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Y Kang

Publications and source records attributed to Y Kang.

At least 91 records · Page 5Linked to original sources
PubMed1999

The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function.

The human Tap protein mediates the sequence-specific nuclear export of RNAs containing the constitutive transport element and is likely also critical for general mRNA export. Here, we demonstrate that a previously defined arginine-rich nuclear localization signal (NLS) present in Tap acts exclusively via the transportin import factor. Previously, transportin has been shown to mediate the nuclear import of several heterogeneous nuclear ribonucleoproteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, by binding to a sequence element termed M9. Although the Tap NLS and the hnRNP A1 M9 element are shown to compete for transportin binding, they show no sequence homology, and the Tap NLS does not conform to the recently defined M9 consensus. The Tap NLS also differs from M9 in that only the latter is able to act as a nuclear export signal. The Tap NLS is therefore the first member of a novel class of transportin-specific NLSs that lack nuclear export signal function.

Amino Acid Sequence↗
PubMed1999

Brain gamma-glutamyl cysteine synthetase (GCS) mRNA expression patterns correlate with regional-specific enzyme activities and glutathione levels.

The first and rate-limiting reaction in the formation of glutathione is catalyzed by gamma-glutamylcysteine synthetase (GCS), a dimer composed of a catalytic heavy and a regulatory light subunit. We previously found that heavy subunit GCS mRNA appears to be expressed at high levels in the hippocampus, cerebellum, and cortex of murine brain and at lower levels in the neostriatum (Kang et al. [1997] NeuroReport 8:2053). Here we report that variations in expression of light subunit GCS mRNA in murine brain resembles that of the heavy subunit mRNA with a few minor exceptions. Moreover, levels of GCS activity and glutathione levels in various brain regions appear to correspond to levels of expression of both GCS mRNA subunits. Based on these data, differences in the distribution of expression of the GCS subunits in the brain may therefore have major implications for the susceptibility of various brain regions to oxidative stress and/or mitochondrial damage.

Animals↗
PubMed1999

Analysis of the RNA binding specificity of the human tap protein, a constitutive transport element-specific nuclear RNA export factor.

The human Tap protein has been proposed to mediate Mason Pfizer monkey virus constitutive transport element (CTE)-dependent nuclear RNA export and may also play a role in global mRNA export. Here, we have used in vivo assays, in both yeast and human cells, together with in vitro assays, to further characterize the RNA binding properties of Tap, which has been proposed to contain a novel leucine-rich RNA binding motif. Using the yeast three hybrid assay, we selected RNA molecules that retain Tap binding activity from a pool of randomized CTE sequences. The recovered RNA sequences differed only minimally from the wild-type CTE yet all displayed lower affinity for Tap both in vivo and in vitro. Analysis of the RNA export activity of the recovered CTE variants revealed that Tap affinity was highly predictive of CTE biological activity. Together, these observations provide additional evidence supporting the identification of Tap as the direct cofactor for CTE function and demonstrate that RNA binding by Tap is highly sequence specific.

Animals↗
PubMed1999

A new autoantigen reactive with prediabetic nonobese diabetic mice sera.

The identification and characterization of new autoantigens would widen the knowledge of the pathogenic mechanism of insulin dependent diabetes mellitus. Screening of lambda gt11 mouse insulinoma (MIN6N8a) cell cDNA library with prediabetic nonobese diabetic (NOD) mice sera resulted in the isolation of a strong positive clone, named the clone 3-5, of 1579 nucleotides without a poly A region. After 5'-rapid amplification of the cDNA end (RACE), complete nucleotide sequence of the clone 3-5 gene consisting of 2231 nucleotides showed that the 3-5 gene had the theoretical open reading frame of 634 amino acids. However, the real antigenic protein of the clone 3-5 was only 21 amino acids long encoded by only 63 nucleotides. The 21 amino acids were expressed as a fusion protein in E. coli and purified by affinity chromatography. The purified 3-5 recombinant protein was examined for its reactivity with prediabetic NOD mice sera by immunoblotting. The only non-denatured form of the 3-5 protein showed a binding reactivity with NOD mice sera, demonstrating that the conformational epitope of 3-5 protein was important for antibody recognition. The prevalence of autoantibody reactive to the 3-5 protein was about 78% (14/18) and 46% (11/24) in prediabetic and acute diabetic NOD mice sera, respectively. However, the sera from other mouse strains such as BALB/c, ICR, C57BL/6, SJL/J, and NOD/SCID did not show a positive reactivity to the 3-5 protein, which indicated that immune reactivity against the 3-5 protein was autoimmune diabetic mouse-specific.

