Biomedical subjects

S Kim

Publications and source records attributed to S Kim.

At least 181 records · Page 10Linked to original sources

PubMed2001

Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study.

A phase I dose-escalation clinical trial of peritumoral injections of interleukin 12 (IL-12)-transduced autologous fibroblasts was performed in patients with disseminated cancer for whom effective treatment does not exist. The goals of this study were to assess the safety and toxicities as well as the efficacy, and ancillarily the immunomodulatory effects, of peritumoral IL-12 gene transfer. Primary dermal fibroblasts cultured from the patients were transduced with retroviral vector carrying human IL-12 genes (p35 and p40) as well as the neomycin phosphotransferase gene (TFG-hIL-12-Neo). Patients received four injections at intervals of 7 days. Nine patients were enrolled in this dose-escalation study, with secreted IL-12 doses ranging from 300 ng/24 hr for the first three patients to 1000, 3000, and 5000 ng/24 hr for two patients in each subsequent dosage level. Although a definite statement cannot be made, there appears to be perturbation of systemic immunity. Also, the locoregional effects mediated by tumor necrosis factor alpha (TNF-alpha) and CD8+ T cells were observed with tumor regression. Treatment-related adverse events were limited to mild to moderate pain at the injection site; clinically significant toxicities were not encountered. Transient but clear reductions of tumor sizes were observed at the injected sites in four of nine cases, and at noninjected distant sites in one melanoma patient. Hemorrhagic necrosis of tumors was observed in two melanoma patients. These data indicate that gene therapy by peritumoral injection of IL-12-producing autologous fibroblasts is feasible, and promising in patients with advanced cancer.

Adult↗

PubMed2001

Structural basis of the redox switch in the OxyR transcription factor.

The Escherichia coli OxyR transcription factor senses H2O2 and is activated through the formation of an intramolecular disulfide bond. Here we present the crystal structures of the regulatory domain of OxyR in its reduced and oxidized forms, determined at 2.7 A and 2.3 A resolutions, respectively. In the reduced form, the two redox-active cysteines are separated by approximately 17 A. Disulfide bond formation in the oxidized form results in a significant structural change in the regulatory domain. The structural remodeling, which leads to different oligomeric associations, accounts for the redox-dependent switch in OxyR and provides a novel example of protein regulation by "fold editing" through a reversible disulfide bond formation within a folded domain.

Bacterial Proteins↗

PubMed2001

A cofactor of tRNA synthetase, p43, is secreted to up-regulate proinflammatory genes.

An auxiliary factor of mammalian multi-aminoacyl-tRNA synthetases, p43, is thought to be a precursor of endothelial monocyte-activating polypeptide II (EMAP II) that triggers proinflammation in leukocytes and macrophages. In the present work, however, we have shown that p43 itself is specifically secreted from intact mammalian cells, while EMAP II is released only when the cells are disrupted. Secretion of p43 was also observed when its expression was increased. These results suggest that p43 itself should be a real cytokine secreted by an active mechanism. To determine the cytokine activity and active domain of p43, we investigated tumor necrosis factor (TNF) and interleukin-8 (IL-8) production from human monocytic THP-1 cells treated with various p43 deletion mutants. The full length of p43 showed higher cytokine activity than EMAP II, further supporting p43 as the active cytokine. p43 was also shown to activate MAPKs and NFkappaB, and to induce cytokines and chemokines such as TNF, IL-8, MCP-1, MIP-1alpha, MIP-1beta, MIP-2alpha, IL-1beta, and RANTES. Interestingly, the high level of p43 was observed in the foam cells of atherosclerotic lesions. Therefore, p43 could be a novel mediator of atherosclerosis development as well as other inflammation-related diseases.

Amino Acyl-tRNA Synthetases↗

PubMed2001

Mice resistant to experimental autoimmune encephalomyelitis have increased thymic expression of myelin basic protein and increased MBP specific T cell tolerance.

The relationship between expression of the autoantigens in thymi and susceptibility to autoimmune disease was determined in the experimental autoimmune encephalomyelitis (EAE) model. In two different sets of MHC congenic strains of mice characterized by differential susceptibility to EAE, levels of expression of MBP were shown to be higher in the more resistant strain. These data raised the possibility that more central tolerance to MBP may occur in more resistant strains. Differential tolerance was then evidenced by a decrease in T cell responses to MBP 83-102 in the more resistant strains. Together, these data indicate that the list of non-MHC genes involved in susceptibility to autoimmune disease should include genes which regulate expression of autoantigens in thymi.

