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B C Kim

Publications and source records attributed to B C Kim.

51 records · Page 3Linked to original sources
PubMed1997

Dietary antioxidant inhibits lipoprotein oxidation and renal injury in experimental focal segmental glomerulosclerosis.

Lipid peroxidation may be involved in the pathogenesis of focal segmental glomerulosclerosis (FSGS). In the present study we examined whether lipid-soluble antioxidants, probucol and vitamin E, could inhibit renal injury in rats with chronic puromycin aminonucleoside (PA) nephrosis and dietary hypercholesterolemia by protecting lipoproteins from oxidation. Male Sprague-Dawley rats received six intraperitoneal injections of PA over a 10 week period and were fed a high cholesterol (HC) diet (PA-HC) or the same diet supplemented with either 1% probucol or vitamin E (100 IU/kg) for 32 weeks. For comparison, a group of rats received PA injections and a normal diet (PA-normal) with or without probucol or vitamin E. Another group rats received saline injections instead of PA and were fed a HC diet (Sal-HC) with or without probucol or vitamin E. At the end of the experiment, proteinuria, FSGS and tubulointerstitial lesions were present in the untreated rats with PA-HC or PA-normal. The magnitude of these lesions was significantly greater in the PA-HC rats than the PA-normal. In contrast to the PA-HC group with hypercholesterolemia, the PA-normal group did not show hypercholesterolemia from week 16 onwards. The rats with PA-HC alone showed significantly higher renal cortical malondialdehyde (MDA) levels and greater susceptibility of plasma very low density lipoprotein (VLDL) + low density lipoprotein (LDL) to the copper-mediated oxidation than the rats with PA-normal or Sal-HC alone. The administration of probucol or vitamin E in the rats with PA-HC significantly reduced the susceptibility of plasma VLDL + LDL to in vitro oxidation, renal cortical MDA level, proteinuria, mesangial volume density and magnitude of FSGS and interstitial lesions. Immunohistochemical staining of renal tissue showed focal segmental distribution of oxidized LDL (Ox-LDL) in the glomeruli of rats with PA-HC. Administration of probucol or vitamin E reduced the intensity of Ox-LDL staining. The staining with ED1 demonstrated that infiltrating glomerular macrophages were significantly more prevalent in the untreated rats with PA-HC than PA-normal or Sal-HC. Treatment with probucol or vitamin E significantly reduced the number of glomerular macrophages in the rats with PA-HC. These results suggest that alimentary hypercholesterolemia aggravates the renal damage in association with increased renal lipid peroxides in chronic PA nephrosis, and that dietary probucol or vitamin E attenuates renal injury in rats with PA-HC possibly by making lipoproteins resistant to oxidation and by inhibiting intraglomerular macrophage infiltration.

Animals↗
PubMed1996

Synthesis of human globin polypeptides mediated by recombinant adeno-associated virus vectors.

Adeno-associated virus, serotype 2 (AAV2)-based chimeric plasmids that harbored a near-full-length human alpha- or beta-globin cDNA were constructed. The cDNAs were spliced into an AAV plasmid, pAAV delta K, downstream from the viral P40 promoter, substituting the capsid gene region. The correctness of the insertion with regard to the transcription polarity was ascertained by both restriction enzyme analysis and DNA sequencing. One of the constructs, pAAVcHBBLCR, contained the erythroid-specific enhancer elements, the locus control region, HS1 and HS2, to ensure an efficient and tissue-specific gene expression. Use of a defective complementing helper, pAVXB (Dixit, M.; et al. Gene 1991, 104, 253-257.) and adenovirus 2 made it possible to prepare recombinant AAVs (rAAVs). Infection of human 293 cells (embryonal kidney cell line) with the resultant rAAV (AAVcHBB) and cotransfection of mouse erythroleukemia (MEL) cells with the beta-globin construct (pAAVcHBBLCR) and an alpha-globin construct (pAAVcHAB) triggered efficient synthesis of human globin polypeptides in the cells, as analyzed by biochemical and immunohistochemical means. The LCR made the construct respond to an inducer, N,N-hexamethylenebisacetamide, the amount of expressed human beta-globin reaching a similar level as the endogenous mouse beta-globin in MEL cells. Electrotransfection of mouse bone marrow hematopoietic stem/progenitor cells with the constructs dramatically increased the number of benzidine-positive cells in liquid suspension culture, indicating expression and synthesis of a human hemoglobin in these cells. Thus, the rAAV constructs may be useful for gene therapy of hemoglobinopathies.

