Biomedical subjects

K J Kim

Publications and source records attributed to K J Kim.

At least 91 records · Page 5Linked to original sources

PubMed1999

Staphylococcus lugdunensis--a potential pathogen in oral infection.

OBJECTIVE: The purpose of this study was to investigate the pathogenicity of Staphylococcus lugdunensis in acute oral infection. STUDY DESIGN: S. lugdunensis was isolated from patients with acute oral infections and from healthy control subjects. Antibiotic susceptibility, in vitro cellular toxicity, in vivo virulence, and hemolytic activity testing and dot blot analysis were performed. The statistical significance of in vitro cellular toxicity was determined by means of analysis of variance. RESULTS: Isolated from the infected patients, S. lugdunensis showed resistance to penicillin, ampicillin, methicillin, cephalothin, and clindamycin, exhibited virulence in vivo, and showed delta-like hemolysin activity. Four of the 6 strains of S. lugdunensis gave synergistic hemolysis. In dot blot analysis, S. lugdunensis showed a positive reaction to the probe of the delta-hemolysin gene in S. aureus. CONCLUSIONS: The results suggest that S. lugdunensis may be a potential pathogen in acute oral infection.

Abscess↗

PubMed1999

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation.

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca2+ uptake by Na+-Ca2+ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 microg/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 microM), 25 microM Zn2+ and 50 microM Mn2+. Dopamine-inhibited enzyme activity was reversed by 10 microg/ml SOD and 10 microg/ml catalase. Pretreatment of dopamine decreased Ca2+ transport in synaptosomes, which was restored by 10 microg/ml SOD and 10 mM DMSO. Dopamine (1-100 microM) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.

Animals↗

PubMed1999

Organic cation transport in rabbit alveolar epithelial cell monolayers.

PURPOSE: To characterize organic cation (OC) transport in primary cultured rabbit alveolar epithelial cell monolayers, using [14C]-guanidine as a model substrate. METHODS: Type II alveolar epithelial cells from the rabbit lung were isolated by elastase digestion and cultured on permeable filters precoated with fibronectin and collagen. Uptake and transport studies of [14C]-guanidine were conducted in cell monolayers of 5 to 6 days in culture. RESULTS: The cultured alveolar epithelial cell monolayers exhibited the characteristics of a tight barrier. [14C]-Guanidine uptake was temperature dependent, saturable, and inhibited by OC compounds such as amiloride, cimetidine, clonidine, procainamide, propranolol, tetraethylammonium, and verapamil. Apical guanidine uptake (Km = 129 +/- 41 microM, Vmax = 718 +/- 72 pmol/mg protein/5 min) was kinetically different from basolateral uptake (Km = 580 +/- 125 microM, Vmax = 1,600 +/- 160 pmol/mg protein/5 min). [14C]-Guanidine transport across the alveolar epithelial cell monolayer in the apical to basolateral direction revealed a permeability coefficient (Papp) of (7.3 +/- 0.4) x 10(-7) cm/sec, about seven times higher than that for the paracellular marker [14C]-mannitol. CONCLUSIONS: Our findings are consistent with the existence of carrier-mediated OC transport in cultured rabbit alveolar epithelial cells.

Animals↗

PubMed1999

Monolayers of human alveolar epithelial cells in primary culture for pulmonary absorption and transport studies.

PURPOSE: To develop a cell culture model of human alveolar epithelial cells in primary culture for the in vitro study of pulmonary absorption and transport. METHODS: Type II pneumocytes isolated from normal human distal lung tissue by enzyme treatment and subsequent purification were plated on fibronectin/collagen coated polyester filter inserts, and cultured using a low-serum growth medium. Characterization of the cell culture was achieved by bioelectric measurements, cell-specific lectin binding, immunohistochemical detection of cell junctions, and by assessment of transepithelial transport of dextrans of varying molecular weights. RESULTS: In culture, the isolated cells spread into confluent monolayers, exhibiting peak transepithelial resistance of 2,180 +/- 62 ohms x cm2 and potential difference of 13.5 +/- 1.0 mV (n = 30-48), and developing tight junctions as well as desmosomes. As assessed by lectin-binding, the cell monolayers consisted of mainly type I cells with some interspersed type II cells, thus well mimicking the situation in vivo. The permeability of hydrophilic macromolecular FITC-dextrans across the cell monolayer was found to be inversely related to their molecular size, with Papp values ranging from 1.7 to 0.2 x 10(-8) cm/sec. CONCLUSIONS: A primary cell culture model of human alveolar epithelial cells has been established, which appears to be a valuable in vitro model for pulmonary drug delivery and transport studies.

