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Biomedical subjects

K S Kim

Publications and source records attributed to K S Kim.

At least 55 records · Page 3Linked to original sources
PubMed2002

An efficient motion estimation and compensation method for ultrasound synthetic aperture imaging.

This paper describes a method for overcoming motion artifacts in synthetic aperture imaging. The method is based on a computer simulation study on the influence of target motion on synthetic aperture techniques. A region-based motion compensation approach is used in which only the axial motion is estimated and compensated for a given region of interest under the assumption that the whole ROI moves uniformly. The estimated axial motion is calculated with a crosscorrelation method at the point where the focused signal has the maximum energy within the ROI. We also present a method for estimating axial motion using the autocorrelation method that is widely used to estimate average Doppler frequency. Both computer simulations and in vivo experiments show that the proposed crosscorrelation-based method can greatly improve the spatial resolution and SNR of ultrasound imaging by implementing SA techniques for two-way dynamic focusing without motion artifacts. In addition, the autocorrelation-based motion compensation method provides almost the same results as the crosscorrelation-based method, but with a dramatically reduced computational complexity.

Adult↗
PubMed2001

Activation of mouse macrophage by soluble endogenous murine leukemia virus (MuLV) envelope protein.

We identified recently an endogenous murine leukemia virus (MuLV) envelope protein as a new autoantigen reactive with autoimmune diabetic mouse sera and observed immunosuppressive activity of this envelope protein. In the present study, to elucidate the mechanism involved, we treated macrophages with the envelope protein and investigated activation of macrophage. We found enhancements of iNOS mRNA and nitrite in envelope protein-treated RAW264.7 cells and peritoneal macrophages. The stimulation was highly envelope protein-specific, and also time- and dose-dependent. The activation pattern was similar to that elicited by lipopolysaccharide (LPS) since the envelope protein showed a synergistic effect on macrophage activation in conjunction with interferon gamma (IFN-gamma). Furthermore, deacylated LPS as a competitive inhibitor of LPS showed inhibition of envelope protein-mediated macrophage activation. These data show that MuLV envelope protein can be a new macrophage activator and suggest that the retroviral envelope protein may elicit immunosuppressive activity through macrophage activation.

Animals↗
PubMed2001

Cocaine activates redox-regulated transcription factors and induces TNF-alpha expression in human brain endothelial cells.

Cocaine abuse is frequently associated with cerebrovascular pathology. Although the cellular and molecular mechanisms of these alterations are not fully understood, they may involve oxidative injury or dysfunction of brain microvascular endothelial cells. To test this hypothesis, total glutathione levels, activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), as well as induction of the TNF-alpha gene expression were determined in human brain microvascular endothelial cells (HBMEC) exposed to cocaine. Exposure of HBMEC to cocaine resulted in a dose-dependent depletion of total glutathione levels. In addition, cocaine markedly activated redox-regulated transcription factors, NF-kappaB and AP-1. Activation of these transcription factors was accompanied by induction of AP-1- or NF-kappaB-dependent transcription, as measured by dual luciferase assay in HBMEC transfected with the AP-1- or NF-kappaB-responsive reporter constructs. Furthermore, HBMEC treatment with cocaine induced a dose-dependent expression of the tumor necrosis factor-alpha (TNF-alpha) gene. These results indicate that exposure to cocaine can trigger inflammatory pathways via activation of redox-sensitive transcription factors and induction of expression of the inflammatory genes in HBMEC. These events may contribute to the cerebrovascular insults observed in cocaine-abused patients.

Brain Chemistry↗
PubMed2001

Oxidative modification of human ceruloplasmin by peroxyl radicals.

Ceruloplasmin (CP), the blue oxidase present in all vertebrates, is the major copper-containing protein of plasma. We investigated oxidative modification of human CP by peroxyl radicals generated in a solution containing 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). When CP was incubated with AAPH, the aggregation of proteins was increased in a time- and dose-dependent manner. Incubation of CP with AAPH resulted in a loss of ferroxidase activity. Superoxide dismutase and catalase did not protect the aggregation of CP, whereas hydroxyl radical scavengers such as ethanol and mannitol protected the protein aggregation. The aggregation of proteins was significantly inhibited by the copper chelators, diethyldithiocarbamate and penicillamine. Exposure of CP to AAPH led to the release of copper ions from the enzyme and the generation of protein carbonyl derivatives. Subsequently, when the amino acid composition of CP reacted with AAPH was analyzed, cysteine, tryptophan, methionine, histidine, tyrosine, and lysine residues were particularly sensitive.

Amidines↗
PubMed2001

Apoptosis-linked gene 2 binds to the death domain of Fas and dissociates from Fas during Fas-mediated apoptosis in Jurkat cells.