Amino Acid Sequence↗
PubMed1999

Induction of hyporesponsiveness to intact foreign protein via retroviral-mediated gene expression: the IgG scaffold is important for induction and maintenance of immune hyporesponsiveness.

IgG molecules can be highly tolerogenic carriers for associated antigens. Previously, we reported that recipients of bone marrow or lipopolysaccharide-stimulated B-cell blasts, both of which were retrovirally gene-transferred with an immunodominant peptide in-frame with the variable region of a murine IgG heavy chain, were rendered profoundly unresponsive to that epitope. To further investigate whether tolerance to larger molecules can be achieved via this approach and whether the IgG scaffold is important for induction and maintenance of immunological tolerance, we engineered two retroviral constructs encoding the cI lambda repressor (MBAE-1-102 and MBAE-1-102-IgG) for gene transfer. Our results show that recipients of bone marrow or peripheral B cells, transduced with the MBAE-1-102-IgG recombinant, are hyporesponsive to p1-102. In addition, the self-IgG scaffold enhanced the induction and maintenance of such an immune hyporesponsiveness. Thus, our studies demonstrate that in vivo-expressed IgG heavy chain fusion protein can be processed and presented on the appropriate MHC class II, resulting in hyporesponsiveness to that antigen and offering an additional therapeutic approach to autoimmune diseases.

Animals↗
PubMed1999

Mitogenic signaling by ATP/P2Y purinergic receptors in astrocytes: involvement of a calcium-independent protein kinase C, extracellular signal-regulated protein kinase pathway distinct from the phosphatidylinositol-specific phospholipase C/calcium pathway.

Activation of ATP/P2Y purinergic receptors stimulates proliferation of astrocytes, but the mitogenic signaling pathway linked to these G-protein-coupled receptors is unknown. We have investigated the role of extracellular signal-regulated protein kinase (ERK) in P2Y receptor-stimulated mitogenic signaling as well as the pathway that couples P2Y receptors to ERK. Downregulation of protein kinase C (PKC) in primary cultures of rat cerebral cortical astrocytes greatly reduced the ability of extracellular ATP to stimulate ERK. Because occupancy of P2Y receptors also leads to inositol phosphate formation, calcium mobilization, and PKC activation, we explored the possibility that signaling from P2Y receptors to ERK is mediated by a phosphatidylinositol-specific phospholipase C (PI-PLC)/calcium pathway. However, neither inhibition of PI-PLC nor chelation of calcium significantly reduced ATP-stimulated ERK activity. Moreover, a preferential inhibitor of calcium-dependent PKC isoforms, Gö 6976, was significantly less effective in blocking ATP-stimulated ERK activity than GF102903X, an inhibitor of both calcium-dependent and -independent PKC isoforms. Furthermore, ATP stimulated a rapid translocation of PKCdelta, a calcium-independent PKC isoform, but not PKCgamma, a calcium-dependent PKC isoform. ATP also stimulated a rapid increase in choline, and inhibition of phosphatidylcholine hydrolysis blocked ATP-evoked ERK activation. These results indicate that P2Y receptors in astrocytes are coupled independently to PI-PLC/calcium and ERK pathways and suggest that signaling from P2Y receptors to ERK involves a calcium-independent PKC isoform and hydrolysis of phosphatidylcholine by phospholipase D. In addition, we found that inhibition of ERK activation blocked extracellular ATP-stimulated DNA synthesis, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y receptors.

Animals↗
PubMed1999

Control of autoimmune diabetes in NOD mice by GAD expression or suppression in beta cells.

