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Biomedical subjects

S I Chung

Publications and source records attributed to S I Chung.

At least 37 records · Page 2Linked to original sources
PubMed1998

Tissue transglutaminase is not increased during apoptosis of HT-1080 human fibrosarcoma cells.

Tissue transglutaminase (tTGase), a cytosolic enzyme which catalyzes the covalent cross-linking of proteins, is thought to be involved in the apoptosis. Here, we tested whether tTGase is involved during HT-1080 fibrosarcoma cell apoptosis induced by the YIGSR (Tyr-Ile-Gly-Ser-Arg) peptide. This sequence is derived from the laminin alpha1 chain, and its potency is increased by the formation of a 16mer polymerization using a lysine tree structure. Cells were treated with several different concentrations of Ac-Y 16 for 16 hours, and apoptosis was increased in dose-dependent manner. When assayed by incorporation of [14C] putrescine into succinylated casein, total transglutaminase activity was decreased in parallel with the change in the number of attached cells. Western blot analysis using polyclonal antibody against tTGase showed that the tTGase protein level had not been significantly changed when equal amounts of the protein were applied. To confirm this result, we induced apoptosis of these cells by coating the tissue culture plates with non-adhesive poly-hydroxyethyl methacrylate (HEMA). Western blot analysis showed that the tTGase protein level did not change during this process of apoptosis. Although it has been suggested that tTGase is involved in the process of apoptosis of various cells in vitro and in vivo, our data demonstrate that tTGase is not involved in the process of apoptosis of HT-1080 human fibrosarcoma cell induced by either Ac-Y 16 or a non-adhesive culture surface.

Apoptosis↗
PubMed1998

Serodiagnosis of Helicobacter pylori infection in Korean patients using enzyme-linked immunosorbent assay.

Helicobacter pylori (H. pylori) is a gram-negative spiral bacteria that are associated with gastritis, peptic ulcer and gastric cancer. We have developed enzyme-linked immunosorbent assay (ELISA) that detects serum anti-H. pylori immunoglobulin G antibodies using H. pylori strains isolated from Korean patients. To assess the sensitivity and specificity of our assay system with different commercial kits, serum samples from 249 Korean patients with a variety of gastrointestinal diseases were tested. Among 249 Korean patients, 178 (71.5%) were positive in culture and/or urease test. The sensitivity and specificity between our assay system and four other commercial kits (Bio-Rad, DAKO, ROCHE, and IPR) were as follows: 97.8% and 92%, 94.3% and 53%, 56.5% and 92%, 83.3% and 96%, 58.2% and 92%, respectively. All sera showing discordant immunoassay results between different ELISA kits were confirmed by immunoblot analysis. These results indicate that our assay system showed a highly accurate and reliable results in diagnosis of H. pylori infection in Korean patients.

Adolescent↗
PubMed1997

The fate of trichohyalin. Sequential post-translational modifications by peptidyl-arginine deiminase and transglutaminases.

Trichohyalin (THH) is a major structural protein of the inner root sheath cells and medulla layer of the hair follicle and, to a lesser extent, of other specialized epithelia. THH is a high molecular weight insoluble alpha-helix-rich protein that forms rigid structures as a result of postsynthetic modifications by two Ca2+-dependent enzymes, transglutaminases (TGases) (protein cross-linking) and peptidyl-arginine deiminase (conversion of arginines to citrullines with loss of organized structure). The modified THH is thought to serve as a keratin intermediate filament matrix protein and/or as a constituent of the cell envelope. In this paper, we have explored in vitro the order of processing of THH to fulfill these functions, using an expressed truncated, more soluble form THH-8. THH-8 is a complete substrate for three known TGases expressed in epithelia, but the kinetic efficiency with TGase 3 is by far the greatest. Following maximal conversion of its arginines to citrullines, THH-8 is cross-linked even more efficiently by TGase 3, using most glutamines partially and all lysines. In addition, we show that insoluble aggregates of THH-8 or native pig tongue THH can be solubilized following peptidyl-arginine deiminase modification. Together, these data suggest an in vivo model in which THH located in insoluble cytoplasmic droplets is first modified by peptidyl-arginine deiminase which denatures it and makes it more soluble. This renders it available for efficient cross-linking by TGase 3 to form highly cross-linked rigid structures in the cells. This temporal order of reaction is supported by the observation that THH is expressed in hair follicle cells before the TGase 3 enzyme.

