Biomedical subjects

K Kim

Publications and source records attributed to K Kim.

At least 109 records · Page 6Linked to original sources

PubMed2001

A functional retinoic acid response element (RARE) is present within the distal promoter of the rat gonadotropin-releasing hormone (GnRH) gene.

We previously demonstrated that all-trans-retinoic acid (all-trans-RA) regulates gonadotropin-releasing hormone (GnRH) release and gene expression in rat hypothalamic fragments and GT1-1 neuronal cells. Promoter analysis of rat GnRH gene revealed that the enhancing effect of all-trans-RA on GnRH transcription is mediated by cis-elements localized within --1640/--1438 of the rat GnRH promoter. In the present study, we attempted to localize functional retinoic acid response elements (RAREs) within the all-trans-RA-responsive region of the rat GnRH gene. Sequence analysis showed that there exist three putative repeats of AGGTCA-related sequences (--1637/--1617, --1579/--1562, and --1494/--1470) within this promoter sequence. Among them, only the --1494/--1470 sequence could compete the specific binding of GT1-1 nuclear extracts to the consensus RARE (direct repeat of AGGTCA with a 5-bp spacer, DR-5) and vice versa in electrophoretic mobility shift assays. In addition, like consensus RARE, the --1494/--1470 sequence could confer all-trans-RA responsiveness when inserted into the upstream region of SV40 promoter. Treatment of GT1-1 cells with all-trans- or 9-cis-RA increased the specific bindings of GT1-1 nuclear extracts to the consensus RARE and to the --1494/--1470 sequence while not affecting the specific binding to the cAMP response element (CRE). Both retinoids induced RARbeta gene expression in GT1-1 cells. The --1494/--1470 sequence (5'-TCTTAGGACTCTGTGTGACCTAAGA) is similar to the direct repeat of TGACCT (complementary sequence of AGGTCA) with a spacer of 5 bp (i.e. DR-5 in the reverse orientation). A mutation of the second core recognition motif of the --1494/--1470 sequence to a more divergent one from consensus RARE (from TGACCT to TTACAT) abolished the responsiveness to all-trans-RA, whereas a mutation of first core recognition motif to a more TGACCT-like sequence (from AGGACT to TGAACT) increased the responsiveness to all-trans-RA. These results indicate that the --1494/--1470 sequence is indeed a weak but functional RARE of the modified DR-5 type. Taken together, these data indicate that all-trans-RA enhances GnRH transcription via functional RARE present in the distal region of the GnRH promoter.

Animals↗

PubMed2001

Thymic emigrants isolated by a new method possess unique phenotypic and functional properties.

T cells that emigrate from the thymus have primarily been studied in vivo using fluorescent dye injection of the thymus. This study examined the properties of thymocytes that emigrate from cultured thymic lobes in organ culture. Under these conditions, thymic emigrants displayed the expected phenotype, that of mature thymocytes expressing high levels of T-cell receptor (TCR-alphabeta) and either CD4 or CD8, and were observed to emigrate within 24 hours of positive selection. Emigration was inhibited by cytochalasin D, pertussis toxin, or Clostridium difficile toxin B, implicating an active motility process. Most of the surface markers on alphabeta-thymic emigrants (Thy1, CD44, CD69, CD25, leukocyte functional antigen-1, intercellular adhesion molecule-1, alpha(4)-integrin, alpha(5)-integrin, CD45, and CD28) were expressed at a surface density similar to that on mature intrathymic cells and peripheral splenic T cells. Heterogeneous expression of L-selectin and heat-stable antigen (HSA) suggested that subsets emerge from the thymus with a commitment to different migration patterns. The only marker on emigrants not found on either intrathymic cells or mature spleen T cells was CTLA-4, which could dampen the response of emigrants to peripheral antigens. Antigen responsivenes measured in vitro against allogeneic dendritic cells showed a proliferative response comparable to that of splenic T cells. In vivo, however, thymic emigrants failed to induce an acute graft-versus-host reaction in allogeneic severe combined immunodeficiency recipients. This suggests that a mechanism operating in vivo, perhaps tolerance or migration pattern, attenuates the response of emigrants against antigens that did not induce their deletion in the thymus.

Abatacept↗

PubMed2001

Analysis of exonic splicing enhancers in the mouse gonadotropin-releasing hormone (GnRH) gene.

We have previously reported that efficient removal of the first intron (intron A) of gonadotropin-releasing hormone (GnRH) pre-mRNA is the prerequisite event for post-transcriptional regulation. In the present study, using an in vitro HeLa splicing system, we examined the enhancing activities of exonic elements for GnRH pre-mRNA splicing. While not excised by exon 2 alone, intron A was efficiently excised when exon 3 and/or exon 4 was combined with exon 2, suggesting the presence of exonic splicing enhancers (ESEs) in exons 3 and 4. Purine-rich sequences located in the border of exons 2 and 3 (denoted ESE3) and in exon 4 (ESE4) revealed strong splicing enhancing activities. Mutation in ESE3 decreased pre-mRNA splicing, while mutation in purine-rich sequences in exon 2 did not. We further analyzed the functional activity of ESE4 by mutations or deletions of the ESE4 sequence that consists of three purine-repeats separated by two spacers and a putative hairpin constructing sequence. An UV cross-linking assay using the RNA sequence of ESE4 examined the presence of ESE4-specific binding proteins in the nuclear extracts from GT1 hypothalamic GnRH neurons. Collectively, this study indicates that a sequence context of ESE4 and its binding proteins may be crucially involved in enhanced GnRH pre-mRNA splicing. However, it should be further clarified as to which splicing factor(s) is responsible for ESE4-dependent GnRH pre-mRNA splicing.

