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Biomedical subjects

B H Chung

Publications and source records attributed to B H Chung.

At least 55 records · Page 3Linked to original sources
PubMed1996

High-level secretion of human alpha 1-antitrypsin from Saccharomyces cerevisiae using inulinase signal sequence.

The use of a proper signal sequence is one of the major determinants for the efficient secretion of heterologous proteins from yeast. The signal sequence derived from inulinase (INU1A) of Kluyveromyces marxianus was evaluated in directing the secretion of a human glycoprotein, alpha 1-antitrypsin (alpha 1-AT), from Saccharomyces cerevisiae. A yeast expression vector for alpha 1-AT was constructed by placing the coding sequence of human alpha 1-AT fused with the INU1A signal sequence downstream of the GAL10 promoter. S. cerevisiae transformants harboring the expression vector secreted about 70% of the total alpha 1-AT synthesized into the culture media. The intracellularly retained form of alpha 1-AT was mostly unglycosylated, whereas the secreted protein had high mannose-type glycosylation. The fed-batch cultivation of the recombinant yeast achieved a high-cell density, leading to the secretion of biologically active alpha 1-AT up to 75 mg l-1. The secreted protein was purified and subjected to N-terminal sequencing, which confirmed that the secreted alpha 1-AT was processed correctly at the Kex2 cleavage site as expected from the sequence of INU1A signal peptide. The results suggest that the inulinase signal sequence is useful for the high-level secretion of relatively large glycoproteins, such as human alpha 1-AT, from S. cerevisiae.

Amino Acid Sequence↗
PubMed1996

A PCR-RFLP method for the detection of activated H-ras oncogene with a point mutation at codon 12 and 61.

To investigate the incidence of the H-ras gene activation in bladder tumor and the feasibility of using urinary washout samples for screening, a series of 33 human bladder tumors and their preoperatively collected urinary washout samples were screened using a mutant specific PCR-RFLP (polymerase chain-restriction fragment length polymorphism) to detect a point mutation of the H-ras gene. Five tumors were found to harbor H-ras mutations where two tumors had a glycine to valine (G-->T) change in codon 12 and three tumors had a glutamine to lysine (C-->A) change in codon 61, respectively. Moreover, we could also detect the same point mutations of the H-ras gene in corresponding urine washout samples. The incidence of H-ras mutation in Korean bladder cancer was estimated at approximately 15.2%. In conclusion, a mutant specific PCR-RFLP method for the detection of H-ras gene mutation is useful for screening or postoperative follow-up of bladder tumor due to its simplicity and high specificity even in urinary samples.

Base Sequence↗
PubMed1996

Effects of nitric oxide on detrusor relaxation.

PURPOSE: Recently we (1994) reported the photo-induced adequate nitric oxide (PIANO) system, in which an NO- or NO2-carrying molecule which has been photoactivated to release NO, could be exploited to investigate the role of NO in various smooth muscle functions. This study was designed to characterize the effect of nitric oxide (NO) exploiting PIANO on rat detrusor relaxation by isometric tension recording and measuring changes in cGMP content. MATERIALS AND METHODS: Exposure to ultraviolet light was used (1 to 60 seconds) to evoke PIANO in the presence of streptozotocin, an NO-carrier, and N omega-nitro-L-arginine (L-NOARG), an NO2-carrier. During relaxation the cyclic guanosine monophosphate (cGMP) content was measured by radioimmunoassay. RESULTS: Rat detrusor strips were reversibly relaxed upon NO generation via PIANO. Pyrogallol, an O2 generator, significantly (p < 0.01) diminished PIANO-mediated relaxation. During PIANO-mediated relaxation, the tissue level of cyclic GMP significantly (p < 0.05) increased over that of the control. Furthermore, methylene blue, a guanylate cyclase inhibitor, significantly (p < 0.01) inhibited both the relaxation and the increase of cGMP. CONCLUSION: We concluded that rat detrusor muscle was capable of responding to NO, and these findings might lead to a treatment for bladder instability and detrusor hyperreflexia, by the use of intravesical instillation of NO donors.