Amino Acid Sequence↗
PubMed1997

Detection, quantitation, and localization of bovine angiogenin by immunological assays.

Bovine angiogenin (bAng) is a potent blood vessel inducing protein found in bovine serum and milk. Antisera have been raised against bAng. Western blot analysis for bAng indicated that the polyclonal antibody recognized bAng specifically, and no cross-reactivity with bovine RNase A, a protein homologous to bAng, was observed. The sandwich enzyme-linked immunosorbent assay for bAng was sensitive to 10 pg of bAng, and this assay was able to quantitate bAng in bovine serum (100-180 ng/mL) and milk (4-8 micrograms/mL). Strong positive immunohistochemical reactions were detected in alveolar cells, the secretion of alveolar cells and excretory ducts in sections of cow mammary gland, epithelial cells of visceral peritoneum and bile-duct in sections of cow liver, and epithelial cells and mucous glands in sections of cow gallbladder. These results suggest that epithelial cells and secretory cells are major sites of angiogenin synthesis.

Angiogenesis Inducing Agents↗
PubMed1996

The transglutaminase 1 enzyme is variably acylated by myristate and palmitate during differentiation in epidermal keratinocytes.

The transglutaminase 1 (TGase 1) enzyme is involved in the formation of a cornified cell envelope in terminally differentiating epidermal keratinocytes. The enzyme is present in proliferating cells but is more abundantly expressed in differentiating cells and exists in several intact or proteolytically processed cytosolic or membrane-anchored forms. We show here that the equilibrium partitioning of TGase 1 between the cytosol and membranes is controlled by variable modification by myristate and palmitate. During synthesis, it is constitutively N-myristoylated. Later, it is modified by an average of two S-myristoyl adducts in proliferating cells or one S-palmitoyl adduct in differentiating cells. The three myristoyl adducts of the former provide more robust anchorage to membranes than the one myristoyl and one palmitoyl adduct of the latter. The half-lives of the S-myristoyl and especially the S-palmitoyl adducts are less than that of the TGase 1 protein, suggesting a mechanism for cycling off membranes. In in vitro overlay assays, the S-acylated 10-kDa anchorage fragment facilitates binding of TGase 1 forms, supporting a mechanism of cycling back onto membranes in vivo. We conclude that differential acylation increases the repertoire of functional TGase 1 forms, depending on the differentiation state of epidermal keratinocytes.

Acylation↗
PubMed1996

Inactive zymogen and highly active proteolytically processed membrane-bound forms of the transglutaminase 1 enzyme in human epidermal keratinocytes.

The transglutaminase 1 enzyme is important for the formation of a cornified cell envelope in terminally differentiating keratinocytes. We show here that it is present in low levels in proliferating foreskin or cultured epidermal cells as an inactive zymogen full length form of 106 kDa, of which >95% is attached to membranes. In terminally differentiating keratinocytes, there is a > or = 100-fold induction of mRNA and protein. In addition to some cytosolic protein, most of the newly expressed protein is attached to membranes, of which about half exist in the zymogen form. Other protein consists of a 67/33/10 kDa complex formed by proteolytic processing at specific sites, and is anchored by way of the 10 kDa fragment. This processed form is very highly active and thus accounts for almost all transglutaminase 1 activity in keratinocytes.

Base Sequence↗
PubMed1996

The infectious bronchitis virus nucleocapsid protein binds RNA sequences in the 3' terminus of the genome.

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a bacterial fusion protein which differed from the native protein only in the addition of six amino terminus histidine residues. Using RNA overlay protein blot assays, the recombinant protein was shown to bind to RNA fragments specific for the positive sense 3' noncoding end of the IBV genome. At greater concentrations of sodium chloride, the native and fusion nucleocapsid proteins similarly bound to G RNA, representing the terminal 1805 3' nt of the genome, whereas bovine serum albumin and allantoic fluid protein did not bind to labeled G RNA. Competitive gel shift assays with labeled G RNA indicated that the protein interacted with several unlabeled RNA representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the small segment cDNA also inhibited the interaction with IBV G RNA to approximately the same extent as homologous unlabeled G RNA, whereas reactions with bovine liver RNA and yeast tRNA were considerably weaker. Whereas yeast tRNA did not inhibit the interaction with the labeled large G RNA, interactions of the fusion protein with EF, a region from 78 to 217 nt from the 3' terminus of the IBV genome, were also apparently weaker than interactions with fragment CD which consisted of the 3' terminal 155 nt. On a molar basis, the latter interacted in an identical nature to a RNA consisting of CD and an additional 1053 nt of plasmid sequences. Compared to bovine liver RNA, unlabeled G specifically inhibited binding to the two smaller labeled IBV fragments in gel shift assays. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present on the genome, and may represent a common mechanism used by similar viral nucleoproteins whose functions depend on binding to RNA.

