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K Ahn

Publications and source records attributed to K Ahn.

54 records · Page 3Linked to original sources
PubMed1995

In vivo assembly of the proteasomal complexes, implications for antigen processing.

The multicatalytic and multisubunit proteasomal complexes have been implicated in the processing of antigens to peptides presented by class I major histocompatibility complex molecules. Two structural complexes of this proteinase, 20 S and 26 S proteasomes, have been isolated from cells. By analyzing in vivo assembly of the proteasomal complexes we show that the 20 S proteasomal complexes are irreversibly assembled via 15 S assembly intermediates containing unprocessed beta-type subunits. The 20 S proteasomes further associate reversibly with proteasome activators PA28 or pre-existing ATPase complexes to form 26 S proteasomal complexes. Our findings that not all of the 20 S proteasomal complexes are assembled into 26 S proteasomal complexes within cells and that all of PA28 and ATPase complexes are associated with 20 S proteasomes strongly suggest that all proteasomal complexes coexist within cells. We further demonstrate that 26 S proteasomal complexes are predominantly present in the cytoplasm and a significant portion of the 20 S proteasomal complexes is associated with the endoplasmic reticulum membrane. Taken together, our findings suggest that depending upon their associated regulatory components, 26 S and 20 S-PA28 proteasomal complexes serve different housekeeping functions within the cells, while they degrade antigens in a cooperative manner in antigen processing.

Animals↗
PubMed1995

The cytoplasmic and N-terminal transmembrane domains of cytochrome P450 contain independent signals for retention in the endoplasmic reticulum.

Microsomal cytochrome P450 is inserted into the membrane of the endoplasmic reticulum (ER) by its N-terminal signal/anchor sequence which also functions as an ER retention signal. To analyze further potential retention signals of cytochrome P450, topological domains of cytochrome P450 2C1 or 2C2, epidermal growth factor receptor, a plasma membrane protein, and bacterial alkaline phosphatase, a secreted protein were exchanged. The N-terminal signal/anchor of cytochrome P450 2C1 functioned as an ER retention signal when placed at the N terminus of several reporter proteins but not when fused at the C terminus of the extracellular domain of epidermal growth factor receptor, with or without a heterologous cytoplasmic domain. Chimeric proteins in which the cytoplasmic domain of cytochrome P450 2C2 was substituted for that of epidermal growth factor receptor were retained in the ER indicating that an independent retention signal is present in the cytoplasmic part of cytochrome P450 2C2. These chimeras were enzymatically active which argues against misfolding as the primary cause of retention. The ER retention signal of the cytoplasmic domain could not be localized to a single amino acid segment by deletion analysis. These results show that cytochrome P450 2C2 contains redundant, complex ER retention signals in its cytoplasmic and N-terminal hydrophobic domains and that the function of the N-terminal signal is context-dependent.

Alkaline Phosphatase↗
PubMed1995

A viral inhibitor of peptide transporters for antigen presentation.

Cytotoxic T lymphocytes lyse target cells after T-cell-receptor-mediated recognition of class I major histocompatibility complex molecules presenting peptides. Antigenic peptides are generated in the cytoplasm by proteasomes and translocated into the lumen of the endoplasmic reticulum (ER) by peptide transporters (TAP). Herpes simplex virus (HSV) expresses a cytoplasmic protein, ICP47, which seems to interfere with such immune surveillance by mediating retention of 'empty' class I molecules in the ER. By expressing ICP47 in HeLa cells under an inducible promoter, we show that ICP47 efficiently inhibits peptide transport across the ER membrane such that nascent class I molecules fail to acquire antigenic peptides. This inhibition was overcome by transfecting murine TAP. Further, we demonstrate that ICP47 colocalizes and physically associates with TAP within the cell. Inhibition of peptide translocation by a viral protein indicates a previously undocumented potential mechanism for viral immune evasion.

ATP-Binding Cassette Transporters↗
PubMed1995

Three-dimensional finite-element simulation of a turbulent push-pull ventilation system.

A finite-element formulation with penalty approach to enforce continuity is employed here to simulate the three-dimensional velocity field resulting from a simple push-pull ventilation configuration. An analytic expression for the length scale and a transport equation for turbulent kinetic energy are coupled with the momentum equations. A coaxial square hood and jet are arranged with cross-draught perpendicular to the common centreline. Numerical predictions of the velocity and turbulence kinetic energy fields are evaluated in the plane of symmetry with hot film anemometry, and smoke-wire flow visualizations. The agreement of the simulated jet trajectories with flow visualizations is reasonable, as are velocities. Predictions of turbulence kinetic energy are not as good, particularly near the hood face. Despite the limitations the numerical approach is useful in assessing the impact of cross-draughts on the push-pull arrangement.

Air↗
PubMed1995

A permanent human cell line (EA.hy926) preserves the characteristics of endothelin converting enzyme from primary human umbilical vein endothelial cells.