Apoptosis-linked gene 2 (ALG-2) is a member of the family of Ca(2+)-binding proteins with penta-EF-hand and is essential for the execution of apoptosis by various signals including Fas activation. We studied the regulation of ALG-2 during Fas-mediated apoptosis in Jurkat cells. The 22-kDa ALG-2 protein is cleaved and becomes a 19-kDa protein after Fas activation. The appearance of 19-kDa ALG-2 protein increases for 4 h after treatment with 200 ng/ml of anti-Fas Ab treatment and gradually degrades afterward. Confocal microscopic analysis showed that ALG-2 translocated from the plasma membrane to the cytosol during Fas-mediated apoptosis. Therefore, we examined if ALG-2 interacts with Fas. The protein-protein interaction of ALG-2 with Fas was demonstrated using yeast two-hybrid assays as well as in vitro GST pull-down assay. Endogenous ALG-2 was immunoprecipitated with anti-Fas Ab in Jurkat cells without Fas activation. However, the endogenous ALG-2 was no longer immunoprecipitated with anti-Fas Ab 2 h after anti-Fas Ab treatment. This study, for the first time, presents a direct molecular connection of ALG-2 to apoptosis by its direct interaction with Fas, and enlists ALG-2 as a new member of posttranslationally modified proteins during Fas-mediated apoptotic process.

Apoptosis↗
PubMed2001

Mutational effects on thermostable superoxide dismutase from Aquifex pyrophilus: understanding the molecular basis of protein thermostability.

We designed two mutants of superoxide dismutase (SOD), one is thermostable and the other is thermolabile, which provide valuable insight to identify amino acid residues essential for the thermostability of the SOD from Aquifex pyrophilus (ApSOD). The mutant K12A, in which Lys12 was replaced by Ala, had increased thermostability compared to that of the wild type. The T(1/2) value of K12A was 210 min and that of the wild type was 175 min at 95 degrees C. However, the thermostability of the mutant E41A, which has a T(1/2) value of 25 min at 95 degrees C, was significantly decreased compared to the wild type of ApSOD. To explain the enhanced thermostability of K12A and thermolabile E41A on the structural basis, the crystal structures of the two SOD mutants have been determined. The results have clearly shown the general significance of hydrogen bonds and ion-pair network in the thermostability of proteins.

Bacterial Proteins↗
PubMed2001

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.

Animals↗
PubMed2001

Mechanistic insight into the inactivation of carboxypeptidase A by alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid, a novel type of irreversible inhibitor for carboxypeptidase A with no stereospecificity.

On the basis of the active site topology and enzymic catalytic mechanism of carboxypeptidase A (CPA), a prototypical zinc-containing proteolytic enzyme, alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid (1), was designed as a novel type of mechanism-based inactivator of the enzyme. All four possible stereoisomers of the inhibitor were synthesized in an enantiomerically pure form starting with optically active aspartic acid, and their CPA inhibitory activities were evaluated to find that surprisingly all of the four stereoisomers inhibit CPA in a time dependent manner. The inhibited enzyme did not regain its enzymic activity upon dialysis. The inactivations were prevented by 2-benzylsuccinic acid, a competitive inhibitor that is known to bind the active site of the enzyme. These kinetic results strongly support that the inactivators attach covalently to the enzyme at the active site. The analysis of ESI mass spectral data of the inactivated CPA ascertained the conclusion from the kinetic results. The values of second-order inhibitory rate constants (k(obs)/[I](o)) fall in the range of 1.7-3.6 M(-1) min(-1). The lack of stereospecificity shown in the inactivation led us to propose that the ring cleavage occurs by the nucleophilic attack at the 2-position rather than at the 5-position and the ring opening takes place in an addition-elimination mechanism. The tetrahedral transition state that would be generated in this pathway is thought to be stabilized by the active site zinc ion, which was supported by the PM3 semiemprical calculations. In addition, alpha-benzyl-2-oxo-1,3-oxazolidine-5-acetic acid (18), a structural isomer of 1 was also found to inactivate CPA in an irreversible manner, reinforcing the nucleophilic addition-elimination mechanism. The present study demonstrates that the transition state for the inactivation pathway plays a critical role in determining stereochemistry of the inactivation.

Carboxypeptidases↗
PubMed2001

A high-efficiency synthetic promoter that drives transgene expression selectively in noradrenergic neurons.