Animals↗
PubMed1996

Involvement of oxidation in LDL-induced collagen gene regulation in mesangial cells.

Oxidized low-density lipoprotein (Ox-LDL) is present in the lesions of focal segmental glomerulosclerosis (FSGS), but the role of Ox-LDL in the disease process is not clear. Recent studies have shown that LDL stimulates the type IV collagen mRNA expression in cultured mesangial cells. Thus, we examined whether oxidative stress is responsible for the stimulation of LDL-induced collagen gene expression in cultured human mesangial cells (HMCs). When quiescent HMCs were exposed to serum-free media containing LDL for 48 hours, peroxidation of LDL was induced as shown by the increased production of thiobarbituric acid-reactive substances (TBARS). LDL stimulated the alpha 1 (I), alpha 1 (III), and alpha 1 (IV) mRNA expression in a dose-dependent manner. At a concentration of 200 micrograms/ml, LDL enhanced the levels of alpha 1 (I), alpha 1 (III), and alpha 1 (IV) mRNA by 3.7-, 3.8- and 3.2-fold, respectively, over the levels in the control cells. These transcripts were further increased 5.4-, 6.7-, and 5.9-fold, respectively, by the addition of 500 micrograms/ml of LDL. Cu(2+)-catalyzed Ox-LDL at a concentration of 200 micrograms/ml also stimulated the alpha 1 (I), alpha 1 (III), and alpha 1 (IV) mRNA expression 4.4-, 5.9-, and 2.8-fold, respectively, compared with the control cells. The addition of monoclonal antibody (mAb) OL-10, which recognizes the malondialdehyde (MDA)-modified peptide epitope, or vitamin E (50 microM) to cultured HMC exposed to LDL markedly inhibited the stimulation of collagen gene expression. When HMCs were incubated with MDA (200 microM), alpha 1 (I), alpha 1 (III), and alpha 1 (IV) mRNA levels increased by two- to threefold compared to control cells. Immunohistochemical staining utilizing mAb OL-10 demonstrated the presence of MDA-modified proteins in the cytoplasm of HMC exposed to either LDL or MDA. These results suggest that peroxidative products of LDL stimulate collagen gene expression possibly via modification of proteins, which are responsible for the expression of collagen genes in cultured HMCs. Given that, lipid peroxidation of LDL may be implicated in the development of glomerulosclerosis by facilitating excessive mesangial matrix generation.

Antibodies, Monoclonal↗
PubMed1996

Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2.

By screening suppressor mutants of the hy2 mutation of Arabidopsis thaliana, two dominant photomorphogenic mutants, shy1-1D and shy2-1D, for two genetic loci designated as SHY1 and SHY2 (suppressor of hy2 mutation) have been isolated. Both of these non-allelic, extragenic suppressor mutations of hy2 are located on chromosome 1 of the Arabidopsis genome. Both mutations suppress the elongated hypocotyl phenotype of hy2 by light-independent inhibition of hypocotyl growth as well as by increasing the effectiveness of light inhibition of hypocotyl elongation. The shy1-1D mutation is partially photomorphogenic in darkness with apical hook opening and reduced hypocotyl elongation. The shy2-1D mutant displays highly photomorphogenic characteristics in darkness such as true leaf development, cotyledon expansion and extremely reduced hypocotyl growth. In regard to hypocotyl elongation, however, the shy2-1D mutation is still light sensitive. Examination of red-far-red light responses shows that the shy1-1D mutation suppresses the hypocotyl elongation of the hy2 mutation effectively in red light but not effectively in far-red light. The shy2-1D suppresses hypocotyl elongation of the hy2 mutation effectively in both red and far-red light. Both mutations can also suppress the early-flowering phenotype of hy2 and have a distinct pleiotropic effect on leaf development such as upward leaf rolling. The data obtained suggest that SHY1 and SHY2 represent a novel class of components involved in the photomorphogenic pathways of Arabidopsis. This is the first report on the identification of dominant mutations in the light signal transduction pathway of plants.