Binding, Competitive↗
PubMed1996

Immunohistochemical evaluation of transglutaminase C in tumours of salivary glands.

Transglutaminase C (TGase C), a family of Ca(2+)-dependent enzymes and an essential component in the cross-linking of peptide bonds, has been found to be a marker of epithelial differentiation with a possible role in cellular apoptosis, extracellular matrix stabilisation and Ca2+ binding, thereby having a potential role in tumour growth, differentiation and invasive behaviour. The expression of TGase C was evaluated in normal human salivary glands and their neoplastic lesions which included pleomorphic adenoma (n = 30), Warthin's tumour (n = 5), adenoid cystic carcinoma (n = 10), acinic cell carcinoma (n = 5), mucoepidermoid carcinoma (n = 5) and control tissue specimens of normal oral mucosa and squamous cell carcinoma, using polyclonal antibody, the specificity of which was determined by Western blotting, generated by immunising rabbits with purified transglutaminase. The TGase C was observed in the epithelial cells in the control tissue specimens examined. Pleiomorphic adenoma revealed reaction products in luminal tumour cells, the non-luminal or modified myoepithelial cells and their plasmacytoid variants, squamous metaplastic cells and chondroid cells. Adenoid cystic carcinomas had tumour cells in the luminal cells of tubular and cribriform structures and the acinic cell carcinoma had from low to moderate immunoreactivity in the tumour cell component and a diffuse immunoreactivity in the stroma for TGase C. Mucoepidermoid carcinoma showed no reaction products in the mucous-producing cells, while intermediate and epidermoid cells had immunoreactivity in the cell cytoplasm. As the presence of TGase C in salivary gland tumours was confined to those tumour cells which form the predominant histomorphology in each tumour subtype, it may be suggested that these enzymes may have a potential role in the regulation of cellular function in neoplastic salivary tissues affecting tumour growth, differentiation and neoplastic behaviour.

Adenolymphoma↗
PubMed1996

Factors that control extravascular fibrinolysis.

The physiological and pathophysiological state of tissues determines the exudation of plasma proteins, hemostasis, and fibrinolysis, i.e., inflammation, injury, and malignancy. The physiological controls of extravascular fibrinolysis ultimately rest on a balance between generation of the fibrinolytic enzyme(s), i.e., plasmin, elastase, cathepsins, etc., and inhibitors of the fibrinolytic enzyme(s), i.e., plasminogen activator inhibitors, alpha-2 plasmin inhibitor, alpha 1-protease inhibitors, etc. Moreover, it is the structural modification of fibrin that determines its stability toward proteolytic enzymes and physical duress. The structural modification of fibrin involves factor XIIIa-mediated cross-linking of interfibrin chains and alpha 2-plasmin inhibitor to fibrin. In turn, there are a number of agents that influence factor XIIIa catalytic activity (e.g., sulfhydryl agents, albumin, erythrocytes). The two key proenzymes, factor XIII and plasminogen, are tightly bound with the circulating fibrinogen molecules. Such high selective affinity for fibrin(ogen) provides the reaction specificity in a complex tissue fluid milieu and governs the kinetics of fibrinolysis. Any agents that interfere with such binding reactions, e.g., autoantibodies, may also affect the fibrinolytic reactions. Understanding these unique biochemical controls of factors involved in fibrinolysis may provide an insight into the complex regulatory process of extravascular fibrinolysis.

Animals↗
PubMed1996

Fibrin degradation in the synovial fluid of rheumatoid arthritis patients: a model for extravascular fibrinolysis.