Animals↗

PubMed2001

IFN-gamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death.

Fas ligand (FasL), perforin, TNF-alpha, IL-1, and NO have been considered as effector molecule(s) leading to beta cell death in autoimmune diabetes. However, the real culprit(s) in beta cell destruction have long been elusive, despite intense investigation. We and others have demonstrated that FasL is not a major effector molecule in autoimmune diabetes, and previous inability to transfer diabetes to Fas-deficient nonobese diabetic (NOD)-lpr mice was due to constitutive FasL expression on lymphocytes from these mice. Here, we identified IFN-gamma/TNF-alpha synergism as the final effector molecules in autoimmune diabetes of NOD mice. A combination of IFN-gamma and TNF-alpha, but neither cytokine alone, induced classical caspase-dependent apoptosis in insulinoma and pancreatic islet cells. IFN-gamma treatment conferred susceptibility to TNF-alpha-induced apoptosis on otherwise resistant insulinoma cells by STAT1 activation followed by IFN regulatory factor (IRF)-1 induction. IRF-1 played a central role in IFN-gamma/TNF-alpha-induced cytotoxicity because inhibition of IRF-1 induction by antisense oligonucleotides blocked IFN-gamma/TNF-alpha-induced cytotoxicity, and transfection of IRF-1 rendered insulinoma cells susceptible to TNF-alpha-induced cytotoxicity. STAT1 and IRF-1 were expressed in pancreatic islets of diabetic NOD mice and colocalized with apoptotic cells. Moreover, anti-TNF-alpha Ab inhibited the development of diabetes after adoptive transfer. Taken together, our results indicate that IFN-gamma/TNF-alpha synergism is responsible for autoimmune diabetes in vivo as well as beta cell apoptosis in vitro and suggest a novel signal transduction in IFN-gamma/TNF-alpha synergism that may have relevance in other autoimmune diseases and synergistic anti-tumor effects of the two cytokines.

Animals↗

PubMed2001

Fibrillar collagen specifically regulates human vascular smooth muscle cell genes involved in cellular responses and the pericellular matrix environment.

Proliferation and alpha(v)beta(3) integrin-dependent migration of vascular smooth muscle cells are suppressed on polymerized type I collagen. To identify genes specifically regulated in human smooth muscle cells by polymerized collagen, we used the suppressive subtraction hybridization technique. Compared with smooth muscle cells cultured on monomer collagen, polymerized collagen suppresses the following: (1) a number of other extracellular matrix proteins, including fibronectin, thrombospondin-1, tenascin-C, and cysteine-rich protein 61; (2) actin binding proteins including alpha-actinin; (3) signaling molecules; (4) protein synthesis-associated proteins; and (5) genes with unknown functions. Some of the identified genes, including cysteine-rich protein 61, show unique kinetics of mRNA regulation by monomer or polymerized collagen distinct from growth factors, suggesting extracellular matrix-specific gene modulation. Moreover, in vivo balloon catheter-mediated injury to the rat carotid artery induces many of the genes that are suppressed by polymerized collagen. Protein levels of thrombospondin-1 and fibronectin are also suppressed by polymerized collagen. Thrombospondin-1-mediated smooth muscle cell migration on vitronectin is significantly inhibited after culture on polymerized collagen for 24 hours, which is associated with decreased alpha-actinin accumulation at focal adhesions. Thus, polymerized type I collagen dynamically regulates gene expression, pericellular accumulation of extracellular matrix molecules, and the response to a given matrix molecule.

Actinin↗

PubMed2001

Controlling duration of contact between T cells and antigen-presenting cells.

A new method which allows precise control of the duration of contact between T cells and antigen-presenting cells (APCs) has been developed. A glass coverslip coated with poly-L-lysine, and then with T cells, was placed at the base of a cylindrical well, and the well was filled with liquid medium. A round coverslip, on which APCs were adhered, was supported on the surface of the medium by surface tension, cell-side down. By withdrawing medium from four capillary holes near the base of the well, the coverslip could be lowered to initiate contact between T cells and APCs at a defined time zero. The contact was broken at desired time points by re-introducing medium into the well in order to separate the two coverslips. Each cell type remained adherent to its original surface after separation for all contact times studied. The T cells were monitored for intracellular calcium mobilization using the fluorescent dye, Fura-2. Contact durations of less than 1 min did not trigger calcium signals. Contact durations of 3 and 5 min induced strong calcium signals. Breaking the contact caused a rapid decrease in intracellular calcium levels. This method of cell manipulation allows precise control of the duration of contact of T cells with APCs, while keeping the cells under continuous observation. The measurements so obtained provide a quantitative understanding of the dynamics of early T cell activation.