Absorption↗

PubMed1999

More characters or more taxa for a robust phylogeny--case study from the coffee family (Rubiaceae).

Using different data sets mainly from the plant family Rubiaceae, but in parts also from the Apocynaceae, Asteraceae, Lardizabalaceae, Saxifragaceae, and Solanaceae, we have investigated the effect of number of characters, number of taxa, and kind of data on bootstrap values within phylogenetic trees. The percentage of supported nodes within a tree is positively correlated with the number of characters, and negatively correlated with the number of taxa. The morphological analyses are based on few characters and weakly supported trees are expected. The percentage of supported nodes is also dependent on the kind of data analyzed. In analyses of Rubiaceae based on the same number of characters, RFLP data give trees with higher percentage of supported nodes than rbcL and morphological data. We also discuss the support values for particular nodes at the familial and subfamilial levels. Two new data sets of ndhF and rbcL sequences of Rubiaceae are analyzed and together with earlier studies of the family we can conclude that the monophyly of the Rubiaceae is supported and within the family there are three well supported, but not easily characterized, large subfamilies, Rubioideae, Cinchonoideae s.s. and Ixoroideae s.l. There are also a few genera (Luculia and Coptosapelta) unclassified to subfamily.

Coffee↗

PubMed1999

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells.

We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na+ pump (Na+-K+-ATPase) and rat epithelial Na+ channel subunits. Rat AEC were cultivated in minimal defined serum-free medium (MDSF) on polycarbonate filters. Beginning on day 5, confluent monolayers were exposed to either 95% air-5% CO2 (normoxia) or 95% O2-5% CO2 (hyperoxia) for 48 h. Transepithelial resistance (Rt) and short-circuit current (Isc) were determined before and after exposure. Na+ channel alpha-, beta-, and gamma-subunit and Na+-K+-ATPase alpha1- and beta1-subunit mRNA levels were quantified by Northern analysis. Na+ pump alpha1- and beta1-subunit protein abundance was quantified by Western blotting. After hyperoxic exposure, Isc across AEC monolayers decreased by approximately 60% at 48 h relative to monolayers maintained under normoxic conditions. Na+ channel beta-subunit mRNA expression was reduced by hyperoxia, whereas alpha- and gamma-subunit mRNA expression was unchanged. Na+ pump alpha1-subunit mRNA was unchanged, whereas beta1-subunit mRNA was decreased approximately 80% by hyperoxia in parallel with a reduction in beta1-subunit protein. Because keratinocyte growth factor (KGF) has recently been shown to upregulate AEC active ion transport and expression of Na+-K+-ATPase under normoxic conditions, we assessed the ability of KGF to prevent hyperoxia-induced changes in active ion transport by supplementing medium with KGF (10 ng/ml) from day 2. The presence of KGF prevented the effects of hyperoxia on ion transport (as measured by Isc) relative to normoxic controls. Levels of beta1 mRNA and protein were relatively preserved in monolayers maintained in MDSF and KGF compared with those cultivated in MDSF alone. These results indicate that AEC net active ion transport is decreased after 48 h of hyperoxia, likely as a result of a decrease in the number of functional Na+ pumps per cell. KGF largely prevents this decrease in active ion transport, at least in part, by preserving Na+ pump expression.

Animals↗

PubMed1999

Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels.

Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 microM) produced approximately 20-30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that alpha-, beta- and gamma-subunits of rat epithelial Na(+) channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations (P(Na(+))/P(K(+)) approximately 1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.

Age Factors↗

PubMed1999

Abnormal glutathione transport in cystic fibrosis airway epithelia.

