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Biomedical subjects

C H Chung

Publications and source records attributed to C H Chung.

At least 127 records · Page 7Linked to original sources
PubMed1995

Requirement of ATP hydrolysis for assembly of ClpA/ClpP complex, the ATP-dependent protease Ti in Escherichia coli.

The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Either ADP or adenosine 5'-(beta,gamma-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.

Adenosine Triphosphatases↗
PubMed1995

Multiple ubiquitin C-terminal hydrolases from chick skeletal muscle.

A new method for assaying ubiquitin C-terminal hydrolases was developed using a 125I-labeled ubiquitin-alpha NH-MHISPPEPESEEEEEHYC was substrate. Since the peptide portion was almost exclusively radiolabeled, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid-soluble products. Using this assay protocol, we identified at least 10 ubiquitin C-terminal hydrolase activities from the extract of chick skeletal muscle, which were tentatively named UCHs 1 through 10. Of these, UCH-6 was purified to apparent homogeneity. Purified UCH-6 behaved as a dimer of 27-kDa subunits. The apparent molecular masses of the other partially purified UCHs ranged from 35 to 810 kDa as determined under a non-denaturing condition. Muscle UCHs, except UCH-1, were activated dramatically by poly-L-Lys but with an unknown mechanism. All of the UCHs were sensitive to inhibition by sulfhydryl-blocking agents such as iodoacetamide. In addition, all of the UCHs were capable of releasing free ubiquitin from a ubiquitin-alpha NH-carboxyl extension protein of 80 amino acids and from ubiquitin-alpha NH-dihydrofolate reductase. Five of the enzymes, UCHs 1 through 5, were also capable of generating free ubiquitin from poly-His-tagged diubiquitin. In addition, UCH-1 and UCH-7 could remove ubiquitin that had been ligated covalently by an isopeptide linkage to a ubiquitin (RGA)-alpha NH-peptide, the peptide portion of which consists of the 20 amino acids of the calmodulin binding domain of myosin light chain kinase. These results suggest that the 10 UCH activities isolated from chick skeletal muscle appear to be distinct from each other at least in their chromatographic behavior, size, and substrate specificity.

Amino Acid Sequence↗
PubMed1995

Primary structures of two homologous subunits of PA28, a gamma-interferon-inducible protein activator of the 20S proteasome.

The primary structures of two proteins that comprise PA28, an activator of the 20S proteasome, have been determined by cDNA cloning and sequencing. These protein subunits, termed PA28 alpha and PA28 beta, are about 50% identical to one another and are highly conserved between rat and human. PA28 alpha and PA28 beta are homologous to a previously described protein, Ki antigen, whose function is unknown. PA28 alpha, but neither PA28 beta nor Ki antigen, contains a 'KEKE motif', which has been postulated to promote the binding of proteins having this structural feature. PA28 alpha and PA28 beta were coordinately regulated by gamma-interferon, which greatly induced mRNA levels of both proteins in cultured cells. The mRNA level of the Ki antigen also increased in response to gamma-interferon treatment, but the magnitude of the increase was less than that for the PA28s, and the effect was transient. These results demonstrate the existence of a new protein family, at least two of whose members are involved in proteasome activation. They also provide the basis for future structure/function studies of PA28 subunits and the determination of their relative physiological roles in the regulation of proteasome activity.

Amino Acid Sequence↗
PubMed1995

Distinctive roles of the two ATP-binding sites in ClpA, the ATPase component of protease Ti in Escherichia coli.