The patterns of degradation and the influence of factor XIII polymerization on fibrin stability were examined in vitro following incubation with leukocyte elastase. In vivo experiments, various factor XIII-polymerized fibrin clots were implanted subcutaneously in mice to evaluate the stability of clots in the extravascular space. Both in vitro and in vivo lysis proceeded faster with nonpolymerized fibrin and was not influenced by the presence of cross-linked alpha 2-plasmin inhibitor. In vivo lysis of implanted clots was prevented by elastatinal, powerful elastase inhibitor, suggesting that granulocyte elastase is chiefly responsible for clot lysis in the extravascular space. To further extend investigations on the mechanisms of fibrinolysis in tissues, we evaluated fibrin and its degradation products in the synovial space. Expression of factor XIII in synovial cells and activities of coagulation factors, fibrinolytic enzymes, and inhibitors were investigated in the synovial fluid of rheumatoid arthritis patients. Immunohistochemical analysis showed deposits of insoluble fibrin on synovial membranes and pannus to an extent related to the progression of the disease. Factor XIII was expressed by fibroblasts and macrophages in the early stages of the disease, whereas in advanced stages factor XIII staining was associated with fibrin. The reduction of certain coagulation factors and high level of thrombin-antithrombin complexes in synovial fluid show a steady activation of the coagulation cascade. The evaluation of fibrinogen degradation products and the pattern of degradation of synovial fibrin(ogen) suggest the participation of leukocyte elastase in fibrin(ogen) lysis in synovial tissue of rheumatoid arthritis.

Arthritis, Rheumatoid↗
PubMed1995

Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein.

Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier. In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a full-length cDNA clone in bacteria and purified it to homogeneity. We have also isolated loricrin from newborn mouse epidermis. By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no alpha or beta structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature. The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different. The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain cross-links involving different lysines and glutamines. Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo. The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo. Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene.

Amino Acid Sequence↗
PubMed1995

Identification of YHR068w in Saccharomyces cerevisiae chromosome VIII as a gene for deoxyhypusine synthase. Expression and characterization of the enzyme.

Deoxyhypusine synthase catalyzes the formation of deoxyhypusine, the first step in hypusine biosynthesis. Amino acid sequences of five tryptic peptides from rat deoxyhypusine synthase were found to match partially the deduced amino acid sequence of the open reading frame of gene YHR068w of Saccharomyces cerevisiae chromosome VIII (AC:U00061). In order to determine whether the product of this gene corresponds to yeast deoxyhypusine synthase,a 1.17-kilobase pair cDNA with an identical nucleotide sequence to that of the YHR068w coding region was obtained from S. cerevisiae cDNA by polymerase chain reaction and was expressed in Escherichia coli B strain BL21 (DE3). The recombinant protein was found mostly in the E. coli cytosol fraction and comprised approximately 20% of the total soluble protein. The purified form of the expressed protein effectively catalyzed the formation of deoxyhypusine in yeast eIF-5A precursors as well as in human precursor and in those from Chinese hamster ovary cells. The molecular mass of the enzyme was estimated to be 172,000 +/- 4,300 Da by equilibrium centrifugation. The mass of its polypeptide subunit was determined to be approximately 43,000 Da, in close agreement with that calculated for the coding region of the YHRO68w gene. These findings show that this gene is a coding sequence for yeast deoxyhypusine synthase and that the product of this gene exists in a tetrameric form.

Amino Acid Sequence↗
PubMed1995

Highly active soluble processed forms of the transglutaminase 1 enzyme in epidermal keratinocytes.

The transglutaminase 1 (TGase 1) enzyme is required for the formation of a cornified cell envelope in epidermal keratinocytes. We show here that in addition to its membrane-anchored form, soluble forms of it are also important in keratinocytes. Proliferating cells contain soluble full-length enzyme of 106 kDa, but terminally differentiating cells contain a soluble 67-kDa form often complexed with a 33-kDa protein as well. The amino terminus of the 67 kDa form is residue 93 of the TGase 1 protein, corresponding to the site of proteolytic activation of the factor XIIIa TGase. The amino terminus of the 33-kDa protein is residue 573, corresponding to the site of a second proteolytic cleavage site of factor XIIIa, and of the site for proteolytic activation of the TGase 3 enzyme. The specific activity of the 67/33-kDa soluble complex is twice that of the soluble 67-kDa form and 10 times that of full-length TGase 1. The half-lives of the 67/33- and 106-kDa forms are about 7 or 20 h, respectively. Thus the TGase 1 enzyme is complex, since it exists in keratinocytes as multiple soluble forms, either intact or proteolytically processed at conserved sites, and which have varying specific activities and likely functions.

Amino Acid Sequence↗
PubMed1995

Deoxyhypusine synthase from rat testis: purification and characterization.