Animals↗
PubMed1996

Probing structure and function of VLDL by synthetic amphipathic helical peptides.

Class A amphipathic helical peptides have been shown to mimic many properties of exchangeable apolipoproteins. The three analogs of the class A amphipathic peptides were used to probe the structure and function of human very low density lipoproteins (VLDL): 1) 18 residue peptide possessing a single helical domain (18A) with the sequence Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe; 2) two domains of 18A separated by a Pro (37pA); and 3) and 18A analog with the end groups protected to increase helicity (Ac-18A-NH2). Upon incubation of the peptides with VLDL at a peptide to VLDL, (protein) ratio of 1:1, the 37pA and Ac-18A-NH2 were able to displace most of apolipoprotein (apo) Cs and E from VLDL without alteration in its lipid composition and morphology while 18A had minimal effect. The extent of displacement was a function of the peptide to VLDL ratio. The rank order of displaceability of apolipoproteins on VLDL was apoE > C-III > C-II. The displacement of apoE and/or Cs from VLDL by peptides variably affected the ability of VLDL to interact with purified bovine milk lipoprotein lipase (LpL) and cultured macrophages. Treatment of VLDL with Ac-18A-NH2 markedly lowered its reactivity to LpL and its ability to induce lipid accumulation in cultured macrophages: however, treatment of VLDL with 37pA or 18A only minimally lowered their abilities. Ac-18A-NH2 treatment of VLDL resulted in the increase of apparent K(m) and a decrease of Vmax for lipoprotein lipase (LpL)-catalyzed hydrolysis of VLDL triglycerides. When an artificial triglyceride emulsion was used as a substrate of LpL, 37pA, but not Ac-18A-NH2, activated LpL. The above data indicate that 1) amphipathic helical peptides can alter the metabolic and functional properties of VLDL by dissociating the functionally important exchangeable apolipoproteins from VLDL as well as by acting as a functional element of VLDL after their incorporation; and 2) the class A amphipathic peptides having different lipid-associating properties exert significantly different effect on VLDL function.

Amino Acid Sequence↗
PubMed1995

Oxidized low-density lipoproteins delay endothelial wound healing: lack of effect of vitamin E.

The purpose of this study was to examine the influence of oxidized low-density lipoprotein (oxLDL) on endothelial regrowth in an in vitro wounding model and the possible protection afforded by vitamin E (E). Endothelial cells grown on micropore filters were wounded by scraping and allowed to reestablish growth on denuded areas in the presence of LDL or oxLDL (25-200 micrograms/ml), linoleic acid (FA, 90 microM) or linoleic acid hydroperoxide (OFA, 15 microM) for 24 h. Some monolayers were pretreated with 25 microM E for 24 h. Transendothelial albumin movement was used as a measure of endothelial barrier function and as an indicator of endothelial monolayer regrowth. Exposure to levels of oxLDL as low as 25 micrograms/ml for 24 h resulted in depressed endothelial monolayer regrowth, whereas native LDL was without effect and pre-enrichment with 25 microM E offered no protection. In comparison, E pre-enrichment improved endothelial regrowth to control levels in FA- and OFA-treated cultures, unlike oxLDL-treated cultures. It is concluded that circulating oxLDL may reduce regrowth of wounded endothelium and supplemental E may not offer protection. Moreover, fatty acids or their hydroperoxides are unlikely to be involved in this effect.

Animals↗
PubMed1995

Lipolysis-induced partitioning of free fatty acids to lipoproteins: effect on the biological properties of free fatty acids.