Arabidopsis↗
PubMed1996

Pollen of a male-sterile mutant of Arabidopsis thaliana isolated from a T-DNA insertion pool is not effectively released from the anther locule.

We have isolated a male-sterile mutant from a pool of T-DNA insertional lines of Arabidopsis thaliana generated by an in planta transformation procedure [Chang et al. (1994) Plant J. 5: 551]. Pollen in this mutant is not effectively released from anther locules after cleavage of the stomium. Most mutant pollen grains are round, in contrast to the tricolpate wild-type pollen, and some pollen grains show an abnormal surface structure. Manually released mutant pollen grains are not fertile and show defects in pollen tube germination in vitro. Genetic analysis disclosed that this lesion is due to a single recessive nuclear mutation located on chromosome 3 closely linked to the gll locus. The mutation locus is tightly linked to the inserted T-DNA.

Arabidopsis↗
PubMed1995

Antisickling effects of 2,3-diphosphoglycerate depletion.

Elevation of 2,3-bisphosphoglycerate (2,3-DPG) in sickle erthrocytes (SS RBCs) and concomitant acidification of the cell interior promote polymerization by decreasing the solubility (csat) of deoxyhemoglobin S. The antisickling effect of 2,3-DPG depletion was evaluated after activation of the 2,3-DPG phosphatase activity of bisphosphoglycerate mutase by glycolate-2-phosphate, leading to rapid loss of intracellular 2,3-DPG. To ensure its maximal reduction in a physiologic medium, isosmotic CO2/bicarbonate-buffered saline, pH 7.0, was used. Substitution of K+ for Na+ as the major extracellular cation suppressed K:Cl cotransport, prevented cell shrinkage, and allowed demonstration of the full antisickling effect of 2,3-DPG depletion. The modest effect on solubility per se of removing intraerythrocytic 2,3-DPG (delta Csat = 1.6 g/dL) was amplified into a much larger antisickling effect by interaction with three other cellular variables affecting solubility and polymer content (intracellular pH, O2 saturation, and mean cell hemoglobin concentration). Acting in concert, these four antisickling effects (three solubilizing, one osmotic) reduced polymer fraction of glycolate-treated SS RBCs by 32% to 63%, with a concomitant decrease in sickling of 46% to 95% at the nominal pO2 of the microcirculation (20 mm Hg). A decrement in sickling of this magnitude should significantly ameliorate the vasoocclusive severity of sickle cell disease.

2,3-Diphosphoglycerate↗
PubMed1993

Sparing effect of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S at physiologic ligand saturations.

Recent interest in therapies for sickle cell anemia based on elevating fetal Hb has made accurate estimates of the sparing effect of fetal Hb (Hb F) and other non-sickle Hbs on sickle Hb (Hb S) polymerization essential. We have developed a technique, using HbCO as surrogate for HbO2, that enables us to assess the solubility of Hb S as a function of ligand saturation under conditions that mimic those of the sickling disorders. Equimolar mixtures of unliganded Hb S with Hb F or normal Hb A2 were isosoluble. Solubilities for equimolar mixtures with normal (Hb A) or abnormal (Hb C) Hbs were also identical but were lower than in the prior case. Thus, the sparing effect of both Hb F and Hb A2 should be considered in therapeutic strategies designed to modify Hb S polymerization. Hemolysates, stripped of 2,3-bisphosphoglycerate, from sickle cell disease patients with Hb (F + A2) levels varying from 6 to 25%, as well as from a sickle trait individual, were used to evaluate equilibrium solubility as a function of ligand saturation over the range of pathophysiologic interest (25-70%). Our results show that the sparing effect of Hb (F + A2) increases relative to that of Hb A as ligand saturation increases, and that in the absence of ligand, approximately 30% Hb (F + A2) is essentially isosoluble with the 60% Hb A of sickle trait. Although detailed knowledge of expected therapeutic benefits is confounded by the heterogeneity of Hb F distribution and other variables, these data should provide a framework for estimating likely clinical benefit from pharmacologic efforts to modulate globin gene expression.