ClpA is the ATPase component of the ATP-dependent protease Ti (Clp) in Escherichia coli and contains two ATP-binding sites. A ClpA variant (referred to as ClpAT) carrying threonine in place of the 169th methionine has recently been shown to be highly soluble but indistinguishable from the wild-type, 84-kDa ClpA in its ability to hydrolyze ATP and to support the casein-degrading activity of ClpP. Therefore, site-directed mutagenesis was performed to generate mutations in either of the two ATP-binding sites of ClpAT (i.e. to replace the Lys220 or Lys501 with Thr). ClpAT/K220T hydrolyzed ATP and supported the ClpP-mediated proteolysis 10-50% as well as ClpAT depending on ATP concentration, while ClpAT/K501T was unable to cleave ATP or to support the proteolysis. Without ATP, ClpAT and both of its mutant forms behaved as trimeric molecules as analyzed by gel filtration on a Sephacryl S-300 column. With 0.5 mM ATP, ClpAT and ClpAT/K501T became hexamers, but ClpAT/K220T remained trimeric. With 2 mM ATP, however, ClpAT/K220T also behaved as a hexamer. These results suggest that the first ATP-binding site of ClpA is responsible for hexamer formation, while the second is essential for ATP hydrolysis. When trimeric ClpAT/K220T was incubated with the same amount of hexameric ClpAT/K501T (i.e. at 0.5 mM ATP) and then subjected to gel filtration as above, a majority of ClpAT/K220T ran together with ClpAT/K501T as hexameric molecules. Furthermore, ClpAT/K501T in the mixture strongly inhibited the ability of ClpAT/K220T to cleave ATP and to support the ClpP-mediated proteolysis. Similar results were obtained in the presence of 2 mM ATP and also with the mixture with ClpAT. On the other hand, the ATPase activity of the mixture of ClpAT and ClpAT/K220T was significantly higher than the sum of that of each protein, particularly in the presence of 2 mM ATP, although its ability to support the proteolysis by ClpP remained unchanged. These results suggest that a rapid exchange of the subunits, possibly as a trimeric unit, occurs between the ClpAT proteins in the presence of ATP and leads to the formation of mixed hexameric molecules.

Adenosine Triphosphatases↗
PubMed1995

Involvement of transglutaminase in myofibril assembly of chick embryonic myoblasts in culture.

Involvement of transglutaminase in myofibrillogenesis of chick embryonic myoblasts has been investigated in vitro. Both the activity and protein level of transglutaminase initially decreased to a minimal level at the time of burst of myoblast fusion but gradually increased thereafter. The localization of transglutaminase underwent a dramatic change from the whole cytoplasm in a diffuse pattern to the cross-striated sarcomeric A band, being strictly colocalized with the myosin thick filaments. For a brief period prior to the appearance of cross-striation, transglutaminase was localized in nonstriated filamental structures that coincided with the stress fiber-like structures. When 12-o-tetradecanoyl phorbol acetate was added to muscle cell cultures to induce the sequential disassembly of thin and thick filaments, transglutaminase was strictly colocalized with the myosin thick filaments even in the myosacs, of which most of the thin filaments were disrupted. Moreover, monodansylcadaverine, a competitive inhibitor of transglutaminase, reversibly inhibited the myofibril maturation. In addition, myosin heavy chain behaved as one of the potential intracellular substrates for transglutaminase. The cross-linked myosin complex constituted approximately 5% of the total Triton X-100-insoluble pool of myosin molecules in developing muscle cells, and its level was reduced to below 1% upon treatment with monodansylcadaverine. These results suggest that transglutaminase plays a crucial role in myofibrillogenesis of developing chick skeletal muscle.

Animals↗
PubMed1994

The 65-kDa protein derived from the internal translational initiation site of the clpA gene inhibits the ATP-dependent protease Ti in Escherichia coli.

The clpA gene that encodes the ATPase subunit of the ATP-dependent protease Ti (Clp) in Escherichia coli contains a putative internal translational initiation site. Here we show that mutagenesis of its 5'-end AUG codon resulted in an exclusive synthesis of the 65-kDa protein (ClpA65), while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produced only the 84-kDa protein (ClpA84T). On the other hand, the cells carrying the wild-type clpA gene produced both the 84- and 65-kDa proteins (ClpA84/65). While the purified ClpA84T and ClpA84/65 hydrolyzed ATP nearly as well as the 84-kDa ClpA alone (ClpA84), ClpA65 cleaved ATP at a rate less than 5% of that by ClpA84. Unlike ClpA84 and ClpA84T, ClpA65 could not support the casein-degrading activity of ClpP. Furthermore, ClpA65 inhibited the proteolysis by the mixture of ClpP with ClpA84 or ClpA84T but not that with ClpA84/65, which could support the proteolytic activity of ClpP only about 40% as well as ClpA84. Nevertheless, ClpA65 showed little or no effect on the basal or protein-activated ATPase activity of ClpA84, ClpA84T, or ClpA84/65 alone or in the presence of ClpP. These results suggest that ClpA65 may interfere the interaction of ClpA84 or ClpA84T with ClpP and, hence, impair their assembly into an active form of the ATP-dependent protease Ti.

Adenosine Triphosphatases↗
PubMed1994

clpX encoding an alternative ATP-binding subunit of protease Ti (Clp) can be expressed independently from clpP in Escherichia coli.