Purification of endothelin converting enzyme (ECE) from endothelial cells has been hindered by the difficulty in obtaining primary endothelial cells in large quantity. We therefore tested transformed human umbilical vein endothelial cells (EA.hy926) for ECE activity. Our data clearly demonstrate that this transformed cell line preserves the ECE properties of the primary cell line. These include: (i) one sharp activity optimum at neutral pH; (ii) characteristics typical of a metalloprotease; (iii) IC50 value for phosphoramidon of 1.8 microM (2.7 microM for HUVEC); (iv) no inhibition by captopril and thiorphan, inhibitors of angiotensin converting enzyme and neutral endopeptidase 24.11. The enzyme showed a substrate specificity for big ET-1:big ET-2:big ET-3 in a ratio of 40:2.5:1. This report presents evidence that a permanent human endothelial cell line, EA.hy926, preserves the ECE activity of HUVEC and is useful for the study of ECE and its regulation of ET-1 production.

Animals↗
PubMed1995

Mycobacterium tuberculosis infection in an orangutan (Pongo pygmaeus).

A respiratory disorder was noted in a 5-year-old female orangutan kept in the Yongin Farmland. Radiographically, multiple radiodense foci ranging from 2 to 6 mm diameter were seen throughout the lung lobes. Grossly, the thoracic cavity revealed a firm texture and grayish-pink discoloration of the left apical lung lobe. Histopathologically, multifocal areas of granulomatous pneumonia present the right and left apical lung lobes. Both primers from IS1081 and IS6110 targeting 196 bp and 245 bp respectively were used in polymerase chain reaction, Mycobacterium tuberculosis was isolated from liver and confirmed by polymerase chain reaction.

Animals↗
PubMed1994

Inverse relationship of cotranslational translocation with the hydrophobic moment of the bovine preproparathyroid hormone signal sequence.

To investigate the dependence of transmembrane translocation on the hydrophobic moment of the hydrophobic core of the preproparathyroid hormone signal sequence, amino acids were switched to maximize or minimize the hydrophobic moment without changing the length or overall hydrophobicity of the core. As assayed in an in vitro translation system with microsomal membranes, the efficiency of translocation of these mutants was inversely related to the hydrophobic moment, indicating the hydrophobic moment or a related property may contribute to translocational activity.

Amino Acid Sequence↗
PubMed1994

Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II.

The RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome, suggests a functional link between transcription and DNA repair. To understand the connection between these two cellular processes, we have extensively purified and functionally analysed TFIIH. We find that TFIIH has a dual role, being required for basal transcription of class II genes and for participation in DNA-excision repair. TFIIH is shown to complement three different cell extracts deficient in excision repair: XPB/ERCC3, XPC and XPD/ERCC2. The complementation of XPB and XPD is a consequence of ERCC3 and ERCC2 being integral subunits of TFIIH, whereas complementation of XPC is due to an association of this polypeptide with TFIIH. We found that the general transcription factor IIE negatively modulates the helicase activity of TFIIH through a direct interaction between TFIIE and the ERCC3 subunit of TFIIH.

DNA Helicases↗
PubMed1993

The amino-terminal 29 amino acids of cytochrome P450 2C1 are sufficient for retention in the endoplasmic reticulum.

Cytochromes P450 are inserted into and anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic signal sequence at the NH2 terminus. To determine whether the NH2-terminal sequence might also have an ER retention function, the NH2-terminal 29 amino acids of cytochrome P450 2C1, with and without an additional 29 amino acids containing an N-glycosylation site, were fused either to a soluble cytoplasmic protein, Escherichia coli beta-galactosidase, or to a secreted protein, E. coli alkaline phosphatase, and the hybrid proteins were expressed in COS1 cells. Subcellular fractionation indicated that both the beta-galactosidase and alkaline phosphatase hybrid proteins cosedimented with marker enzymes for ER membranes, and localization by immunofluorescent staining was consistent with an ER location. Hybrid proteins with the NH2-terminal glycosylation site were glycosylated in COS1 cells, and the carbohydrate moiety was sensitive to endoglycosidase H digestion, providing further evidence that the proteins were retained in the ER. In vitro studies of membrane insertion of the alkaline phosphatase hybrid indicated that fusion to alkaline phosphatase hybrid indicated that fusion to alkaline phosphatase did not alter the topological properties of the cytochrome P450 NH2-terminal sequence. In addition, alkaline phosphatase fused to the extracellular and transmembrane domains of epidermal growth factor receptor was transported to the plasma membrane in COS1 cells, which establishes that alkaline phosphatase as a cytoplasmic domain does not prevent transport from the ER. These observations indicate that the large cytoplasmic domain of cytochrome P450 is not required for retention in the ER and suggest that a specific sequence or structure within the NH2-terminal 29 amino acids functions as an ER retention signal.

Alkaline Phosphatase↗
PubMed1992

The endothelin-converting enzyme from human umbilical vein is a membrane-bound metalloprotease similar to that from bovine aortic endothelial cells.

