/CeUBP130 in the sperm and at the microtubule-organizing centre (MTOC) during early cleavage. Furthermore, the embryonic lethal phenotype was rescued by mating with wild-type males. CONCLUSIONS: Since it is known that the MTOC in the fertilized embryo is contributed by sperm asters in C. elegans, we suggest that UCH/CeUBP130 and ubiquitin protein degradation pathways may be involved in microtubule-based sperm aster formation. Therefore UCH/CeUBP130 is necessary for the formation of a functional MTOC in the fertilized embryo of C. elegans.

Amino Acid Sequence↗
PubMed2001

Effect of occlusal trauma on healing of periapical pathoses: report of two cases.

AIM: To present two clinical cases and demonstrate that occlusal trauma may affect healing of periapical pathoses. SUMMARY: Two teeth with periradicular disease did not respond successfully to conventional root canal treatment or endodontic surgery. Occlusal adjustment was finally performed on both cases. After occlusal adjustment, uncomplicated healing and periapical repair occurred in both cases. The findings in these two cases suggest that occlusal trauma may play a role in the healing of periapical pathoses. KEY LEARNING POINTS: Occlusal trauma is positively correlated with changes in periodontal tissues. Although experiments in animals have shown that application of forces to teeth will not induce further periodontal destruction, we believe that some failures of root canal treatment may be due to the presence of occlusal trauma modulating the responses of inflamed periapical tissues or apical pathoses with persistent infection.

Adult↗
PubMed2001

Maxillary sagittal and vertical displacement induced by surgically assisted rapid palatal expansion.

The purpose of this study was to investigate the sagittal and vertical effects on the maxilla induced by surgically assisted rapid palatal expansion. Twenty patients (average age, 25.6 years) who required a surgically assisted rapid palatal expansion procedure were available for this study. Each patient was banded with a Haas-type palatal expander, maxillary surgery was performed, and the expander was activated. Presurgical and postexpansion lateral cephalograms were taken for each patient. The measurements of SNA, Frankfort horizontal-nasion-A point, sella-nasion-palatal plane, A-point-nasion-perpendicular (in millimeters), 1-nasion-A-point, 1-sella-nasion, 1-nasion-perpendicular (in millimeters) were made on each cephalogram, and the presurgical and postexpansion tracings were superimposed on the cranial base to determine the changes in the anterior nasal spine and posterior nasal spine positions. Results showed that from the presurgical cephalogram to the postexpansion cephalogram SNA, Frankfort horizontal-nasion-A point and A-point-nasion-perpendicular had a mean increase of 0.60 degrees (P <.05), 0.65 degrees (P <.05), and 0.55 mm (P <.05), respectively; 1-nasion-A-point decreased 2.18 degrees (P <.05) and 1-sella-nasion decreased 1.53 degrees (P <.05). No significant maxillary vertical displacement was noted from the first cephalogram to the second. In conclusion, surgically assisted RPE did not significantly affect the maxilla vertically; however, it did induce a slight forward movement of the maxilla and a slight retroclination of the maxillary incisors sagittally (P <.05).

Adolescent↗
PubMed2001

Basic fibroblast growth factor as a growth factor for SRV-2-infected simian retroperitoneal fibromatosis cells, an animal model for AIDS related Kaposi's sarcoma.

Basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) were demonstrated to be important factors sustaining the growth of Kaposi's sarcoma. RF cells were used to provide a model to study the pathogenesis of Kaposi's sarcoma. In this paper, we demonstrated that bFGF is present in the RF cells, cultured media, and tissues from monkey. The biological activities of bFGF on RF cells were also studied in vitro with serum-free media. The bFGF from serum-free-conditioned media is biologically active to stimulate RF cells in certain media condition. The mitogenic effect was abrogated by sheep neutralizing anti-bFGF antibody. Furthermore, the effect of antibody was reversed by the addition of exogenous bFGF. ELISA measurements indicating the growth potency of conditioned media correlated with the amount of bFGF in the conditioned media. The data from flow cytometry demonstrated the co-existence of SRV-2 and bFGF among RF cells and RF tissues. Immunohistochemical staining of RF tissue blocks for bFGF revealed that bFGF was present in the tumor and the presence of bFGF was not caused by the artifact of tissue culture. These results indicate that bFGF is an important growth factor to promote RF cell growth in vitro and RF tumor in vivo. Further studies are required to determine the relationship between the interaction of bFGF, SRV-2, and VEGF. This model also provides an adequate alternative to the model induced by simian immunodeficiency virus (SIV) to study the Kaposi's sarcoma.

Animals↗
PubMed2001

The effect of combined 5-fluorouracil and dexamethasone on cultured human retinal pigment epithelial cells.