Glutathione (GSH) is a potentially important component of antioxidant defense in the epithelial lung lining fluid. Cystic fibrosis (CF) patients have chronic inflammation in which oxidative stress can be a factor. To examine the hypothesis that the transport of GSH content was defective in CF patients, intracellular and extracellular GSH were measured by HPLC. Four cell lines were used: CFT1 cells [with defective CF transmembrane conductance regulator (CFTR), DeltaF508 homozygous, two clones] and one of the CFT1 clones transfected with either normal CFTR (CFTR repleted) or beta-galactosidase. GSH content in the apical fluid was 55% lower in CFTR-deficient cultures than in CFTR-repleted cells (P < 0.001). In contrast, intracellular GSH content was similar in CFT1 cells and CFTR-repleted cells. gamma-Glutamyl transpeptidase activity, which degrades extracellular GSH, did not account for differences in apical GSH. Rather, GSH efflux of CFTR-deficient cells was lower than that of CFTR-repleted cells. These studies suggested that decreased GSH content in the apical fluid in CF resulted from abnormal GSH transport associated with a defective CFTR.

Biological Transport↗

PubMed1999

Neutralizing anti-vascular endothelial growth factor antibody inhibits further growth of established prostate cancer and metastases in a pre-clinical model.

PURPOSE: The formation of new blood vessels from the pre-existing vasculature is necessary for support of primary tumor growth and appears coincident with the development of metastasis. In previous studies, inhibition of vascular endothelial growth factor (VEGF), a potent angiogenic factor and mediator of vascular permeability, inhibited tumor neovascularization with consequent inhibition of both primary tumor growth and micrometastases when administered at the time of tumor inoculation. In the present study, we examined the effect of inhibiting VEGF on primary tumor growth and metastases in an in vivo model of established metastatic prostate cancer. MATERIALS AND METHODS: The human prostate cancer cell line DU-145 was found to secrete VEGF. DU-145.luciferase, a subclone stably transfected with an expression vector encoding the luciferase gene, injected subcutaneously, consistently formed tumors in C.B.-17 scid/scid mice. After 6 weeks, assay of whole lung lysates showed significant luciferase activity, consistent with the presence of micrometastasis. RESULTS: Twice weekly treatment of the animals with a monoclonal anti-VEGF neutralizing antibody, A4.6.1, not only suppressed primary tumor growth, but inhibited metastatic dissemination to the lung. When treatment was delayed until the primary tumors were well-established, further growth was still inhibited, as was the progression of metastatic disease. CONCLUSION: Inhibition of tumor-secreted VEGF by a neutralizing antibody is sufficient to significantly impair prostate tumor growth and its subsequent metastasis in an in vivo model of established advanced prostate cancer. These data suggest a critical role for VEGF in initiation and maintenance of tumor angiogenesis in prostate cancer. Inhibition of VEGF in patients with VEGF-secreting prostate cancers may prove an effective approach for inhibiting disease progression even after micro-metastatic dissemination has occurred.

Animals↗

PubMed1999

Nucleotide stimulation of Cl(-) secretion in the pigmented rabbit conjunctiva.

We evaluated the role of extracellular UTP and other nucleotides in the regulation of active ion transport across the pigmented rabbit conjunctiva. When added to the mucosal side of the conjunctiva, UTP (0.01-1000 microM), increased the short-circuit current by up to 14. 6 +/- 2.1 microA/cm(2). The half-maximal concentration was 11.4 +/- 2.3 microM. The serosal absence of Cl(-), serosal presence of 10 microM bumetanide, and mucosal presence of 0.3 mM N-phenylanthranilic acid significantly reduced the change in the short-circuit current (DeltaIsc) induced by 10 microM UTP by 78, 77, and 42%, respectively. Mucosal 10 microM UTP significantly increased (36)Cl flux in the serosal-to-mucosal direction by 0.17 microEq/cm(2)/h, while not affecting mucosal-to-serosal (36)Cl flux. By contrast, (22)Na transport in either direction was unaffected. The rank order of DeltaIsc elicited by adenosine and nucleotides was consistent with the predominant involvement of P2Y purinergic receptors in the UTP effect on conjunctival ion transport. Moreover, the DeltaIsc elicited by UTP was inhibited by 0.05 and 1 mM suramin (a P2-purinergic receptor antagonist), resulting in a rightward shift of the half-maximal concentration to 106.7 +/- 1.3 microM. In conclusion, the primary effect of UTP on ion transport in the pigmented rabbit conjunctiva is stimulation of Cl(-) secretion, possibly at the P2Y(2) and/or the P2Y(4) receptor on the mucosal side of the tissue. Because of the coupling of fluid flow with Cl(-) secretion, UTP or its analogs may be considered for stimulating transconjunctival fluid flow in the dry-eye state.

