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Biomedical subjects

Y Cho

Publications and source records attributed to Y Cho.

At least 127 records · Page 7Linked to original sources
PubMed1996

Cryptococcus neoformans meningitis in the rat.

The primary clinical manifestation of Cryptococcus neoformans infection in humans is meningoencephalitis. To study the defense mechanisms that participate in the host response against C. neoformans infection of the central nervous system (CNS), we have developed a new model of cryptococcal meningitis in rats. Intracisternal inoculation of C. neoformans produced a granulomatous meningitis with minimal brain parenchymal involvement, resembling cryptococcal meningitis in immunocompetent patients. The granulomas were composed of T cells (CD4+ and CD8+) and macrophages (CD11b/c+); a subpopulation of the macrophages expressed inducible nitric oxide synthase (NOS2). In this model, C. neoformans disseminated to systemic organs early in the course of infection and provoked granuloma formation and NOS2 expression. The temporal profile of inflammation indicated that the CNS inflammatory response is delayed relative to that in the lung and the spleen, which suggests that the effective inflammatory response within the CNS may follow activation of T cells in the periphery and their subsequent entry into the CNS. Inflammation in the meninges was associated with signs of subpial and subependymal glial activation, including enhanced expression of CD11b/c and CD4 in microglia and glial fibrillary acidic protein in astrocytes. Neither cells, however, expressed NOS2. Although C. neoformans invasion to the brain parenchyma was rare, soluble polysaccharide was commonly associated with reactive glial cells. Necrosis was not a feature of C. neoformans granulomas, but, instead, inflammatory cells underwent apoptosis in inflamed organs. The current rat intrathecal cryptococcosis model has several unique advantages for the study of human cryptococcal meningoencephalitis that include close resemblance of histopathologic changes to those in humans, easy accessibility to the cerebrospinal fluid compartment, and no requirement of immunosuppressive agents for establishment of infection.

Animals↗
PubMed1996

[Shone's syndrome].

Explore the source record for details and available documents.

Aortic Coarctation↗
PubMed1995

Probing the roles of active site residues in D-xylose isomerase.

The roles of active site residues His54, Phe94, Lys183, and His220 in the Streptomyces rubiginosus D-xylose isomerase were probed by site-directed mutagenesis. The kinetic properties and crystal structures of the mutant enzymes were characterized. The pH dependence of diethylpyrocarbonate modification of His54 suggests that His54 does not catalyze ring-opening as a general acid. His54 appears to be involved in anomeric selection and stabilization of the acyclic transition state by hydrogen bonding. Phe94 stabilizes the acyclic-extended transition state directly by hydrophobic interactions and/or indirectly by interactions with Trp137 and Phe26. Lys183 and His220 mutants have little or no activity and the structures of these mutants with D-xylose reveal cyclic alpha-D-xylopyranose. Lys183 functions structurally by maintaining the position of Pro187 and Glu186 and catalytically by interacting with acyclic-extended sugars. His220 provides structure for the M2-metal binding site with properties which are necessary for extension and isomerization of the substrate. A second M2 metal binding site (M2') is observed at a relatively lower occupancy when substrate is added consistent with the hypothesis that the metal moves as the hydride is shifted on the extended substrate.

Aldose-Ketose Isomerases↗
PubMed1995

Properties of acetylcholinesterase reconstituted in liposomes of a different charge.

Acetylcholinesterase (AChE) purified from mouse brain was reconstituted in liposomes of a different charge, and the properties of liposome-associated AChE were investigated. Relative to the Km value (38.5 microM) of AChE bound to a neutral liposome, the value of AChE reconstituted in a negatively-charged liposome decreased to 23.3 microM, whereas that of AChE in a positively-charged liposome increased to 90.9 microM. Additionally, AChE bound to a positively-charged liposome expressed a wider range of optimum pH than the enzyme in a negatively-charged liposome. In a stability study, it was found that soluble AChE was unstable at pH 5.5 and 7.4, while it was relatively stable at pH 10. Noteworthy, the immobilization of AChE to liposome enhanced the stability of soluble enzyme at acidic and neutral pH. Moreover, in the stabilization of the enzyme, a neutral liposome was more effective than charged liposomes, of which a positively-charged liposome was more effective than a negatively-charged liposome at acidic pH. Based on these results, it is proposed that while the Km value and the pH dependence of AChE activity are affected by the charge of liposome, the stability of AChE is determined mainly by a hydrophobic binding to a phospholipid membrane.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Nutritional modulation of guinea pig skin hyperproliferation by essential fatty acid deficiency is associated with selective down regulation of protein kinase C-beta.