This study was designed to investigate the inhibitory effect of 5-fluorouracil (5-FU) and dexamethasone (DEX) on the proliferation of human retinal pigment epithelial (RPE) cells in vitro. The human RPE cells (R-50 cell line) were cultured and exposed to various concentrations of combined 5-FU (0, 250, 500, 1000, 2000 ng/ml) and DEX (0, 1, 10, 100, 200 micrograms/ml). The cells were incubated for 96 hr and the medium was changed every 48 hr to replenish the drug action. Cell viability was assessed using cell counting and trypan blue exclusion method. Tetrazolium salt, which can be metabolized by mitochondrial dehydrogenase to form a formazan dye, was used to assay cell proliferation. Treatment with 5-FU alone inhibited cell proliferation in a dose-dependent manner. The concentration of 5-FU that inhibited growth by 50% (IC50) was found to be 704.12 ng/ml. There was a bimodal effect of DEX on RPE cells--stimulation at low concentrations (1, 10 micrograms/ml) and inhibition at high concentrations (100, 200 micrograms/ml). When the two drugs were combined, there was additive inhibition in the concentration of 200 micrograms/ml of DEX. These results indicate that a combination of 5-FU and DEX is no more effective in the inhibition of human RPE cells, except in combination with high concentrations of DEX (> or = 200 micrograms/ml).

Cell Division↗
PubMed2000

Heptameric ring structure of the heat-shock protein ClpB, a protein-activated ATPase in Escherichia coli.

The heat-shock protein ClpB is a protein-activated ATPase that is essential for survival of Escherichia coli at high temperatures. ClpB has also recently been suggested to function as a chaperone in reactivation of aggregated proteins. In addition, the clpB gene has been shown to contain two translational initiation sites and therefore encode two polypeptides of different size. To determine the structural organization of ClpB, the ClpB proteins were subjected to chemical cross-linking analysis and electron microscopy. The average images of the ClpB proteins with end-on orientation revealed a seven-membered, ring-shaped structure with a central cavity. Their side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since the ClpB subunit has two large regions containing consensus sequences for nucleotide binding, each layer of the ClpB heptamer appears to represent the side projection of one of the major domains arranged on a ring. In the absence of salt and ATP, the ClpB proteins showed a high tendency to form a heptamer. However, they dissociated into various species of oligomers with smaller sizes, depending on salt concentration. Above 0.2 M NaCl, the ClpB proteins behaved most likely as a monomer in the absence of ATP, but assembled into a heptamer in its presence. Furthermore, mutations of the first ATP-binding site, but not the second site, prevented the ATP-dependent oligomerization of the ClpB proteins in the presence of 0.3 M NaCl. These results indicate that ClpB has a heptameric ring-shaped structure with a central cavity and this structural organization requires ATP binding to the first nucleotide-binding site localized to the N-terminal half of the ATPase.

Adenosine Triphosphatases↗
PubMed2000

The HslU ATPase acts as a molecular chaperone in prevention of aggregation of SulA, an inhibitor of cell division in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. SulA, which is an inhibitor of cell division and has high tendency of aggregation, is degraded by HslVU protease. Here we show that HslU plays a role not only as a regulatory component for the HslV-mediated proteolysis but also as a molecular chaperone. Purified HslU prevented aggregation of SulA in a concentration-dependent fashion. This chaperone activity required oligomerization of HslU subunits, which could be achieved by ATP-binding or in the presence of high HslU protein concentrations. hsl mutation reduced the SulA-mediated inhibition of cell growth and this effect could be reversed upon overproduction of HslU, suggesting that HslU promotes the ability of SulA to block cell growth through its chaperone function. Thus, HslU appears to have two antagonistic functions: one as a chaperone for promotion of the ability of SulA in cell growth inhibition by preventing SulA aggregation and the other as the regulatory component for elimination of SulA by supporting the HslV-mediated degradation.

ATP-Dependent Proteases↗
PubMed2000

Tissue-specificity, functional characterization and subcellular localization of a rat ubiquitin-specific processing protease, UBP109, whose mRNA expression is developmentally regulated.

A cDNA encoding an ubiquitin-specific processing protease, UBP109, in rat skeletal muscle was cloned and its product was characterized. Northern analysis revealed that UBP109 mRNA is highly expressed in testis and spleen, compared with other tissues. Furthermore, in situ hybridization showed that the level of UBP109 mRNA in liver, spinal cord and brain dramatically changed during embryonic development, indicating that the expression of UBP109 mRNA is developmentally regulated. UBP109 was expressed in Escherichia coli and purified to apparent homogeneity using a (125)I-labelled ubiquitin-peptide fusion as a substrate. The purified enzyme cleaved at the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes. UBP109 also released free ubiquitin from poly-His-tagged penta-ubiquitin. Moreover, it released free ubiquitin from poly-ubiquitinated protein conjugates of rabbit reticulocytes. In addition, UBP109 localized to both the cytoplasm and the nucleus and, among three putative nuclear localization sequences, only the one located near the C-terminus is responsible for nuclear localization. These results suggest that UBP109 may play an important role in generation of free ubiquitin from its precursors and its recycling from poly-ubiquitinated protein conjugates, and hence in regulation of ubiquitin-mediated cellular processes, particularly related to embryonic development.