Adenosine↗

PubMed1998

The amiloride-sensitive epithelial sodium channel alpha-subunit is transcriptionally down-regulated in rat parotid cells by the extracellular signal-regulated protein kinase pathway.

Previous studies have shown that an inducible Raf-1 kinase protein, DeltaRaf-1:ER, activates the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK)-signaling pathway, which is required for the transformation of the rat salivary epithelial cell line, Pa-4. Differential display polymerase chain reaction was employed to search for mRNAs repressed by DeltaRaf-1:ER activation. Through this approach, the gene encoding the alpha-subunit of the amiloride-sensitive epithelial sodium channel (alpha-ENaC) was identified as a target of activated Raf-1 kinases. alpha-ENaC down-regulation could also be seen in cells treated with 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA), indicating that the repression of steady-state alpha-ENaC mRNA level was dependent upon the activity of protein kinase C, the target of TPA, as well. Pretreatment of cells with a specific inhibitor of the ERK kinase pathway, PD 98059, markedly abolished the down-regulation of alpha-ENaC expression, consistent with the hypothesis that the ERK kinase-signaling pathway is involved in TPA-mediated repression. Moreover, through the use of transient transfection assays with alpha-ENaC-reporter and activated Raf expression construct(s), we provide the first evidence that activation of the ERK pathway down-regulates alpha-ENaC expression at the transcriptional level. Elucidating the molecular programming that modulates the expression of the alpha-subunit may provide new insights into the modulation of sodium reabsorption across epithelia.

Animals↗

PubMed1998

Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton- coupled dipeptide transporter (hPepT1).

hPepT1 is a proton-coupled peptide transporter that mediates the absorption of di- and tripeptides. Here we show that tyrosine 167 (Y167) in transmembrane domain 5 (TMD5) of this 12-transmembrane spanning protein contributes to its transport function. We identified this particular amino acid by a computer model of the arrangement of the TMDs of hPepT1 and investigated its role by site-directed mutagenesis and dipeptide uptake studies. [3H]Gly-sar uptake in cells transiently transfected with Y167A-hPepT1 was abolished completely, even though the level of Y167A-hPepT1 expression by Western blot analysis and cell surface expression by immunofluorescence microscopy was similar to those of the wild type. Therefore, mutation affected transport function, but apparently not the steady-state protein level or trafficking of the transporter to the plasma membrane. Moreover, mutation of Y167 into phenylalanine, serine, or histidine all abolished gly-sar uptake in transfected HEK 293 cells. Taken together, these findings suggest that Y167 plays an essential role in hPepT1 function, perhaps due to the unique chemistry of its phenolic side chain.

Blotting, Western↗

PubMed1998

(Ta/Si) multilayer as a wide-bandpass monochromator material.

Specular and non-specular X-ray reflectivity intensities of a (Ta/Si)(60) multilayer sample were measured to characterize its interface structure. Since the multilayer has a good reflectance at its multilayer peaks, its performance as a wide-bandpass monochromator for X-ray scattering experiments of polymers has been tested.

Journal Article↗

PubMed1998

Advanced capabilities for future light sources.

Methods to extend the capabilities of light sources beyond those available at the current generation synchrotron radiation sources based on undulators in electron storage rings are discussed. Taking advantage of the radiation-particle interaction and/or the availability of high-power ultrashort optical lasers, it is possible to develop sources with higher brightness, smaller temporal resolution, or higher photon energy. This paper is a summary of some of these schemes, with an emphasis on new ideas rather than on a comprehensive review.

Journal Article↗

PubMed1998

Production of high-titer retroviral vectors and detection of replication-competent retroviruses.