In a previous study we demonstrated that 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns 4,5-P2) and released as 13-HODE-containing-diacylglycerol (13-HODE-DAG). In vitro, 13-HODE-DAG was shown to selectively inhibit epidermal total protein kinase C (PKC-beta) activity. To determine whether these observations are relevant in vivo, guinea pigs were made essential fatty acid deficient (EFAD) by feeding them a basal diet supplemented with 4% hydrogenated coconut oil for 8 wk. Tissue levels of putative 13-HODE-DAG, protein kinase C (PKC) isozymes and tissue hyperproliferation were determined in the epidermal preparations from skin of control safflower oil-fed guinea pigs, those fed EFAD diet and those fed EFAD diet followed by the control diet for 2 wk. Our data revealed that cutaneous 13-HODE and 13-HODE-DAG were significantly lower in EFAD animals than in safflower-fed controls. These reductions were associated with both elevated epidermal hyperproliferation and elevated expressions and activities of PKC-alpha and beta-isozymes. Refeeding the animals with safflower oil for 2 wk replenished tissue levels of 13-HODE-DAG, which inversely correlated with the selective down regulation of PKC-beta expression and activity and the reversal of hyperproliferation. In contrast, although, the expression and activity of PKC-alpha was elevated in the epidermis of the EFAD guinea pigs, this elevated PKC-alpha expression was not down regulated after refeeding the safflower oil diet to the animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Nutritional Physiological Phenomena↗
PubMed1995

Bactericidal properties of murine intestinal phospholipase A2.

We purified a molecule from the murine small intestine that killed both Escherichia coli and Listeria monocytogenes, and identified it as intestinal phospholipase A2 (iPLA2) by NH2-terminal sequencing and enzymatic measurements. The ability of iPLA2 to kill. L. monocytogenes was greatly enhanced by 5 mM calcium, inhibited by EGTA and abolished after reduction and alkylation, suggesting that enzymatic activity was required for iPLA2-mediated bactericidal activity. A mouse-avirulent phoP mutant, S. typhimurium 7953S, was 3.5-fold more susceptible to iPLA2 than its isogenic virulent parent, S. typhimurium 14028S (estimated minimal bactericidal concentrations 12.7 +/- 0.5 micrograms/ml vs. 43.9 +/- 4.5 micrograms/ml P < 0.001). Overall, these findings identify iPLA2 as part of the antimicrobial arsenal that equips Paneth cells to protect the small intestinal crypts from microbial invasion. Because iPLA2 is identical to Type 2 phospholipase A2 molecules found in other sites, including spleen, platelets and inflammatory exudate cells, this enzyme may also contribute to antibacterial defenses elsewhere in the body.

Amino Acid Sequence↗
PubMed1995

Identification of CG-1, a natural peptide antibiotic derived from human neutrophil cathepsin G.

Cathepsin G is a neutral serine protease of the granzyme B family which is found in human PMN, cells known to be important in the defense of the periodontium against periodontal bacteria. We propose that cathepsin G serves as a "pro-antibiotic" containing peptide domains which express selective antibiotic properties. In this study, we used HPLC to separate the low-molecular-weight peptides derived from the ultrafiltrate of a granule extract from unstimulated PMN. One of the peptides exhibited intense bactericidal activity as determined by radial diffusion overlay assay (against Escherichia coli ML-35P), an amino-terminal sequence "RVSSFLPWIR...", and a 3.1-kDa molecular mass determined by electrospray ionization-mass spectrometry. The sequence and mass are consistent with the C-terminus of cathepsin G deduced by cDNA analysis. These findings support the hypothesis that antibiotic peptides derived from cathepsin G occur naturally in human PMN. Since this is the first naturally occurring antibiotic peptide derived from cathepsin G, we designate it "CG-1".

Amino Acid Sequence↗
PubMed1994

Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations.