Glutamic acid decarboxylase (GAD) is a pancreatic beta cell autoantigen in humans and nonobese diabetic (NOD) mice. beta Cell-specific suppression of GAD expression in two lines of antisense GAD transgenic NOD mice prevented autoimmune diabetes, whereas persistent GAD expression in the beta cells in the other four lines of antisense GAD transgenic NOD mice resulted in diabetes, similar to that seen in transgene-negative NOD mice. Complete suppression of beta cell GAD expression blocked the generation of diabetogenic T cells and protected islet grafts from autoimmune injury. Thus, beta cell-specific GAD expression is required for the development of autoimmune diabetes in NOD mice, and modulation of GAD might, therefore, have therapeutic value in type 1 diabetes.

Adoptive Transfer↗
PubMed1999

Motor perseverative behavior on a line cancellation task.

OBJECTIVES: To study the behavioral and neuroanatomic characteristics of perseverative behavior encountered on a target cancellation task in patients with neglect. METHODS: Motor perseverative behavior during line cancellation task was evaluated retrospectively in 60 patients with left hemispatial neglect from right hemispheric stroke. RESULTS: More than 30% of the patients (21 of 60) showed perseveration, manifested as either repetitive cancellation of the same target (18/21) or cancelling extra lines created by the patients themselves (3/21). Neglect severity correlated positively with the frequency of perseverative errors. Perseveration was most prominent in the rightmost portion of the array. Anterior lesions or massive lesions involving anterior and posterior regions were more likely to be associated with motor perseveration than were lesions restricted to posterior areas. CONCLUSIONS: Although the mechanism of motor perseveration remains to be elucidated, our findings suggest that the combination of aberrant approach behaviors associated with frontal lobe injury and an attentional or intentional bias toward the rightsided targets might explain the behavior.

Adolescent↗
PubMed1999

The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences.

The constitutive transport element (CTE) encoded by simian type D retroviruses directs unspliced viral RNAs into a nuclear export pathway that is congruent with the pathway used by cellular mRNAs. Here, we show that quail cells are refractory to CTE function but become highly permissive upon expression of the human Tap protein, a candidate CTE cofactor. Tap contains a novel sequence-specific RNA binding domain that is sufficient for CTE binding but inadequate to support CTE function. Using microinjection assays, we have defined two NLSs and one NES in Tap. Mutational inactivation of the Tap NES, which lies outside the RNA-binding domain, not only blocks Tap function but also generates dominant-negative forms of Tap. Whereas replacement of the Tap NES with the well-defined Rev NES rescues the ability of Tap to support CTE function, this substitution also confers sensitivity to agents that block the activity of Crm1, the Rev NES cofactor. Together, these data validate Tap as the first human sequence-specific nuclear mRNA export factor and identify a novel type of NES that can support nuclear mRNA export but does not act via Crm1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Hyaluronan suppresses fibronectin fragment-mediated damage to human cartilage explant cultures by enhancing proteoglycan synthesis.

Hyaluronic acid, recently renamed hyaluronan, has been used as a therapeutic intervention in the treatment of osteoarthritis. We have reported that high-molecular-weight (800 kDa) hyaluronan is effective in blocking the catabolic action of fibronectin fragments in explant cultures of bovine cartilage and in an experimental in vivo model of damage to the rabbit knee joint. The fibronectin fragments induce catabolic cytokines in human cartilage, which, in turn, suppress proteoglycan synthesis and induce matrix metalloproteinases to decrease the proteoglycan content. Since the clinical target of high-molecular-weight hyaluronan is human cartilage, which may differ in certain ways from bovine cartilage, we tested the effect on human knee cartilage. We found that 1 mg/ml hyaluronan completely blocked fibronectin fragment-mediated decreases in proteoglycan content in five of five specimens of cartilage from the human knee. This was associated with binding of exogenous hyaluronan to the superficial surface, suppressed penetration of the fibronectin fragment into the cartilage, decreased expression for the first week in culture of one of the matrix metalloproteinases involved in cartilage degradation, matrix metalloproteinase-3, and proteoglycan synthesis rates that increased to supernormal levels. However, the appearance of the NITEGE and VDIPEN neoepitopes, indices of cartilage degradation, was not blocked but was delayed by 1 week. The addition of hyaluronan to cartilage previously damaged by the fibronectin fragments or to osteoarthritic cartilage fully restored the proteoglycan content to control levels. We conclude that hyaluronan blocked damage at least partly by blocking penetration of the fibronectin fragments and slowing matrix metalloproteinase expression. However, the major effect on blocking damage and promoting repair may be through enhanced proteoglycan synthesis, a mechanism that requires further study. Nonetheless, these data clearly demonstrate that hyaluronan completely protected human cartilage in explant culture and facilitated a full restoration of proteoglycan in damaged cartilage.