Amino Acid Sequence↗
PubMed2000

A new SUMO-1-specific protease, SUSP1, that is highly expressed in reproductive organs.

A full-length cDNA encoding a SUMO-1-specific protease, named SUSP1, was identified and cloned for the first time from the human brain. Nucleotide sequence analysis of the cDNA containing an open reading frame of 3336 base pairs revealed that the protease consists of 1112 amino acids with a calculated molecular mass of 126,116 Da. Like yeast Ulp1, SUSP1 is a cysteine protease containing the well conserved His/Asp/Cys catalytic triad. SUSP1 expressed in Escherichia coli cells efficiently released SUMO-1 from SUMO-1. beta-galactosidase fusion but not from other ubiquitin-like protein fusions, including Smt3.beta-galactosidase, suggesting its role in the generation of matured SUMO-1 specifically from its precursors. Interestingly, reproductive organs, such as testis, ovary, and prostate, contained much higher amounts of SUSP1 mRNA than colon and peripheral blood leukocyte, whereas other tissues, such as heart and spleen, had little or none. In addition, confocal microscopy using green fluorescent protein.SUSP1 fusion showed that SUSP1 is exclusively localized to the cytoplasm of NIH3T3 and HeLa cells. These results suggest that SUSP1 may play a role in the regulation of SUMO-1-mediated cellular processes particularly related to reproduction.

Amino Acid Sequence↗
PubMed2000

Retinoic acid and its geometrical isomers block both growth and fusion of L6 myoblasts by modulating the expression of protein kinase A.

All-trans retinoic acid (RA) and its geometrical isomers, such as 9-cis RA, 13-cis RA, and 9,13-di-cis RA, strongly inhibited both growth and fusion of L6 myoblasts. However, illumination of white light diminished their inhibitory activity on membrane fusion with little effect on cell growth. During myogenic differentiation, the intracellular level of cAMP decreased whereas the total activity of protein kinase A as well as the protein level of its regulatory subunit Ialpha (RIalpha) and catalytic subunit (Calpha) increased. RAs raised the intracellular level of cAMP by over 3-fold, but decreased the total activity of protein kinase A. Like RAs, dibutyryl-cAMP inhibited myoblast fusion and reduced the expression of both RIalpha and Calpha subunits. These results suggest that RAs negatively modulate the differentiation of L6 myoblasts by increasing the intracellular level of cAMP, which may in turn down-regulate the expression of protein kinase A and hence its activity.

Animals↗
PubMed2000

Involvement of the theta-type protein kinase C in translocation of myristoylated alanine-rich C kinase substrate (MARCKS) during myogenesis of chick embryonic myoblasts.

The phosphorylation pattern of numerous proteins in the soluble extracts of chick embryonic muscle cells changes dramatically during myogenesis. One of these proteins, the 63 kDa protein, whose phosphorylation state declines during the differentiation process, was identified as the myristoylated alanine-rich C kinase substrate (MARCKS), a major, specific substrate of protein kinase C (PKC). This decrease in the phosphorylation state of MARCKS was due to a decrease in the level of protein in the cytosol with a simultaneous increase in its level in the membrane fraction. Immunostaining of the cultured myoblasts also revealed that MARCKS translocated from the cytosol to the plasma membrane and to the peripheral region of nuclei as the mononucleated myoblasts fused to form multinucleated myotubes. Immunoprecipitation with an anti-PKC-theta antibody, but not with the antibodies against the other PKC isoforms, such as conventional PKC-alpha, novel PKC-delta, and novel PKC-epsilon, inhibited phosphorylation of MARCKS. Moreover, expression of PKC-theta was found to be down-regulated during the course of myogenic differentiation. In addition, treatment of the cells with PMA, which activates PKC-theta and hence increases the phosphorylation state of MARCKS, reversibly inhibited both MARCKS translocation and myoblast fusion. These results suggest that MARCKS is preferentially phosphorylated by PKC-theta in cultured myoblasts and that the down-regulation of PKC-theta; is partly responsible for MARCKS translocation during myogenesis. These results also suggest that PKC-theta-controlled MARCKS translocation is associated with, or a requisite event for, myoblast fusion.

Amino Acid Sequence↗