Free fatty acids (FFA) released during the lipolysis of triglyceride (TG)-rich lipoproteins in vivo are generally believed to be bound to serum albumin. When hypertriglyceridemic (HTG) sera were lipolyzed in vitro by purified bovine milk lipoprotein lipase (LpL), there was an 11- to 18-fold increase in serum FFA levels, and a major portion (> 80%) of the FFA in serum was partitioned to lipoprotein fractions. The greatest portion (33%) of FFA in lipolyzed HTG serum was associated with newly formed flocculent remnants that banded just below low density lipoproteins (LDL) in the density gradient tube. Very low density lipoprotein (VLDL), LDL, and high density lipoprotein (HDL) fractions in lipolyzed HTG serum contained 18- to 29-times more FFA molecules than those in prelipolysis serum. Analysis of the fatty acyl chain composition of FFA in lipolyzed HTG serum showed that the extent of partitioning of saturated FFA into the lipoprotein fractions relative to that of polyunsaturated FFA was about 4.5- to 11-times greater than that partitioned into the free protein fraction; most (84%) of FFA partitioned into flocculent remnants were saturated fatty acids. In vivo lipolysis of TG-rich lipoproteins in HTG subjects, induced by heparinization, resulted in only a small (2.8-fold) increase in serum FFA and little or no increase in the partitioning of FFA to lipoproteins. However, in vitro incubation of the postheparin serum at 37 degrees C for 90 min resulted in a 2.9- to 6.8-fold increase in the serum FFA level and the partitioning of > 66% of total serum FFA into lipoprotein fractions. Studies of the interaction of various plasma fractions from control and in vitro lipolyzed HTG serum with cultured mouse peritoneal macrophages (MPM) showed that FFA partitioned to lipoprotein fractions were highly cytotoxic to cultured MPM, whereas FFA partitioned to albumin at a 10 x greater concentration were not cytotoxic. The cytotoxic potencies of FFA bound to lipoproteins and albumin were further compared after in vitro incorporation of FFA (oleic acids) into LDL and to albumin. FFA bound to LDL but not to albumin were cytotoxic to cultured MPM; the cytotoxicity of FFA bound to LDL was more closely related to the FFA to LDL-cholesterol molar ratio than to the total FFA concentration in the culture dish. The ability of FFA bound to LDL and albumin to induce foam cell formation was studied in THP-1 monocyte-derived macrophages, which were less susceptible to cytotoxicity produced by FFA bound to LDL than MPM.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Photo-induced adequate nitric oxide (PIANO)-mediated relaxation in isolated rabbit corpus cavernosum.

1. We further investigated our earlier proposal that NO- and NO2-carrying molecules potentiate photorelaxation using isolated rabbit corpus cavernosum. 2. Corporal smooth muscle, in the presence or absence of endothelium, relaxed only slightly upon u.v. light (366 nm) irradiation. But, NO- and/or NO2-containing compounds such as streptozotocin and NG-nitro-L-arginine methyl ester significantly (P < 0.01) enhanced photorelaxation in this tissue. In addition, NG-nitro-D-arginine methyl ester, known to lack inhibitory action on NO synthase, showed concentration-dependent potentiation of the photorelaxation. 3. Oxygen radical generating system via xanthine+xanthine oxidase and guanylate cyclase inhibitor, methylene blue, significantly (P < 0.05) inhibited the streptozotocin-potentiated photorelaxation. 4. Nitrite was accumulated by photolysis of streptozotocin, NG-nitro-L-arginine methyl ester and NG-nitro-D-arginine methyl ester, in a concentration and exposure time-dependent manner. 5. These observations indicate that NO is a potent relaxant of rabbit corpus cavernosum and further support our hypothesis that NO is released by photolysis from NO- and NO2-carrying molecules.

Animals↗
PubMed1994

Liposome-like particles isolated from human atherosclerotic plaques are structurally and compositionally similar to surface remnants of triglyceride-rich lipoproteins.