Adolescent↗
PubMed1999

Is there occult tissue ischemia in chronic end-stage liver disease?

Whether pathological oxygen supply dependency exists in patients with chronic end-stage liver disease (CESLD) is unknown, although the frequently occurring multiorgan dysfunction seen in these patients may be the result of occult tissue ischemia. In this study, 15 adult patients with CESLD were evaluated for the presence of pathological oxygen supply dependency and, thus, occult tissue ischemia before undergoing orthotopic liver transplantation. Whole-body oxygen consumption (VO2) was measured using indirect calorimetry at baseline, at reduced oxygen delivery (DO2) using positive end-expiratory pressure, and at increased DO2 using volume infusion. As a group, no significant increase or decrease in VO2 was observed with changes in DO2. However, 4 patients showed increases in VO2 of 14%, 10.8%, 9.6%, and 8.2% when DO2 was increased. The study results suggest that pathological oxygen supply dependency is present in a subset of patients with CESLD, and the existence of occult tissue ischemia is speculated.

Adult↗
PubMed1999

Soluble expression in Escherichia coli of murine endogenous retroviral transmembrane envelope protein having immunosuppressive activity.

Recently, we cloned a fragment of the endogenous murine leukemia viral envelope gene from beta cell line (MIN6N8a) as a new autoantigen gene, whose product was reactive with nonobese diabetic (NOD) mice sera. As a result of determination of nucleotide sequence, this envelope protein had the CKS-17 peptide sequence having immunosuppressive activity. To investigate the role of our cloned transmembrane envelope protein in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM) as an autoantigen or immunosuppressive modulator, a high amount of transmembrane envelope protein was essentially required. Therefore, the expression of our cloned retroviral transmembrane envelope gene was tried in Escherichia coli by a pET vector. However, the expression rate was very low (less than 2%) and most of the expressed protein was insoluble. To improve solubility, the hydrophobic transmembrane anchor domain of our envelope gene was deleted and then the expression of the hydrophilic transmembrane envelope protein was carried out by using the same pET expression system. The expressed protein was completely soluble and the expression yield was dramatically improved by around 25-fold increase. The hydrophilic transmembrane envelope protein was purified by one-step Ni-NTA affinity chromatography and then the fusion tag consisting of the six-histidine peptide and S peptide was removed by cleavage with enterokinase. The processed hydrophilic retroviral transmembrane envelope protein was still immune reactive with NOD sera and also showed immunosuppressive activity by down-regulating the Th1-type cytokine (interferon-gamma) and up-regulating the Th2-type cytokine (interleukin 10).

Amino Acid Sequence↗
PubMed1999

The hepatopulmonary syndrome.

The hepatopulmonary syndrome is a disease entity seen in association with liver failure and other disease entities. It is a devastating consequence of liver failure that results in a significant morbidity for affected patients. Currently, there are no identified medications that ameliorate the symptoms of hypoxemia in this disease state. Recent research, however, has begun to unravel the pathobiology of the vascular dilations that arise in the lungs of patients with liver failure. In this article, a compendium of current knowledge is presented, as well as the contemporary methods for identifying and treating patients.

Animals↗
PubMed1999

Li+ and muscarine cooperatively enhance the cationic tail current in rat cortical pyramidal cells.