Mutations in the p53 tumor suppressor are the most frequently observed genetic alterations in human cancer. The majority of the mutations occur in the core domain which contains the sequence-specific DNA binding activity of the p53 protein (residues 102-292), and they result in loss of DNA binding. The crystal structure of a complex containing the core domain of human p53 and a DNA binding site has been determined at 2.2 angstroms resolution and refined to a crystallographic R factor of 20.5 percent. The core domain structure consists of a beta sandwich that serves as a scaffold for two large loops and a loop-sheet-helix motif. The two loops, which are held together in part by a tetrahedrally coordinated zinc atom, and the loop-sheet-helix motif form the DNA binding surface of p53. Residues from the loop-sheet-helix motif interact in the major groove of the DNA, while an arginine from one of the two large loops interacts in the minor groove. The loops and the loop-sheet-helix motif consist of the conserved regions of the core domain and contain the majority of the p53 mutations identified in tumors. The structure supports the hypothesis that DNA binding is critical for the biological activity of p53, and provides a framework for understanding how mutations inactivate it.

Amino Acid Sequence↗
PubMed1994

13-Hydroxyoctadecadienoic acid reverses epidermal hyperproliferation via selective inhibition of protein kinase C-beta activity.

13-Hydroxyoctadecadienoic acid (13-HODE) is a major lipoxygenase metabolite of linoleic acid in epidermis. Employing a docosahexaenoic acid (22:6n-3) induced model of hyperproliferative guinea pig epidermis, we demonstrated reversal of hyperproliferation by topical 13-HODE. To delineate a possible mechanism for 13-HODE effect, we demonstrated that topical 13-HODE was incorporated into 13-HODE-containing diacylglycerol (13HODE-DAG). This novel substituted-DAG which was markedly depleted in the hyperproliferative skin paralleled the increased activities of PKC-alpha and beta. Replenishment of the hyperproliferative epidermis with topical 13-HODE resulted in the accumulation of tissue 13HODE-DAG and the selective suppression of PKC-beta activity. These data taken together suggest that the generation of putative 13-HODE-DAG and the selective suppression of PKC-beta isozyme activity may play a role in modulating epidermal hyperproliferation.

Administration, Topical↗
PubMed1994

Probing the retinol-binding site of bovine beta-lactoglobulin.

The retinol-binding site of beta-lactoglobulin has been located by selective modification of amino acid residues which reside in the two putative binding sites. Based upon two separate crystallographic analyses of bovine beta-lactoglobulin, different binding sites for retinol have been proposed: one proposal favors an interior cavity, the other a surface cleft. To discriminate between these two models, we have made four separate site-directed mutations introducing a W19A or a K70M in the interior pocket and a F136A or K141M in the surface pocket. The K70M beta-lactoglobulin exhibited a marked decrease in its binding of retinoic acid compared to the F136A, K141M, and wild-type proteins. Retinyldenepropylamine, a retinyl Schiff base analog of retinol, was synthesized and its absorption spectrum when bound to the wild-type, K70M, and K141M proteins was examined to probe its interaction with the respective lysine residues. The retinylidenepropylamine bound in the K70M beta-lactoglobulin exhibited a kinetic red shift as distinct from the blue shift observed when it is bound to either the K141M or wild-type beta-lactoglobulins. The blue shift indicates protonation of the Schiff base. The resulting tagged peptide was isolated after cyanogen bromide cleavage and found to be the Ala25-Met107 peptide, consistent with the Lys70 being the residue which interacts with the bound retinol. These results support the proposal that retinol binds to an evolutionarily conserved interior cavity rather than the surface pocket.

Amino Acid Sequence↗
PubMed1994

Perturbing the metal site in D-xylose isomerase. Effect of mutations of His-220 on enzyme stability.

The histidine residue at position 220 in the Streptomyces rubiginosus D-xylose isomerase is conserved in all D-xylose isomerases. The three-dimensional structure of D-xylose isomerase reveals that His-220 is part of the octahedral coordination sphere of M2, one of two metal ions (Mn2+) in the active site. This work describes the effects of replacing His-220 with Ser, Glu, Asn, and Lys. The consequences of these amino acid substitutions on enzyme activity, thermostability, and structure were analyzed by kinetic, denaturation, and crystallographic methods. The kcat values H220S, H220N, and H220E are only 0.3-0.5% of the wild-type values, and the Km for each of these mutant enzymes increased by 30-40-fold over the wild-type value. The mutant enzyme H220K did not exhibit any measurable activity. Thermal denaturation studies (Tm values) indicate that the H220S and H220N mutant enzymes are approximately 5-8 degrees C less stable than the wild-type enzyme, whereas H220E and H220K are 13-24 degrees C less stable than the wild-type enzyme. To analyze the molecular basis for this decreased thermostability, the crystal structures of the H220S, H220N, and H220E mutant enzymes complexed with Mn2+ have been determined at 1.95, 1.90, and 1.75 A, respectively. In the H220S structure, a water molecule effectively replaces the N epsilon-2 atom of the imidazole ring of His-220 and mediates the interaction between Mn2+ at the M2 site and Ser-220. A similar water-mediated interaction between the metal ion and Asn-220 is observed in H220N. No direct or water-mediated interactions between the carboxyl group of Glu-220 and the metal are observed in H220E. Whereas octahedral coordination is maintained for the metal at the M2 site in H220S and H220N, a pentahedral coordination with the metal at the M2 site is observed in H220E. Metal activation measurements support the observation that metal binding is perturbed and is responsible for thermal lability of His-220 mutants.