Gene promoter systems that drive high-level, long-term, and cell-specific transgene expression are of great interest because of their potential utility for gene therapy. To generate an efficient promoter system specific for noradrenergic (NA) neurons, we multimerized an NA-specific cis-regulatory element (PRS2) identified in the human dopamine beta-hydroxylase (hDBH) promoter, and combined it with a minimal promoter (containing a TATA box and transcription start site). Forms of this synthetic promoter that contain 8 or more copies of PRS2 were >50 times more effective than the 1.15-kb hDBH promoter at driving reporter gene expression in cell lines originated from NA neurons. Neither the synthetic promoter nor the 1.15-kb hDBH promoter drove reporter gene expression in nonneuronal cells. Microinjections of an adenoviral vector containing the synthetic promoter directly into rat brain caused more strict NA-specific reporter gene expression than that caused by a vector containing the 1.15-kb hDBH promoter when the targeted region contained large numbers of NA neurons (locus coeruleus). Furthermore, the vector containing the synthetic promoter caused less nonspecific ("leaky") reporter gene expression than that caused by the vector containing the 1.15-kb hDBH promoter when the targeted region was devoid of NA neurons (cerebellum, dentate gyrus). Together, these studies provide in vitro and in vivo evidence that this novel synthetic promoter can target transgene expression to NA neurons even more efficiently and selectively than the naturally occurring, 1.15-kb hDBH promoter.

Adenoviridae↗
PubMed2001

Ultrathin single-crystalline silver nanowire arrays formed in an ambient solution phase.

We report the synthesis of single-crystalline silver nanowires of atomic dimensions. The ultrathin silver wires with 0.4 nanometer width grow up to micrometer-scale length inside the pores of self-assembled calix[4]hydroquinone nanotubes by electro-/photochemical redox reaction in an ambient aqueous phase. The present subnanowires are very stable under ambient air and aqueous environments, unlike previously reported metal wires of approximately 1 nanometer diameter, which existed only transiently in ultrahigh vacuum. The wires exist as coherently oriented three-dimensional arrays of ultrahigh density and thus could be used as model systems for investigating one-dimensional phenomena and as nanoconnectors for designing nanoelectronic devices.

Journal Article↗
PubMed2001

2-(Hydroxycarbonyl)benzyl glycosides: a novel type of glycosyl donors for highly efficient beta-mannopyranosylation and oligosaccharide synthesis by latent-active glycosylation.

2-(Benzyloxycarbonyl)benzyl (BCB) glycosides were prepared by coupling of the corresponding tetraacetylglycosyl bromides and benzyl 2-(hydroxymethyl)benzoate. The BCB glycosides were converted almost quantitatively into the corresponding 2-(hydroxycarbonyl)benzyl (HCB) glycosides by selective hydrogenolysis of the benzyl ester functionality without affecting the benzylidene acetal and the benzyl ether. Treatment of the HCB 4,6-O-benzylidenemannopyranoside 4 with triflic anhydride in the presence of di-tert-butylmethylpyridine and subsequent addition of the glycosyl acceptor having a primary hydroxyl group afforded exclusively the disaccharide of the beta-mannopyranosyl linkage. Glycosylation of the compound 4 with secondary and tertiary alcohols also provided beta-mannopyranosides as the major products. Glycosylation of the HCB 4,6-O-cyclohexylidenemannoside 5 with primary alcohols was also highly beta-selective, and the HCB 2,3-O-cyclohexylidenemannoside 6 exhibited the moderate beta-selectivity. On the other hand, unlike the HCB mannosides, the HCB 4,6-O-benzylideneglucoside 7 gave exclusively the disaccharides of the alpha-glycopyranosyl linkage in the glycosylation with primary alcohols. The latent BCB-disaccharide 23, which was obtained from the HCB mannoside 4 as the donor and the BCB glucoside 12 as the acceptor by the present glycosylation method, was converted into the active HCB-disaccharide 39 by selective hydrogenolysis. Repetitive glycosylation of the donor 39 with the same acceptor 12 afforded the BCB-trisaccharide 40. Other BCB-trisaccharides 42 and 46 were also efficiently synthesized by employing the present methodology.

Carbohydrate Sequence↗
PubMed2001

Effect of N-glycosylation on ligand binding affinity of rat V1a vasopressin receptor.