Retroviral infection or calcium phosphate-mediated DNA transfection has been used for the generation of retrovirus producing cell lines through the introduction of vector DNA into the chromosomes of packaging cells. To compare the ability of the methods for DNA delivery to produce high-titer virus, we generated stable retroviral vector producing cell lines by the transfection or infection of a LN-based vector DNA into PA317 cells and assayed individual clones for production of virus. Of eight randomly chosen G418-resistant clones generated by transfection, only one clone produced the vector at up to > 10(7) cfu/ml. Two of the five clones generated by infection yielded higher-titer viruses in the absence of helper virus--up to 5 x 10(7) more than the transfected clones. The titer of retroviral vectors can be increased by multiple rounds of infection through long-term incubation of amphotropic virus producing cells with ecotropic virus vectors. Such amplification of vector copy number resulted in increase in vector titer of up to 20-fold. For the experiments presented here, we have used an improved vector/packaging system designed for minimizing the possibilities for the generation of an replication-competent retrovirus (RCR). However, the potential of RCR generation was detected in the culture medium harvested from the highest-titer virus producing PA317 clonal cells generated by amplification of the vector through the modified cocultivation technique, although the generation of RCR is very infrequent in the system.

3T3 Cells↗

PubMed1998

Structure-function study of the extracellular domain of the human IFN-alpha receptor (hIFNAR1) using blocking monoclonal antibodies: the role of domains 1 and 2.

We have performed a structure-function analysis of extracellular domain regions of the human IFN-alpha receptor (hIFNAR1) using mAbs generated by immunizing mice with a soluble hIFNAR1-IgG. Five mAbs described in this study recognize different epitopes as determined by a competitive binding ELISA and by alanine substitution mutant analyses of the hIFNAR1-IgG. Two mAbs, 2E1 and 4A7, are able to block IFN-stimulated gene factor 3 (ISGF3) formation and inhibit the antiviral cytopathic effect induced by several IFN-alpha (IFN-alpha 2/1, -alpha 1, -alpha 2, -alpha 5, and -alpha 8). None of these anti-IFNAR1 mAbs were able to block activity of IFN-beta. mAb 4A7 binds to a domain 1-hIFNAR1-IgG but not to a domain 2-hIFNAR1-IgG, which suggests that its binding region is located in domain 1. The binding of the most potent blocking mAb, 2E1, requires the presence of domain 1 and domain 2. The most critical residue for 2E1 binding is a lysine residue at position 249, which is in domain 2. These findings suggest that both domain 1 and domain 2 are necessary to form a functional receptor and that a region in domain 2 is important. IFN-beta recognizes regions of the hIFNAR complex that are distinct from those important for the IFN-alpha.

Alanine↗

PubMed1998

Joint fluid from patients with failed total hip arthroplasty stimulates pit formation by mouse osteoclasts on dentin slices.

Periprosthetic bone resorption has been implicated in the failure of total joint arthroplasty. Osteolysis is reported to be associated with bone resorption induced by bone-resorbing cytokines, which are released from macrophages and fibroblasts in periprosthetic tissues after stimulation by wear debris generated in the joint cavity. Recent reports have suggested the concept of the effective joint space, which includes all periprosthetic regions that are accessible to joint fluid and wear debris. In this study, we examined the levels of interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), and tartrate-resistant acid phosphatase (TRAP) in joint fluid after failed total hip arthroplasty (THA) with osteolysis and investigated whether the joint fluid could activate osteoclastic bone resorption using unfractionated mouse bone cells cultured on dentin slices. Histochemical analysis showed the presence of more TRAP-positive cells in synovial capsules from failed THA patients when compared with osteoarthritis (OA) patients (controls). The levels of IL-6, sIL-6R, and TRAP in joint fluid from failed THA patients were significantly higher than in OA patients. Mouse osteoclasts cultured on dentin slices with joint fluid from failed THA patients with osteolysis produced a significant increase of pit area, whereas cells cultured with joint fluid from OA patients did not. Interestingly, osteoclastic bone resorption on dentin slices was significantly correlated with TRAP activity in joint fluid (p < 0.0001). These results suggest that joint fluid containing bone-resorbing cytokines is produced by synovial capsules in failed THA patients with osteolysis and may activate osteoclasts around the prosthesis in combination with those produced by interface tissues, thus contributing to periprosthetic bone resorption.

Acid Phosphatase↗

PubMed1998

Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells.

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.

Apoptosis↗