Anemia, Sickle Cell↗
PubMed1990

2,3-Diphosphoglycerate and intracellular pH as interdependent determinants of the physiologic solubility of deoxyhemoglobin S.

We have established that 2,3-diphosphoglycerate (2,3-DPG) content and intracellular pH exert separate, but interdependent, effects on the equilibrium solubility (csat) of deoxyhemoglobin S (deoxy-Hb S) that act in concert to modulate intraerythrocytic polymer formation. In a nonphysiologic csat assay system, a steep dependence of csat on pH in the physiologic range 7.0 to 7.6 was shown for both stripped (Hb) and DPG-saturated deoxy-Hb S (Hb-DPG). The solubility-pH profile for Hb under near-physiologic buffer conditions also showed that csat increased steeply in the same pH range (6.8 to 7.6). The effect of 2,3-DPG on csat under near-physiologic conditions was evaluated separately. At pH 7.20, the pH of the human red blood cell, csat values for Hb and Hb-DPG were 19.56 +/- 0.14 and 17.95 +/- 0.45 g/dL, respectively, indicating that the solubility of Hb-DPG is lower than that of Hb by 8.2% +/- 2.3%. Thus, binding of 2,3-DPG in the beta-cleft promotes the polymerization of deoxy-Hb S, the ultimate determinant of cell sickling. Furthermore, because of the abnormal Bohr effect of sickle blood (approximately double that of normal blood), the intracellular pH of deoxygenated sickle erythrocytes should be approximately 0.28 pH unit higher than that of oxygenated cells (ie, 7.41 v 7.13). At the higher pH, the corresponding csat for Hb-DPG is 20.22 g/dL, which is the best estimate of the intrinsic solubility of T-state Hb S under conditions that approximate closely those of pH, temperature, ionic strength, and 2,3-DPG saturation in the fully desaturated sickle erythrocyte.

2,3-Diphosphoglycerate↗
PubMed1988

Effects of macromolecular polyanions on the solubility of deoxyhemoglobin S.

The effects of four macromolecular polyanions on the equilibrium solubility of deoxy-Hb S and oxygen affinity of Hb A were evaluated. The order of molar effectiveness as gelation inhibitors was: poly-L-aspartate approximately equal to heparin (high M.W.) greater than dextran sulfate greater than heparin (low M.W.). The linear solubility profiles for the two most potent polyanions (poly-L-aspartate and high M.W. heparin) were nearly identical and reached a plateau at roughly the same sub-stoichiometric molar ratio. By contrast, poly-L-lysine, a polycation, strongly promoted gelation, while its succinylated derivative promoted gelation only marginally, as did dextran, a neutral polysaccharide. Among the seven species examined, only the four polyanions affected oxygen affinity appreciably, demonstrating the polyanions exert their solubilizing and oxygen affinity lowering effects via specific interaction with the cluster of cationic groups at the 2,3-diphosphoglycerate binding site in the central cavity of deoxyhemoglobin.

Anions↗
PubMed1986

The effect of 2,3-diphosphoglycerate on the solubility of deoxyhemoglobin S.