Recent studies have demonstrated the presence of unesterified cholesterol-rich, liposome-like vesicles in the extracellular space of atherosclerotic lesions in humans and animals. Liposome-like vesicles accumulate in the subendothelial space in rabbits within 2 weeks of initiation of cholesterol feeding, well before foam cells appear. These observations suggest that extracellular liposome-like vesicles may play a pivotal role in atherogenesis. The origin of these particles is unknown. We report a combination of in vivo and in vitro experiments that suggest a novel origin for these liposome-like vesicles. We demonstrate that the liposome-like particles isolated from postmortem human atherosclerotic plaques are rich in intact apolipoprotein (apo) A-I, C apolipoproteins, and sphingomyelin. We show that the in vivo derived particles are virtually identical, structurally and compositionally, to liposome-like lipolytic surface remnants of triglyceride (TG)-rich lipoproteins produced during in vitro lipolysis of hypertriglyceridemic serum. In vitro lipolysis of isolated very-low-density lipoprotein has shown that the lipolytic surface remnants remain attached to the core remnants in the absence of high-density lipoprotein (HDL), dissociate to form liposome-like vesicles in the presence of low levels of HDL, and are assimilated into HDL to form larger HDL particles in the presence of excess HDL. Thus, the in vitro produced, liposome-like particles represent a complex of lipolytic surface remnants of TG-rich lipoproteins and apo A-I derived from HDL. Two possible origins have been suggested for the extracellular liposome-like vesicles in atherosclerotic plaques: (1) modified, aggregated, and/or degraded LDL particles entrapped in an intimal matrix and (2) intracellular lipid products of arterial wall cells. Neither possibility directly explains the presence of A-I and C apolipoproteins and excess sphingomyelin that we observe. We propose as an alternate explanation that the in vivo liposome-like particles are lipolytic surface remnants of TG-rich lipoproteins. We further suggest that these remnants are produced in the intimal space by undefined processes and/or are transcytosed into the intima from the plasma compartment as a product of normal lipolysis gone awry. We conjecture that one role of HDL may be to assimilate the highly atherogenic liposome-like particles in a (1) "mop-up" fashion to remove them from the artery wall and/or (2) preventive fashion in the plasma compartment to prevent their transcytosis into the artery wall. The suggestion that elevated concentrations of surface remnants act as a "sink" for apo A-I can also account for the well-established but poorly understood link between hypertriglyceridemia and low HDL.

Animals↗
PubMed1993

Enoxacin photosensitivity.

A 71-year-old Korean farmer had pruritic erythematous patches on sun-exposed areas after enoxacin treatment for prostatitis. Suggestive features of photoallergy, such as moderate blurring of the margin and possible ectopic flare phenomenon, were reproduced by oral provocation test with enoxacin. Because all quinolones have structural similarities, and photosensitivity was observed in nalidixic acid and enoxacin, it would be advisable to warn all patients taking quinolones about photosensitivity.

Aged↗
PubMed1992

Disruption of endothelial barrier function by lipolytic remnants of triglyceride-rich lipoproteins.

Remnants, resulting from the lipolysis of triglyceride-rich lipoproteins, injured cultured endothelial cells and resulted in decreased barrier function of the vascular endothelium. Endothelial cells were cultured on micropore filters. Albumin transfer across endothelial cell monolayers was measured after a 24-h exposure to media enriched with control or in vitro-lipolyzed samples of various hypertriglyceridemic (HTG) sera and its isolated lipoprotein (VLDL, LDL and HDL) and serum free protein (d greater than 1.21 g/ml) fractions. Compared with control cultures, neither control HTG serum nor its isolated lipoprotein and serum-free protein fractions had any effect on albumin transfer. In contrast, lipolyzed HTG (L-HTG) serum and all of its isolated lipoprotein fractions (L-VLDL, L-IDL, L-LDL and L-HDL) caused a marked decrease in endothelial barrier function, evidenced by a significant increase in albumin transfer across endothelial monolayers. The L-IDL and L-HDL fractions were more effective in increasing albumin transfer than the L-VLDL and L-LDL fractions. The extent of the L-IDL and L-HDL mediated increases in albumin transfer was concentration dependent. An exposure of 12 h was required for L-HDL to increase albumin transfer. The L-HDL mediated increase in albumin transfer was reversible only after a 12-h exposure at low concentrations. The free protein fraction from L-HTG serum had no significant effect on the barrier function of endothelial cells. The presence of normolipidemic HDL in culture medium prevented disruption of the endothelial barrier induced by L-IDL but not by L-HDL. The decrease in endothelial barrier function induced by lipolyzed samples of HTG serum or lipoproteins appeared to be correlated with the level of free fatty acids contained in lipolytic remnants. Enrichment of LDL, and in particular HDL, with fatty acid significantly increased albumin transfer. Compared with lipolyzed samples, sera/lipoproteins oxidized in vitro by Cu2+ ions had little effect on endothelial barrier function, which did not correlate with their respective thiobarbituric acid-reacting substance (TBARS) values. TBARS remained within normal range after L-HDL incubation with endothelial cells for up to 48 h. At most concentrations tested, exposure to lipolyzed but not oxidized lipoproteins resulted in morphological perturbations of cell monolayers. These data suggest that lipolytic remnants of triglyceride-rich lipoproteins may play an important role in the development of atherosclerosis by decreasing the barrier function of the vascular endothelium. The remnant-induced injury of the arterial wall may permit the entry of cholesterol-rich lipolytic remnants as well as LDL into the arterial wall.