Li+ is known to facilitate the onset of status epilepticus induced by cholinergic stimulation, although the underlying mechanisms are not clear. Under whole-cell current clamp conditions with a CsCl-based internal solution, cortical pyramidal cells display a single plateau-spike followed by a slow depolarizing afterpotential (DAP) in response to injection of a short current pulse. However, the same current pulse generated a burst of plateau-spikes after application of Li+ (2 mM) and muscarine (10 microM). As similar bursts of plateau-spikes were generated through an enhancement of the slow DAP when [K+]o was raised (Kang et al. 1998), we have investigated the effects of Li+ and muscarine on the Ca2+-dependent cationic current underlying the slow DAP, measured as the slow tail current evoked after the offset of depolarizing voltage pulses. Muscarine enhanced the amplitudes of both early and late components of the slow tail current. This effect of muscarine was markedly potentiated by Li+, while Li+ by itself affected the slow tail current only slightly. Intracellular application of heparin (0.5-1 mg/mL) suppressed the effect of muscarine in the presence of Li+. These results suggest that inositol-trisphosphate-induced Ca2+ release is involved in the cooperative enhancement of the slow tail current, and this cooperation may be one of the mechanisms underlying facilitation of the onset of epilepsy induced by these agents.

Animals↗
PubMed1999

Normative study of the Korean-California Verbal Learning Test (K-CVLT).

As a normative study of the Korean version of the California Verbal Learning Test (K-CVLT), the present study examined the K-CVLT performances of 357 neurologically intact Koreans (181 males and 176 females) who were selected by stratified sampling reflecting the recent Korean census data. The factor analysis of the K-CVLT showed that the K-CVLT was a valid and useful tool for qualitatively assessing a number of theoretically meaningful processes and strategies underlying verbal memory. Norms were developed on the 22 memory indices separately for gender and age groups. Implications for the K-CVLT's factor analytic results are discussed, and the K-CVLT's normative data is compared with that of the CVLT.

Adult↗
PubMed1999

Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum.

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.

Fluorescent Antibody Technique↗
PubMed1999

Serotonergic modulation of the hyperpolarizing spike afterpotential in rat jaw-closing motoneurons by PKA and PKC.

Intracellular recordings were obtained from rat jaw-closing motoneurons (JCMNs) in slice preparations to investigate the effects of serotonin (5-HT) on the postspike medium-duration afterhyperpolarization (mAHP) and an involvement of protein kinases in the effects. Application of 50 microM 5-HT caused membrane depolarization and increased input resistance in the most cells without affecting the mAHP, whereas not only membrane depolarization and an increase in input resistance, but also the suppression of the mAHP amplitude was induced by higher dose of 5-HT (100 or 200 microM). On the other hand, when the mAHP amplitude was increased by raising [Ca(2+)](o) from 2 to 6 mM, 5-HT-induced attenuation of the mAHP amplitude was enhanced, and even 50 microM 5-HT reduced the mAHP amplitude. This 5-HT-induced suppression of the mAHP could be mimicked by application of membrane-permeable cAMP analogue 8-Bromo-cAMP, potentiated by the cAMP-specific phosphodiesterase inhibitor Ro 20-1724 and antagonized by protein kinase A (PKA) inhibitor H89. The enhancement of the mAHP attenuation induced by 50 microM 5-HT under raised [Ca(2+)](o) was blocked by a protein kinase C (PKC) inhibitor chelerythrine, suggesting an involvement of PKC in this enhancement. On the other hand, the attenuation of the mAHP induced by PKC activator phorbol 12-myristate 13-acetate was blocked almost completely by H89, suggesting that the PKC action on the mAHP requires PKA activation. Neither 5-HT(1A) antagonist NAN-190 or 5-HT(4) antagonist SB 203186 blocked 5-HT-induced attenuation of the mAHP. We conclude that 5-HT induces dose-dependent attenuation of the mAHP amplitude through cAMP-dependent activation of PKA and that PKC-dependent PKA activation is also likely to be involved in the enhancement of 5-HT-induced attenuation of the mAHP under raised [Ca(2+)](o). Because the slope of the linear relationship between firing frequency and injected current was increased only when the mAHP amplitude was decreased by 5-HT, it is suggested that the relation between incoming synaptic inputs and firing output in JCMNs varies according to serotonergic effects on JCMNs and calcium-dependent modulation of its effects.

Animals↗