Animals↗

PubMed2001

Studies of impurities in magnetic semiconductors: an example of important XAFS applications.

The x-ray absorption fine structure (XAFS) technique has been employed to investigate the local structure and valency about Mn and Fe ions in the III-V diluted magnetic semiconductors In(1-x)Mn(x)As and Ga(1-x)Fe(x)As, prepared by molecular-beam-epitaxy under various growth conditions. These new systems are promising magnetic materials of considerable current interest and with important technical applications including photo-carrier induced magnetism and spin-polarized current devices. The local structure around the magnetic ions can play a pivotal role in affecting the magnetic properties of these semiconductors. Local structure information obtained from XAFS has provided the first direct evidence that the magnetic impurities can indeed substitute for the cation host atoms in samples prepared under appropriate conditions.

Journal Article↗

PubMed2001

Effective treatment of established murine collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express IL-4.

Dendritic cells (DC) are APCs that are able to stimulate or inhibit immune responses, depending on levels of expression of MHC class I and II costimulatory molecules and cytokines. Our previous studies have suggested that the observed contralateral effect, where injection of a vector carrying certain immunomodulatory genes into one joint resulted in inhibition of arthritis in untreated joints, is mediated by in vivo modification of DC. Therefore, we have examined the ability of genetically modified DC to suppress established murine collagen-induced arthritis (CIA) after i.v. delivery. IL-4 has been shown to partially reduce the severity of CIA after repeated injection of recombinant protein or by injection of an adenoviral vector expressing IL-4. Here we demonstrate that i.v. injection of immature DC, infected with an adenoviral vector expressing IL-4, into mice with established CIA resulted in almost complete suppression of disease, with no recurrence for up to 4 wk posttreatment. Injection i.v. of fluorescently labeled DC demonstrated that the cells rapidly migrated to the liver and spleen after 6 h and to the lymph nodes by 24 h. In culture, spleen cells from DC/IL-4-treated mice produced less IFN-gamma after stimulation by collagen than did control groups. In addition, DC/IL-4 administration decreased the level of specific Abs against type II collagen, in particular the IgG2 Th1 isotype 14 days posttreatment. These results demonstrate the ability to treat effectively established murine arthritis by systemic administration of DC expressing IL-4.

Adenoviridae↗

PubMed2001

Molecular characterization of the cDNA encoding an acidic isoform of PR-1 protein in rice.

Rice cDNA encoding an acidic type of pathogenesis-related protein-1 (PR-1a) was cloned and characterized. The deduced PR-1a protein consisted of 168 amino acid residues, including 24 hydrophobic signal sequences at the N-terminus. The predicted molecular mass of the PR-1a was 15,728 Da with a theoretical pI of 4.5, an indication of an acidic protein. The PR-la showed high homology to an acidic PR-1 of Zea mays (74%) and a previously identified basic type PR-1 of rice (64%). Both rice PR-1 and PR-1a genes were found to exist as small gene families through Southern blot hybridization analyses. The PR-1 mRNA was accumulated only in leaves, while the PR-1a transcript was accumulated throughout the plant at a low level. Expression of both PR-1 genes was induced by infections of the rice blast fungus, Magnaporthe grisea, or the bacterial leaf blight pathogen, Xanthomonas oryzae pv. oryzae, and the treatment of benzo (1, 2, 3) thiadiazole-7-carbothioic acid S-ethyl ester, H2O2, or CuSO4. The expression of both PR-1 genes was higher and more rapidly induced in an incompatible interaction than in a compatible interaction in the rice M. grisea interactions.

Acids↗

PubMed2001

NO as an autocrine mediator in the apoptosis of activated microglial cells: correlation between activation and apoptosis of microglial cells.