Although highly charged polyanions, such as inositol hexaphosphate, have been clearly shown to decrease the solubility of deoxyhemoglobin S, the effect of 2,3-diphosphoglycerate (DPG), the endogenous allosteric effector within the red cell, has been more controversial. In this work we have compared the effect of DPG on the solubility of native deoxyhemoglobin S and a derivative in which the DPG binding site is blocked by cross-linking the two beta 82 lysine residues. At pH 6.6 and 30 degrees C the solubility of deoxyhemoglobin S was found to be decreased by 15% (i.e., from 18.8 to 16.0 g/dl) in the presence of saturating concentrations of DPG. Under the same conditions DPG had no effect on the solubility of the cross-linked derivative. This result establishes unequivocally that the binding of DPG within the beta cleft directly facilitates the polymerization of deoxyhemoglobin S. Under physiological conditions, the solubility of deoxyhemoglobin S was found to be decreased by 6% in the presence of an equimolar concentration of DPG. A solubility decrease of this magnitude is sufficient to enhance the tendency of SS cells to sickle and may exacerbate the clinical symptoms of sickle cell disease.

2,3-Diphosphoglycerate↗
PubMed1985

Deoxygenated sickle hemoglobin. Modulation of its solubility by 2,3-diphosphoglycerate and other allosteric polyanions.

The effects of 2,3-diphosphoglycerate (DPG) and other allosteric polyanions of the phosphate or sulfate ester class (inositol hexaphosphate (IHP), ATP, pyridoxamine-5'-phosphate (PMP), and inositol hexasulfate (IHS] on the solubility of deoxyhemoglobin S, and the oxygen affinity of Hb A were evaluated. Their effects on the saturation concentration (csat) indicated promotion of gelation in each case, according to the following order of molar effectiveness: IHP greater than IHS greater than DPG greater than ATP much greater than PMP. Four polybasic carboxylic acids (benzenetricarboxylate (trimesic acid), benzenetetracarboxylate (BTC), benzenepentacarboxylate (BPC), and benzenehexacarboxylate (BHC] were evaluated as well. Their order of molar effectiveness was: BHC greater than BPC greater than BTC much greater than trimesic acid. Both classes of polyanions influenced oxygen affinity in the same order as solubility. Overall, a good correlation existed between the negative charges of these nine allosteric polyanions at neutral pH and their effects on solubility and oxygen affinity. Because of its possible role in the pathophysiology of sickle cell disease, the effect of DPG on csat was examined over the pH range 6.5-7.6. While a decrease in csat was observed for DPG-saturated deoxyhemoglobin S throughout this range, the decrement observed in the physiological pH range (1.8 g/dl) was somewhat lower than that below neutral pH (3.0 g/dl); in either case the sickling tendency of SS red cells would be enhanced. Inasmuch as the intracellular concentration of DPG in sickle cell anemia may be elevated as much as 2-fold, maneuvers aimed at its reduction could be therapeutically beneficial.

2,3-Diphosphoglycerate↗
PubMed1984

Noncovalent inhibitors of sickle hemoglobin gelation: effects of tetrasubstituted ammonium salts.

The effects of various tetrasubstituted ammonium compounds on the solubility of deoxygenated sickle hemoglobin were evaluated. These conclusions were drawn from the slopes of the solubility profiles obtained: (1) for the homologous series of tetraalkylammonium chloride salts (R4NCl), antigelling potency increased with increasing chain length of the R group: Ch3 less than C2H5 less than C3H7 less than C4H9; (2) for halide salts of the tetrabutylammonium cation there was no difference in effectiveness among the anions examined (Br-, Cl-, F-), presumably because of the extremely potent salting-in effect of this cation; (3) substitution of a benzyl for an alkyl group in three tetraalkylammonium chloride salts, C6H5CH2(R)3NCl, where R = CH3, C2H5, or C4H9, potentiated the antigelling capacity by as much as eight-fold. Because they are substantially more water soluble than any other class of noncovalent inhibitors examined to date, further manipulation of the aryl substituent on these compounds may contribute to the design of an effective antisickling agent.

Amines↗