Animals↗
PubMed1991

Cytotoxicity of remnants of triglyceride-rich lipoproteins: an atherogenic insult?

None of more widely accepted theories of atherogenesis can explain all the more pertinent features of atherosclerosis: a) foam dell formation; b) endothelial cell stress/injury; c) protective effect of HDL; d) atherogenicity of triglyceride-rich lipoproteins; e) the vesicular nature of early lipid deposits in atherosclerosis, f) dissociation of diet risk from the risk due to elevation in plasma cholesterol; or g) correlation of postprandial lipemia with CAD risk. The data obtained from our studies provide a new theory of atherogenesis. This theory is that: a) lipolytic surface remnants of TG-rich lipoproteins may represent a major class of atherogenic lipoproteins which are exacerbated during postprandial hyperlipidemia; b) clearance of these surfaces remnants by HDL in vivo may be one important way that HDL prevents atherosclerosis; c) excess surface remnants may be linked to delayed clearance of potentially atherogenic core remnants, directly linked to atherogenicity via surface remnant-mediated cytotoxicity to cells of the artery wall and/or linked to the deposit of unesterified cholesterol-rich vesicles in early atherosclerosis. An appealing aspect of this hypothesis is that it can account for several unexplained features of atherosclerosis, such as anatomic differences in susceptibility to atherosclerosis in the vascular tree, the preference of early atherosclerosis in humans to the region of the coronary artery subjected to low hemodynamic shear stresses, and the vesicular nature of lipid deposits in early atherosclerosis.

Animals↗
PubMed1990

Lipolyzed hypertriglyceridemic serum and triglyceride-rich lipoprotein cause lipid accumulation in and are cytotoxic to cultured human endothelial cells. High density lipoproteins inhibit this cytotoxicity.

The cytotoxic effect of hypertriglyceridemic (HTG) serum and triglyceride-rich lipoprotein (TG-rich lipoprotein) lipolyzed in vitro by purified lipoprotein lipase on cultured human umbilical vein endothelial cells (HUVECs) was studied. When confluent cultures of HUVECs (8.4 x 10(4)/cm2) were incubated in the presence of control (non-lipolyzed HTG serum) or lipolyzed HTG serum or TG-rich lipoprotein, the lipolyzed HTG serum or TG-rich lipoprotein was cytotoxic to the HUVECs as indicated by their detachment from the culture dish; the lipolyzed serum at 10% of the culture medium or lipolyzed TG-rich lipoprotein at 75 micrograms cholesterol/ml caused the detachment of all (100%) of the cells from the culture dish after a 24 h incubation. Control (non-lipolyzed) HTG serum or non-lipolyzed TG-rich lipoprotein at the same or higher concentration was not cytotoxic to the cells. The HUVECs incubated for 48 h with low (sublethal) doses of lipolyzed TG-rich lipoprotein (10-50 micrograms cholesterol/ml) contained massive lipid inclusions; no lipid inclusions were seen within the cells when the culture medium contained control non-lipolyzed TG-rich lipoproteins. Finally, when high density lipoprotein (HDL) was added to the culture medium at the same concentration as the cytotoxic lipolyzed TG-rich lipoprotein (75 micrograms cholesterol/ml), the cytotoxic effect of the lipolyzed TG-rich lipoprotein was inhibited. These data suggest that the interaction of endothelial cells with lipolytic remnants of TG-rich lipoprotein may play a role in the pathogenesis of atherosclerosis and that HDL may play an important role in inhibition of the endothelial cell injury produced by the lipolytic remnants of TG-rich lipoprotein.