Aldose-Ketose Isomerases↗
PubMed1994

Differential inhibition of soluble and membrane-bound acetylcholinesterase forms from mouse brain by choline esters with an acyl moiety of an intermediate size.

Differential inhibitions of soluble and membrane-bound acetylcholinesterase forms purified from mouse brain were examined by the comparison of kinetic constants such as a Km value, a Kss value (substrate inhibition constant), and IC50 values of active site-selective ligands including choline esters. Membrane-bound acetylcholinesterase form (solubilized only in the presence of detergent) showed lower Km and Kss values than soluble acetylcholinesterase form (easily solubilized without detergent). Edrophonium expressed a slightly but significantly (p < 0.01) higher inhibition of detergent-soluble acetylcholinesterase form than aqueous-soluble acetylcholinesterase form, while physostigmine inhibited both forms with a similar potency. A remarkable difference in inhibition was observed using choline esters; although choline esters with acyl chain of a short size (acetyl- to butyrylcholine) or a long size (heptanoyl- to decanoylcholine) showed a similar inhibitory potency for two forms of acetylcholinesterase, pentanoylcholine and hexanoylcholine inhibited more strongly aqueous-soluble acetylcholinesterase than detergent-soluble acetylcholinesterase. Thus, it is suggested that the two forms of AChE may be distinguished kinetically by pentanoyl- or hexanoylcholine.

Acridines↗
PubMed1994

Thermostable variants of bovine beta-lactoglobulin.

The thermal stability of bovine beta-lactoglobulin (BLG) has been enhanced by the introduction of an additional disulfide bond. Wild-type BLG has two disulfide bonds, C106-C119 and C66-C160, with a free cysteine at position 121. We have designed, with the aid of molecular modeling calculations, two mutants of a recombinant BLG (rBLG), L104C and A132C. Molecular dynamics simulations were performed at 300K to study the effect of these alterations on the conformation of the protein. These mutants were then created by site-directed mutagenesis and purified from Escherichia coli carrying a tac expression vector using a two-step renaturation method. Formation of disulfide linkages in the correct arrangement, as designed, was confirmed by peptide mapping. In contrast to wild-type rBLG, which polymerizes at temperatures > 65 degrees C, neither of the mutant proteins polymerized. The conformational stability of the L104C and A132C mutant proteins against thermal denaturation has been substantially increased (8-10 degrees C) as compared with wild-type rBLG. Furthermore, the A132C rBLG exhibits an enhanced stability against denaturation by guanidine hydrochloride as compared with the wild-type or L104C rBLG.

Amino Acid Sequence↗
PubMed1994

[The surgical treatment of coarctation of the aorta and interruption of the aortic arch in the first three months of life--effectiveness of temporary bypass between the pulmonary artery and the descending aorta].

From February 1987 to January 1994, we operated on 24 patients with coarctation of the aorta (CoA) and 9 patients with interruption of the aortic arch (IAA). A policy of staged repair was followed, consisting of reconstruction of the aortic arch with pulmonary artery banding and ligation of the ductus arteriosus in the first stage and intracardiac repair with pulmonary artery band removal in the second stage. In recent cases an extended aortic arch anastomosis was performed using a heparin-coated "shunt tube" between the pulmonary artery and the descending aorta to maintain blood flow to the lower half of the body during aortic cross-clamping. The use of the shunt increased intraoperative urine output (p < 0.05). This technique may allow patients whose condition is poor to undergo aortoplasty more safely. One patient died. This patient had CoA and total anomalous pulmonary venous return, who underwent a one-stage repair, in violation of our policy (early mortality 3.0%). There were two interim deaths before the second stage repair. At present, 18 patients have undergone staged intracardiac repair, including VSD closure (14 cases), Jatene's procedure (1), Ratelli's procedure (1), Damus-Kaye-Stansel (DKS) procedure (1), total cavo-pulmonary connection (TCPC) + DKS procedure+annuloplasty of a common atrioventricular valve (1). There were two early deaths and one late death following intracardiac repair, all in patients with IAA plus VSD.