Abnormal activation of microglial cells has been implicated in various neurodegenerative diseases. Microglial activation needs to be tightly regulated for physiological maintenance and normal functioning of the central nervous system. Potential mechanisms for the down-regulation of activated microglial cells are the deactivation or elimination of activated cells. We hypothesized that the elimination of activated microglial cells by apoptosis is one of the key mechanisms of auto-regulation of activated microglial cells. To test this hypothesis, we utilized BV-2 mouse microglial cells and rat primary microglial cultures exposed to activating agents such as lipopolysaccharide and interferon-gamma, and investigated a possible correlation between apoptosis and activation of these cells. We found that the activation of microglial cells led to apoptotic death, and the activation state of microglial cells inversely correlated with cell viability. We have also demonstrated that: (i) NO was produced by activated microglial cells in a manner dependent on time and dose of activating agents; (ii) inhibition of NO synthesis by iNOS inhibitor blocked the apoptosis of activated microglial cells; (iii) an exogenous NO donor induced apoptosis of microglial cells; and (iv) inhibition of TNFalpha or FasL using neutralizing antibodies did not affect activation-induced apoptosis of microglial cells. These results indicated that activation of microglial cells leads to the production of NO, which in turn acts as the major mediator of cellular apoptosis in an autocrine fashion. Our work suggests the presence of auto-regulatory mechanism for microglial activation, which may have relevance in the pathogenesis of various neurodegenerative diseases possibly resulting from 'over-activation' of microglial cells.

Animals↗

PubMed2001

Cloning of an acidic laccase gene (clac2) from Coprinus congregatus and its expression by external pH.

Coprinus congregatus synthesized and secreted high amounts of laccase under acidic culture condition (pH 4.1) transiently. We have cloned a genomic DNA fragment between the copper binding regions I and II of a laccase by PCR from C. congregatus. This fragment was used as a probe to clone a laccase cDNA from the acidic culture. The cDNA (clac2) consisted of 1817 bp which contains 1086 bp encoding 362 amino acids. The N-terminal sequence of the purified CLAC2 revealed that the first 16 amino acids were removed by the modification. The purified protein had two copper binding regions, although other fungal laccases had four. According to a Northern blotting analysis the clac2 was expressed not in neutral culture but in acidic culture only. The transcription of the clac2 as well as the laccase activity reached a maximum after 24 h upon transferring to an acidic culture, and then decreased very rapidly.

Amino Acid Sequence↗

PubMed2001

In vitro selection of the RNA aptamer against the Sialyl Lewis X and its inhibition of the cell adhesion.

Sialyl Lewis X (sLeX) is a tetra-saccharide glycoconjugate of membrane proteins. It acts as a ligand for the selectin proteins during cell adhesion of inflammatory process. Aberrant overexpression of sLeX is also a characteristic of various cancer cells, especially for highly malignant ones. In this paper, the sLeX-specific RNA aptamer was selected using a random RNA library and its affinity and specificity were measured by Surface Plasmon Resonance technique. Affinity of the selected RNA was increased about 1000-fold as compared with the original RNA pool. RNA aptamer bound more specifically to its cognate sugar than to any other similar sugars. Inhibition of the cell adhesion was also shown by in vitro static assay of sLeX-expressing HL60 cells to the E- and P-selectins. It suggests that the high affinity carbohydrate specific RNA aptamer could be used as an alternative to the antibody.

Base Sequence↗

PubMed2001

Cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma.

Replication-competent, attenuated herpes simplex viruses (HSV) have been demonstrated to be effective oncolytic agents in a variety of malignant tumors. Cytokine gene transfer has also been used as immunomodulatory therapy for cancer. To test the utility of combining these two approaches, two oncolytic HSV vectors (NV1034 and NV1042) were designed to express the murine GM-CSF and murine IL-12 genes, respectively. These cytokine-carrying variants were compared with the analogous non-cytokine-carrying control virus (NV1023) in the treatment of murine SCC VII squamous cell carcinoma. All three viruses demonstrated similar infection efficiency, viral replication, and cytotoxicity in vitro. SCC VII cells infected by NV1034 and NV1042 effectively produced GM-CSF and IL-12, respectively. In an SCC VII subcutaneous flank tumor model in immunocompetent C3H/HeJ mice, intratumoral injection with each virus caused a significant reduction in tumor volume compared with saline injections. The NV1042-treated tumors showed a striking reduction in tumor volume compared with the NV1023- and NV1034-treated tumors. On subsequent rechallenge in the contralateral flank with SCC VII cells, 57% of animals treated with NV1042 failed to develop tumors, in comparison with 14% of animals treated with NV1023 or NV1034, and 0% of naive animals. The increased antitumor efficacy seen with NV1042 in comparison with NV1023 and NV1034 was abrogated by CD4(+) and CD8(+) lymphocyte depletion. NV1042 is a novel, attenuated, oncolytic herpesvirus that effectively expresses IL-12 and elicits a T lymphocyte-mediated antitumor immune response against murine squamous cell carcinoma. Such combined oncolytic and immunomodulatory strategies hold promise in the treatment of cancer.