Azo Compounds↗
PubMed1989

Lipolytic surface remnants of triglyceride-rich lipoproteins are cytotoxic to macrophages but not in the presence of high density lipoprotein. A possible mechanism of atherogenesis?

Hypertriglyceridemic (HTG) serum, lipolyzed in vitro by purified bovine milk lipoprotein lipase, was found to be cytotoxic to cultured macrophages. Surviving macrophages contained numerous lipid inclusions similar to those found in foam cells. Individual lipoprotein fractions isolated from the lipolyzed HTG serum, including HDL, were also cytotoxic. Lipolysis of isolated lipoprotein fractions (either HTG or normal) allowed localization of cytotoxicity to postlipolysis remnant VLDL and chylomicron particles. The presence of a critical concentration of HDL in either the lipolysis mixture or the culture dishes inhibited the cytotoxicity. Below this critical concentration HDL itself became cytotoxic, producing lipid inclusions in surviving macrophages. The lipid fraction of the cytotoxic remnants contained the cytotoxic factor(s); neither FFA nor lysolecithin alone could account for this cytotoxicity. Postprandial lipemic sera from subjects with a brisk chylomicron response, when lipolyzed in vitro, were cytotoxic to cultured macrophages; neither fasted sera from these subjects, nor postprandial sera from normolipidemic subjects with a normal chylomicron response, were cytotoxic. Postheparin (in vivo lipolyzed) serum and its isolated lipoprotein fractions obtained 30 min after heparin injection in subjects with HTG were shown to be cytotoxic to macrophages; by 60 min most of the cytotoxicity had disappeared. The postprandial and postheparin observations support an in vivo significance for remnant-associated cytotoxicity. We hypothesize that cytotoxic remnants of lipolyzed VLDL and chylomicrons may be one of the major atherogenic lipoproteins. Further, we suggest that inhibition of the cytotoxicity of these remnants may be one important way that HDL prevents atherosclerosis.

Animals↗
PubMed1988

Rapid quantitative apolipoprotein analysis by gradient ultracentrifugation and reversed-phase high performance liquid chromatography.

A new methodology for the analysis of lipoprotein composition using a combination of gradient ultracentrifugation and high performance liquid chromatography was used to determine the differences in lipoprotein composition between non-hyperlipidemic men and women. Lipoproteins from each subject were separated into six subfractions: VLDL, IDL, LDL, and three subfractions of HDL by a single gradient ultracentrifugation spin of less than 5 hr. The HDL subfractions were designated HDL-L (the lightest density subfraction, rich in apoCs and poor in apoA-II), HDL-M (the middle subfraction, rich in apoA-II), and HDL-D (the most dense, relatively poor in both the apoCs and apoA-II). The concentrations of the water-soluble apolipoproteins in each subfraction were determined using reversed-phase HPLC. The concentrations of apoB and the lipid components of the lipoproteins were determined by chemical and enzymatic methods. This methodology proved to be highly reproducible when performed on fresh plasma samples and we were able to identify many sex-associated differences in lipoprotein composition. This methodology is the only nonimmunological technique available for analyzing lipoprotein composition that offers such a combination of accuracy, speed, and completeness.

Adult↗
PubMed1987

Dissolution of theophylline from sustained-release dosage forms and correlation with saliva bioavailability parameters.

Correlation between in vitro dissolution characteristics and in vivo salivary bioavailability parameters of four commercial sustained-release and one immediate-release theophylline (TP) or aminophylline dosage forms were examined. Area under the saliva concentration-time curve up to 6 h (AUC0----6), peak saliva concentration (Cmax), and fraction absorbed in 1 h (F1), based on saliva concentration following oral administration of TP or aminophylline dosage forms to five volunteers, were closely correlated with percents dissolved in pH 6.8 buffer in 30 min, D30 (6.8), or 60 min, D60 (6.8). Dissolution study in pH 6.8 buffer seemed to be a useful tool for development, evaluation, and quality control of sustained-release dosage forms of TP, since the saliva concentration was reported to represent the blood concentration of TP indirectly.

Adult↗