Deoxyhypusine synthase is the first enzyme involved in the post-translational formation of hypusine, a unique amino acid that occurs at one position in a single cellular protein, eukaryotic translation initiation factor 5A (eIF-5A). This NAD-dependent enzyme catalyzes the formation of deoxyhypusine by transfer of the butylamine portion of spermidine to the epsilon-amino group of a specific lysine residue in the eIF-5A precursor. Its purification from rat testis was accomplished by ammonium sulfate fractionation and successive ion-exchange chromatographic steps, followed by chromatofocusing on a hydrophilic resin (Mono P). A pI of 4.7 was determined by isoelectric focusing. Amino acid sequences of five tryptic peptides of the pure enzyme did not correspond to any sequences in the protein data banks. The enzyme migrates as a single band on SDS-polyacrylamide gel electrophoresis with an apparent monomer molecular mass of approximately 42,000 Da. Matrix-assisted laser desorption mass spectrometry gave a monomer mass of 40,800 Da. There is evidence, however, that the active enzyme exists as a tetramer of this subunit. Rabbit polyclonal antibodies to the 42-kDa protein precipitated deoxyhypusine synthase activity. The enzyme shows a strict specificity for NAD. Purified deoxyhypusine synthase catalyzes the overall synthesis of deoxyhypusine and, in the absence of the eIF-5A precursor, catalyzes the cleavage of spermidine.

Amino Acid Sequence↗
PubMed1995

Interactions between the IBV nucleocapsid protein and RNA sequences specific for the 3' end of the genome.

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a fusion protein in bacteria. The coding sequence differed from the native protein only in the addition of six histidine residues at the amino terminus which were used for enrichment with a nickel affinity column. In gel shift assays, the mobility of labelled G RNA was decreased with increasing concentrations of the fusion protein. Competitive gel shift assays with labelled G RNA indicated that the protein interacted with relatively high avidities to several unlabelled RNAs representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the smaller segment cDNA also inhibited the interaction with IBV G RNA to the same extent as homologous unlabelled G RNA. In contrast, interactions of the fusion proteins with a region from 99 to 249 bases from the 3' terminus of the IBV genome and bovine liver RNA were relatively weak. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present at a number of sites on the genome, and may represent a common mechanism used by similar viral proteins whose functions depend on binding to RNA.

Animals↗
PubMed1995

Expression of transglutaminase 1 in human epidermis.

To explore the expression and function of the membrane-associated or type I transglutaminase (TGase1) in human epidermis, we have made a new antihuman TGase1 antibody in goats elicited against a purified active recombinant protein expressed in bacteria. By use of Western blotting and immunoprecipitation methods, the antibody reacted with high specificity with only the TGase1 activity of the epidermis and in cultured keratinocytes. By indirect immunofluorescence, the antibody decorated the entire epidermis, including the basal layer, with some potentiation of the granular layer. However, these staining properties are quite different from those of a widely used, commercially available TGase1 monoclonal antibody (termed B.C1), which decorates the granular layers of the epidermis. By Western blotting, it identifies the TGase1 protein band only weakly, but recognizes strongly a group of bands of 15-20 kDa, two of which by amino acid analysis and amino acid sequencing are the small proline-rich (SPR) 1 and SPR2 proteins, also expressed in epidermal and epithelial tissues. Together with a series of blocking experiments with TGase1 proteins and synthetic peptides, these data reveal that the major epitope of the B.C1 antibody most likely resides on the amino-terminus of these two SPR proteins rather than on TGase1. Further studies will now be necessary to determine the role(s) of TGase1 during the different stages of development and differentiation in the epidermis.

Adult↗
PubMed1995

Expression of transglutaminase C during the prenatal development of human submandibular glands.

The involvement of transglutaminase C (TGase C) in morphogenesis and cytodifferentiation during glandular tubule formation was addressed by immunolocalization of the protein at different stages of prenatal human submandibular gland development in 100 fetuses and 20 adult salivary glands. Immunocytochemical detection was carried out using a monospecific antibody to TGase C. The results showed TGase C reactivity in both acini and ducts early in development (from 10 to 14 weeks), followed by a marked increase in ductal activity and a decline in acinar activity up to 32 weeks. During the peak of reactivity at 25 to 32 weeks, staining was concentrated in the apical ends of the columnar cells. In the adult, staining was weakly and diffusely distributed in the striated and excretory ducts. Western blot analysis of the cellular extracts of pooled samples from various stages of salivary gland development showed a single strong band at 76 kDa early in development. This band became weaker after 32 weeks of prenatal development and in the adult. These findings of transient high expression of TGase C, which coincide with the development of tubulo-alveolar structure, suggest that TGase C may play a role in morphogenesis in human salivary gland development.

Adult↗