Animals↗

PubMed2001

A 2.1 A resolution structure of an uncleaved alpha(1)-antitrypsin shows variability of the reactive center and other loops.

Serpin (serine protease inhibitor) proteins are involved in diverse physiological processes including inflammation, coagulation, matrix remodeling, and cell differentiation. Deficiency of normal serpin functions leads to various hereditary diseases. Besides their clinical importance, serpin proteins draw much attention due to the large conformational changes that occur upon interaction with proteases. We present here the crystal structure of an uncleaved alpha(1)-antitrypsin determined by the multiple isomorphous replacement method and refined to 2.1 A resolution. The structure, which is the first active serpin structure based on experimental phases, reveals novel conformations in the flexible loops, including the proximal hinge region of the reactive center loop and the surface cavity region in the central beta-sheet, sheet A. The determined loop conformation explains the results of recent mutagenesis studies and provides detailed insights into the protease inhibition mechanism. The high-resolution structure of active alpha(1)-antitrypsin also provides evidence for the existence of localized van-der-Waals strain in the central hydrophobic core.

Amino Acid Sequence↗

PubMed2001

Binding of serum response factor to CArG box sequences is necessary but not sufficient to restrict gene expression to arterial smooth muscle cells.

Serum response factor (SRF) plays an important role in regulating smooth muscle cell (SMC) development and differentiation. To understand the molecular mechanisms underlying the activity of SRF in SMCs, the two CArG box-containing elements in the arterial SMC-specific SM22alpha promoter, SME-1 and SME-4, were functionally and biochemically characterized. Mutations that abolish binding of SRF to the SM22alpha promoter totally abolish promoter activity in transgenic mice. Moreover, a multimerized copy of either SME-1 or SME-4 subcloned 5' of the minimal SM22alpha promoter (base pairs -90 to +41) is necessary and sufficient to restrict transgene expression to arterial SMCs in transgenic mice. In contrast, a multimerized copy of the c-fos SRE is totally inactive in arterial SMCs and substitution of the c-fos SRE for the CArG motifs within the SM22alpha promoter inactivates the 441-base pair SM22alpha promoter in transgenic mice. Deletion analysis revealed that the SME-4 CArG box alone is insufficient to activate transcription in SMCs and additional 5'-flanking nucleotides are required. Nuclear protein binding assays revealed that SME-4 binds SRF, YY1, and four additional SMC nuclear proteins. Taken together, these data demonstrate that binding of SRF to specific CArG boxes is necessary, but not sufficient, to restrict transgene expression to SMCs in vivo.

3' Untranslated Regions↗

PubMed2001

Estrogen prevents oxidative stress-induced endothelial cell apoptosis in rats.

BACKGROUND: Estrogen replacement attenuates the increased risk of cardiovascular disease in postmenopausal women. Recent studies using an in vitro culture system have shown that estrogen inhibits endothelial cell (EC) apoptosis. The in vivo relevance of this finding, however, is not defined. To do so, we have developed a rat vascular injury model in which EC apoptosis induced by hydrogen peroxide plays a role. METHODS AND RESULTS: Intracarotid arterial administration of 0.01 mmol/L hydrogen peroxide for 5 minutes evoked EC apoptosis after 6 to 24 hours, determined by nuclear staining with Hoechst 33342, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and electron microscopy. Apoptosis was associated with EC loss and was followed by EC regeneration at 72 hours and neointima formation at 1 to 2 weeks. Estradiol replacement in ovariectomized female Wistar rats decreased the rate of apoptotic ECs by approximately 50%, assayed by nuclear morphology of en face specimens, resulting in increased remaining ECs and decreased neointima formation. Progesterone did not influence the effects of estradiol on EC apoptosis. CONCLUSIONS: These results provide new insight into the cardioprotective action of estrogen as well as a paradigm of the response-to-injury hypothesis.

Animals↗

PubMed2001

Effect of potential energy distribution on the melting of clusters.

We find that the potential energy distribution of atoms in clusters can consistently explain many important phenomena related to the phase changes of clusters, such as the nonmonotonic variation of melting temperature with size, the dependence of melting, boiling, and sublimation temperatures on the interatomic potentials, the existence of a surface-melted phase, and the absence of a premelting peak in heat capacity curves. We also find a new type of premelting mechanism in double icosahedral Pd19 clusters, where one of the two internal atoms escapes to the surface at the premelting temperature.

Journal Article↗