A phosphoramidon-sensitive, membrane-bound metalloprotease that cleaves big endothelin 1 (big-ET-1) to ET-1 was obtained from human umbilical vein endothelial cells and also from bovine aortic endothelial cells by isolation of plasma-membrane vesicles free of lysosomes. The enzyme was characterized by RIA with an antibody specific for ET-1 and also by reverse-phase HPLC. For both sources, the pH rate profile of the membrane fraction had a very sharp maximum at pH 7.0; little or no activity was seen at more acidic pH values. In contrast, the cytosolic fraction had a major peak at acidic pH values, as well as a broad peak in the neutral region. The activity at pH 7.0 in the membrane fraction was shown by reverse-phase HPLC to produce ET-1 and C-terminal fragment as products. This activity was abolished by phosphoramidon, EDTA, and 1,10-phenanthroline but was not inhibited by pepstatin A, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, leupeptin, or E-64--consistent with the characteristics of a metalloprotease. These results suggest that this activity is from the physiologically relevant, phosphoramidon-inhibitable, endothelin-converting enzyme. The activity found at neutral pH values in the cytosolic fraction was only partially inhibited by EDTA and 1,10-phenanthroline but was not inhibited by phosphoramidon. The membrane-bound endothelin-converting enzyme from human umbilical vein endothelial cells and bovine aortic endothelial cells showed marked similarities, including IC50 values for phosphoramidon of 2.7 and 1.8 microM and Km values for big-ET-1 of 45.4 and 20.9 microM, respectively. The apparent molecular mass by gel filtration was approximately 300-350 kDa for the enzyme from either source. This report characterizes human endothelin-converting enzyme, which may be an important therapeutic target for cardiovascular disease.

Animals↗
PubMed1990

Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate.

Polyphosphate kinase (PPK) polymerizes the terminal phosphate of ATP to a long chain polyphosphate (poly(P) or (Pi)n) in a freely reversible reaction (Kornberg, S. R. (1957) Biochim. Biophys. Acta 26, 294-300), nATP in equilibrium nADP + (Pi)n, PPK, now purified to homogeneity, is a tetramer of 69-kDa subunits. Addition of a primer in the synthetic reaction is not required, nor does ATP or inorganic orthophosphate (Pi) serve in this role. PPK is autophosphorylated under the conditions of poly(P) synthesis; Pi is linked by a nitrogen-phosphate bond as judged by its acid lability and alkali stability. Incorporation of phosphate from the isolated phosphoenzyme into poly(P) upon the addition of ATP in the synthetic reaction and its incorporation into ATP upon the addition of ADP indicate phosphoenzyme to be an intermediate in the reaction. At an ATP level of 5 microM, well below its Km of 2 mM, a pronounced lag in poly(P) synthesis can be removed by tetrapolyphosphate but not by Pi, PPi, or tripolyphosphate. The basis for this stimulatory effect is not clear inasmuch as tetrapolyphosphate does not promote the dephosphorylation of the presumed phosphoenzyme intermediate.

Autoradiography↗
PubMedDate not supplied

Ammonia removal using hepatoma cells in mammalian cell cultures.

It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2-3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells.

Ammonia↗
PubMedDate not supplied

Diabetes mellitus induces vaginal tissue fibrosis by TGF-beta 1 expression in the rat model.

The commonly reported sexual problem in women with diabetes mellitus is lack of vaginal lubrication. It is our hypothesis that reduced vaginal lubrication in diabetic women may result from the structural changes of the vagina. The aim of this study was to investigate in the diabetic rat model the vaginal structures using histochemistry and the expression of TGF-beta 1 using immunohistochemistry. Twenty female Sprague-Dawley rats (200-210 g) were divided into two groups: control and experimental. The experimental group (n = 10) received intravenous injection of streptozotocin (50 mg/kg). After 4 weeks, blood glucose levels were measured, and the vagina of the rat was excised. Serial sections of the vagina were used to perform hematoxylin and eosin (H & E) and Masson's trichrome stains, and for immunohistochemistry to identify TGF-beta 1 expression. The mean blood glucose concentrations were 67 +/- 11 mg/dL (range; 50-85) in the control group and 522 +/- 61 mg/dl (range; 429-590) in the experimental group. In the diabetic animals, vaginal tissue revealed reduced epithelial layers and decreased vaginal submucosal vasculatures compared to the control animals. The collagen connective tissue in the submucosal area of the diabetic animal tissue showed a dense and irregular, distorted arrangement. The immunoreactivity of TGF-beta 1 in the diabetic animals was prominent in the collagen connective tissue, fibroblasts, and smooth muscle fibers, whereas no immunoactivity was detected in the vaginal structures of the control animals. Diabetes mellitus may induce vaginal tissue fibrosis by TGF-beta 1 expression in the rat model. This implies that reduced vaginal lubrication in the diabetic women may result from the structural changes of the vagina.

Animals↗