ClpX, an alternative ATP-binding subunit for protease Ti (also called Clp), has been shown to support the ATP-dependent hydrolysis of lambda O-protein by ClpP. clpX has also been reported to be in an operon with clpP, and therefore both are co-transcribed in a single mRNA using the promoter proximal to clpP. Here, we show that clpX can be expressed independently from clpP using its own promoter. The cells carrying clpX alone on a multicopy plasmid successively produced the 46-kDa ClpX protein. Moreover, in vitro translation analysis revealed that the recombinant plasmid containing clpX generates the 46-kDa protein that can be immunoprecipitated with anti-ClpX antibody. In addition, it has recently been reported that CipX, but not ClpP, is required for normal replication of bacteriophage Mu. Thus, it appears that clpX can be expressed alone and/or co-expressed with clpP in cells depending on physiological conditions.

ATP-Dependent Proteases↗
PubMed1994

The P1 reactive site methionine residue of ecotin is not crucial for its specificity on target proteases. A potent inhibitor of pancreatic serine proteases from Escherichia coli.

The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis. The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin. Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin. On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect. Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr. Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75. In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase. However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor. Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control. These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability.

Amino Acid Sequence↗
PubMed1994

Nitric oxide as a messenger molecule for myoblast fusion.

Nitric oxide (NO) is a messenger molecule of vascular endothelial cells, macrophages, and neurons. Here, we demonstrate that the activity of NO synthase increases transiently but dramatically in chick embryonic myoblasts that are competent for fusion. This activity requires Ca2+, calmodulin, and NADPH. In addition, the increase in NO synthase activity coincides with an increase in cellular cGMP level. Furthermore, NO generated by treatment with sodium nitroprusside induces precocious myoblast fusion, while treatment with NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, or methylene blue, an inhibitor of guanylate cyclase, delays fusion. These results provide the first evidence for a strong association of NO with myoblast fusion.

Amino Acid Oxidoreductases↗
PubMed1994

A candidate molecule for the matrix assembly receptor to the N-terminal 29-kDa fragment of fibronectin in chick myoblasts.

Myoblast surface proteins with binding activity toward the N-terminal 29-kDa fragment of fibronectin were identified by two different experimental techniques: one involves radioiodination of the cell surface proteins, followed by solubilization with Triton X-100 and affinity purification on a Sepharose column conjugated with the 29-kDa fragment, and the other involves cross-linking of the 29-kDa fragment to the cells metabolically labeled with [35S]methionine, followed by immunoprecipitation with anti-29-kDa IgG. Both approaches revealed that primary cultures of chick myoblasts contain the 66- and 48-kDa proteins that bind to the 29-kDa fragment. These binding proteins were then purified to apparent homogeneity by two successive chromatographies of the solubilized extracts of 12-day-old embryonic muscle on wheat germ agglutinin-agarose and 29-kDa fragment-Sepharose columns. However, the 48-kDa protein was found to be derived from contaminating fibroblasts upon immunoblot analysis of the myogenic cell lines, rat L8E63 and mouse C2A3, and cultured fibroblasts using the antibody raised against the 66-kDa protein. Anti-66-kDa IgG inhibited the binding of the 125I-29-kDa protein to the primary culture of myoblasts in a dose-dependent manner. On the other hand, the same antibody showed little or no effect on the initial binding of 125I-fibronectin to the cell surface, but dramatically inhibited its incorporation into deoxycholate-insoluble matrices. Furthermore, Fab fragments of anti-66-kDa IgG completely blocked the incorporation of fluoresceinated fibronectin into matrices but not its binding to the cell surface. These results suggest that fibronectin matrix assembly is mediated at least in part by the interaction of the 66-kDa protein with the N-terminal type I domain of fibronectin.

Amino Acid Sequence↗
PubMed1994

The 100-kDa protein, whose phosphorylation precedes the fusion of chick embryonic myoblasts, is the eukaryotic elongation factor-2.

We have previously shown that Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein dramatically increases during the early period of myoblast fusion and inhibition of the protein phosphorylation prevents the fusion. Here, we show that the protein phosphorylation occurs exclusively at Thr residue(s) and the purified 100-kDa protein can be ADP-ribosylated upon treatment with diphtheria toxin. Furthermore, the 13 N-terminal amino acid sequence of the 100-kDa protein, N-Val-Asn-Phe-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp-Lys, exactly matches with that of elongation factor-2 from rat and hamster. These results indicate that the 100-kDa protein in chick embryonic myoblasts is identical to the eukaryotic elongation factor-2.

Adenosine Diphosphate Ribose↗
PubMed1994

Cyclic AMP negatively modulates both Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein and membrane fusion of chick embryonic myoblasts.