Anastomosis, Surgical↗
PubMed1994

[A case of successful surgical treatment for coarctation of the aorta associated with ruptured cerebral aneurysm].

We present a case of a sixteen year old boy with coarctation of the aorta who suffered ruptured cerebral aneurysm. Both cerebral and aortic lesions were successfully corrected. He complained of sudden headache and nausea. Subarachnoid hemorrhage was diagnosed by computed tomography of the head. Cerebroangiography demonstrated coarctation of the aorta. Clipping of cerebral aneurysm was done at first, then three months later, complete resection of the aortic coarctation and artificial graft replacement was done by using femoral veno-arterial cardiopulmonary bypass.

Adolescent↗
PubMed1994

Expression of protein kinase C isozymes in guinea pig epidermis: selective inhibition of PKC-beta activity by 13-hydroxyoctadecadienoic acid-containing diacylglycerol.

Prompted by the reversal of skin hyperproliferation to normal by 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid, we investigated a possible mechanism for this antiproliferative action. To address this we first demonstrated that 13-HODE is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns4,5-P2) and released as 13-HODE-containing diacylglycerol by epidermal phospholipase C. Secondly, we tested the possibility whether this putative 13-HODE-containing DAG (13HODE-DAG) could exert a modulatory effect on epidermal protein kinase C (PKC) activity which previously has been associated with skin hyperproliferation. Unlabeled 13HODE-DAG was generated from 13-HODE-containing phosphatidylcholine after phospholipase C hydrolytic cleavage. The effects of the 13HODE-DAG were determined on: i) total epidermal PKC activity; ii) diolein-activated PKC activity; and iii) the two identified epidermal PKC-isozymes (PKC-beta and PKC-alpha). Our data revealed over a twofold activation of total basal PKC activity by diolein. In contrast, replacement of diolein (1,2-dioleoylglycerol) with 13HODE-DAG (1-palmitoyl,2-13HODE-glycerol) in the incubation mixture exerted no effect on total basal PKC activity. In an another experiment, 13HODE-DAG inhibited diolein-activated PKC activity in a dose-dependent manner. To determine whether the effects of 13HODE-DAG are selective, we tested its effects on DEAE-Sephacel-purified and Western blot-confirmed PKC isozymes. Our data revealed that 13HODE-DAG selectively inhibited the activity of PKC-beta isozyme, while exerting negligible effect on the PKC-alpha isozyme. This selective inhibitory effect of 13HODE-DAG on a major epidermal PKC isozyme activity suggests that 13HODE-containing DAG seemingly can modulate epidermal PKC activity, which purportedly is associated with epidermal hyperproliferation.

Animals↗
PubMed1994

Incorporation of 13-hydroxyoctadecadienoic acid (13-HODE) into epidermal ceramides and phospholipids: phospholipase C-catalyzed release of novel 13-HODE-containing diacylglycerol.

Ceramides and phospholipids constitute two important structural lipids of normal skin that are notably rich in polyunsaturated fatty acids. Although linoleic acid (LA) is high in the ceramides, the localization of its 15-lipoxygenase product, 13-hydroxyoctadecadienoic acid (13-HODE) in the epidermis is unknown. In this study, we investigated the relative incorporation of [14C]LA and [14C]13-HODE into ceramides and phospholipids in isolated epidermal slices. Our data revealed minor incorporation of [14C]LA and [14C]13-HODE into ceramides. In contrast, both [14C]LA and [14C]13-HODE are markedly incorporated into phospholipids, particularly, phosphatidylcholine (PC) and phosphatidylinositol (PtdIns). The incorporation of 13-HODE into the PtdIns pool in particular prompted us to investigate into its fate in the signal transduction process and its possible incorporation into diacylglycerol. Our data revealed that 13-HODE is incorporated into epidermal phosphatidylinositol 4,5-bisphosphate (PtdIns4,5-P2) resulting in epidermal phospholipase C-catalyzed release into a novel 13-HODE-containing diacylglycerol (1-acyl-2-13-HODE-glycerol). The possibility now exists that this novel 13-HODE-containing diacylglycerol could function to modulate the activity of epidermal protein kinase C and hyperproliferation/differentiation.

Animals↗