A rat Vla vasopressin (rVla) receptor has two putative N-glycosylation sites at 14th and 27th amino acid asparagine in the extracellular N-terminus. In the present study, we examined the possible roles of N-glycosylation of the N-terminus in the receptor function. Three point mutants for deglycosylated rVla receptor were generated in which the 14th and/or the 27th asparagine was replaced with glutamine, namely N14Q, N27Q, and N14:27Q, each tagged with an enhanced green fluorescent protein (EGFP) at their C-termini, and transfected to COS-7 or HEK292 cells. The two single mutants and a double mutant have progressively smaller molecular mass compared to the wild-type receptor as determined by immunoblot analysis, indicating that the two sites are effectively glycosylated in vivo. The maximal ligand binding capacities of three mutant receptors were comparable to that of wild-type (17.02 +/- 1.32 pmol/g protein) with modest changes in ligand binding affinities: N27Q and N14:27Q had decreased binding affinities compared to N14Q and wild-type receptors. The reduced binding affinities of the deglycosylated mutants are not likely due to the impaired intracellular transport since their traffickings were indistinguishable from one another. Taken together, these results suggest that the N-glycosylation at the two sites of the N-terminus of rV1a receptor minimally affects the surface expression and trafficking of the receptor.

Animals↗
PubMed2001

Post-initiation treatment of Indole-3-carbinol did not suppress N-methyl-N-nitrosourea induced mammary carcinogenesis in rats.

The consumption of cruciferous vegetables (the Family of Cruciferae) such as cabbage, broccoli and Brussels sprouts has been shown to have cancer chemopreventive effects in humans and experimental animals. Indole-3-carbinol (I3C), one component of cruciferous vegetables, has been shown to exert cancer chemopreventive influence in liver, colon, and mammary tissue when given before or concurrent with exposure to a carcinogen. However in some reports, there has been evidence that consumption of I3C after carcinogen treatment might be associated with tumor promotion in some tissues. There have been no reports, to our knowledge, of post-initiation effects of I3C in the N-methyl-N-nitrosourea (MNU)-induced mammary tumor model in rats. Our studies were performed to examine this question. Ninety-six, 4-week-old female Sprague-Dawley rats were randomly divided into five groups. The animals of groups 1, 2 and 3 received an intraperitoneal injection of MNU at the age of 50 days. The animals of groups 4 and 5 were injected with saline only at the same time. Animals of groups 1 and 2 were given diet containing 100 ppm and 300 ppm I3C from week 1 until week 25 after MNU treatment. The animals of group 4 were given basal diet containing 300 ppm I3C without MNU treatment. All animals were killed at week 25. The incidences of mammary tumors in the groups 1, 2 and 3 were 95.8% (23/24), 83.3% (20/24) and 82.4% (28/34), respectively. The average number of tumors in the tumor bearing rats of the MNU and I3C 300 ppm group (group 2; 3.85+/-0.63) was higher than that in the MNU alone group (group 3; 2.46+/-0.31). These results represented that exposure to I3C after carcinogen treatment did not suppress development of mammary tumors.

Animals↗
PubMed2001

Differential role of cytosolic phospholipase A2 in the invasion of brain microvascular endothelial cells by Escherichia coli and Listeria monocytogenes.

Invasion of brain microvascular endothelial cells (BMECs) is a key step in the pathogenesis of meningitis due to Escherichia coli and Listeria monocytogenes. Although host cell actin cytoskeletal rearrangements are essential in BMEC invasion by E. coli K1 and L. monocytogenes, the underlying signaling mechanisms remain unclear. This study demonstrates that host cell cytosolic phospholipase A2 (cPLA2) contributes to E. coli K1 invasion of BMECs but not to L. monocytogenes invasion of BMECs. This difference was observed with 4-bromophenacyl bromide, a nonselective PLA2 inhibitor, and arachidonyl trifluoromethyl ketone, a selective cPLA2 inhibitor, and was confirmed with BMEC derived from cPLA2 knockout mice. Activation of cPLA2 leads to generation of intracellular arachidonic acid, which is metabolized via cyclooxygenase (COX) and lipo-oxygenase (LOX) pathways into eicosanoids. COX and LOX inhibitors also significantly inhibit E. coli K1 invasion of BMECs.

Alkaloids↗
PubMed2001

Activation of mouse B lymphocyte by proteins containing hexahistidine.

Many recombinant proteins are produced as a fusion protein tagging hexahistidine, which has been used for studying their biological function both in vitro and in vivo. Unexpectedly, we observed activation of BALB/c mouse splenocytes when treated with hexahistidine-tagged recombinant proteins. This activation was hexahistidine-specific since the anti-pentahistidine antibody completely neutralized the effect, and the bovine serum albumin conjugated with the hexahistidine peptides also showed a similar activation effect. The B cells seemed to be the activated splenocytes, since the cell population stained with the anti-immunoglobulin antibody and anti-CD80 antibody was increased after the treatment. However, the activation signal by hexahistidine was insufficient to fully differentiate the B lymphocytes. This result suggests that caution must be taken in the use of hexahistidine-tagged recombinant proteins, due to their nonspecific activation of B lymphocyte.

Animals↗