We have previously shown that Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein dramatically increases during the early period of myoblast fusion and treatment of calmodulin antagonists, such as trifluoperazine, blocks the fusion. Here, we show that cAMP treatment of primary cultures of chick embryonic myoblasts blocks 100-kDa protein phosphorylation. This effect is dose-dependent and can be reversed upon removal of the nucleotide from the culture media. However, cAMP shows little or no effect on accumulation of the 100-kDa protein. Furthermore, phosphorylation of the 100-kDa protein by the partially purified Ca2+/calmodulin-dependent protein kinase (CaM kinase III) from cAMP-treated cells occurs to a much lower extent than that from untreated cells. Nevertheless, cAMP-sensitive protein kinase does not seem to be directly involved in phosphorylation and inactivation of CaM kinase III, because preincubation of cAMP with the myoblast extracts lacking the endogenous 100-kDa protein does not show any effect on activity of CaM kinase III. Similar to its effect on 100-kDa protein phosphorylation, cAMP reversibly inhibits the fusion of cultured myoblasts. Moreover, treatment with forskolin or theophylline, which is known to elevate the intracellular cAMP level, also reversibly blocks both protein phosphorylation and myoblast fusion. On the other hand, cAMP shows little or no effect on accumulation of muscle-specific proteins, such as creatine kinase and tropomyosin. These results suggest that cAMP is involved in down-regulation of both 100-kDa protein phosphorylation and membrane fusion of cultured myoblasts. These results also suggest that the cAMP-mediated inhibition of 100-kDa protein phosphorylation may be associated with its inhibitory effect on myoblast fusion.

Animals↗
PubMed1994

IGF-I of serum and vitreous fluid in patients with diabetic proliferative retinopathy.

Vitreous fluid and serum were obtained at the time of vitrectomy from 15 subjects with diabetic proliferative retinopathy and 6 control subjects. The mean serum IGF-I (IGF-I level: 1 nM/l = 7.7 ng/ml) concentrations were 15.40 +/- 4.97 nM/l in controls and 16.24 +/- 4.32 nM/l in diabetic patients. The mean vitreous IGF-I concentration was 1.05 +/- 0.30 nM/l in controls and 0.91 +/- 0.50 nM/l in diabetic patients. There were no significant differences between the two groups, and serum IGF-I concentrations were not correlated with vitreous IGF-I concentrations in the patients with diabetic proliferative retinopathy. These data suggest that IGF-I may not be a risk factor for the development of proliferative diabetic retinpathy.

Adult↗
PubMed1994

[Continuing hospice care of cancer--a three-year experience].

The hospice at Mackay Memorial Hospital was established in February 1990. A group of team workers including physicians, nurses, social workers and the clergy were involved in this holistic care program for terminal cancer patients. Four hundred and seventy-nine patients were eligible for the program up to February 1993. Regarding duration of stay, 62.5% of patients resided for 14 days. Those surviving under 90 days constituted 75.5% of patients. Fifty-one point eight percent of patients died in the hospice and 18.2% died at home soon after being discharged from the hospice. Pain is the most common symptom among the patients. Treatment strategies vary according to the three-step-ladder protocol designed by WHO. Total pain relief was achieved in 80% of patients. Opportune private talking and family conferences formed the basis of the "peer model". Through this model, treatment decisions including physical, psychosocial and spiritual issues were made. Before the peer model, only 36 (10.3%) patients agreed with the idea of hospice care, while 257 (73.6%) patients agreed after the model was established. Awareness of dying was evident in 412 (86%) patients. Two hundred and eighty (68%) patients became aware of the prospect of death through guessing, while the other 132 (32%) patients were informed by medical staff. Problems encountered by the team workers included 1) needs in education and training, 2) psychological pressure, 3) management of loss and grief, 4) needs in supportive system and 5) troubles caused by families' lying to patients. The team workers were satisfied with the quality of care in 38.4% of patients and fairly satisfied with 30.7% of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Tissue-specific expression of the subunits of chick 20S proteasomes.

The subunit patterns of the proteasomes, that were purified from muscle, liver and brain, were found to be significantly different from one another. Furthermore, the proteasomes from adult and embryonic tissues of the same types also differed from each other in their subunit patterns. In addition, the specific activities of the purified proteasomes for peptide-cleavage, but not for casein-hydrolysis, appeared to be varied among the enzymes isolated from the different tissues. Thus, expression of a large number of proteasome subunits appears to be tissue-specific and under developmental control, although its relation with the multicatalytic activities of the proteasomes remains unclear.

Animals↗