Animals↗

PubMed2001

Proteasome inhibitors sensitize human vascular smooth muscle cells to Fas (CD95)-mediated death.

It was investigated whether proteasome activity was implicated in susceptibility of human vascular smooth muscle cells (VSMCs) to Fas-mediated death. Human fetal aorta smooth muscle cells were treated with agonistic anti-Fas antibody (CH11) and proteasome inhibitors (MG115 or MG132) and then cell death was determined by morphology, viability, and DNA fragmentation. The present study reports that: (a) crosslinking of Fas receptor with anti-Fas antibody in the presence of proteasome inhibitor-induced death and DNA degradation in human VSMCs that were blocked by caspases inhibitor z-DEVD.fmk; (b) cotreatment with anti-Fas antibody and proteasome inhibitor activated caspase-3; (c) proteasome inhibitors did not influence expression of procaspase-8, procaspase-3, c-FLIP, and Bcl-2; and (d) proteasome inhibitors up-regulated Fas and FADD. The data indicate that proteasome activity is important in survival of VSMCs and provide the first evidence that proteasome is involved in Fas signal transduction. The present study proposes novel mechanism(s) by which VSMCs become susceptible to FasL.

Antibodies↗

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

Central venous device-related infection and thrombosis in patients treated with moderate dose continuous-infusion interleukin-2.

BACKGROUND: This study was performed to determine the incidence of central venous device-related blood stream infection and thrombosis in patients treated with moderate dose continuous-infusion interleukin-2 (IL-2). METHODS: The records of 160 consecutive patients treated at the University of Wisconsin Hospital and Clinics, between June 1990 and June 1997, with moderate dose continuous-infusion IL-2 (IL-2 [1.5-3.0 x 10(6) U/m(2)/day] Hoffman-LaRoche, Nutley, NJ or IL-2 [4.5 x 10(6) U/m(2)/day] Chiron Corporation, Berkley, CA) were reviewed retrospectively. The majority of patients had metastatic melanoma (78 patients) or renal cell carcinoma (70 patients). All of the patients had a surgically implanted central venous device placed before starting IL-2 therapy; 89% of these were cuffed Hickman catheters. Eighty-four patients received 1 mg of warfarin per day as prophylaxis against device-related thrombosis; none received periinsertion prophylactic antibiotics. RESULTS: Twenty-one patients (13%) developed central venous device-related bloodstream infection (DRBSI) during the study period, a rate of 2 DRBSI per 1000 device-days. DRBSIs were associated with the type of immunotherapy given with IL-2 (P = 0.01) and with thrombosis (odds ratio, 4.1; 95% confidence interval, 1.5-11.4; P = 0.008) but not with patient gender, type of cancer, duration of the central device, or site of device placement. Twenty-six patients (16%) developed central venous device-related thrombosis (DRT) during immunotherapy. Low dose warfarin did not appear to prevent thrombosis. Device-related thrombosis was associated with DRBSI but not with patient gender, type of cancer, type of device, duration or location of device, or concomitant immunotherapy. CONCLUSIONS: Central venous DRBSI and DRT are significant complications that can occur during moderate dose continuous-infusion IL-2 therapy. The risk of DRBSI appears lower than the risk reported with high dose IL-2 therapy by previous investigators. The risk of DRT appears to be higher than the risk reported for patients with similar devices but not given IL-2. Low dose warfarin did not prevent DRT when started after device placement.

Adult↗

PubMed2001

Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel.

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.

Animals↗

PubMed2001

Regiocontrol of radical cyclization by Lewis acids. Efficient synthesis of optically active functionalized cyclopentanes and cyclohexanes.

[figure: see text] Treatment of alpha-alkylidenelactones 3a-d with Bu3SnH and a catalytic amount of Et3B effected a 5-exo radical cyclization preferentially to provide the corresponding 1 and 2 in a ratio of 70:30 to 100:0. Meanwhile, the reaction of 3a and 3b in the presence of Et2AlCl proceeded via a 6-endo cyclization pathway predominantly to afford 2a and 2b with 90% and 92% regioselectivity, respectively.

Journal Article↗

PubMed2001

Metabolism of flupyrazofos in the isolated perfused rat liver.

To investigate the hepatic metabolism of the new insecticide flupyrazofos [O,O-diethyl O-(1-phenyl-3-trifluoromethylpyrazol-5-yl) phosphorothioate], isolated rat liver was perfused with flupyrazofos under single-pass conditions. In outflow perfusate and bile, 1-phenyl-3-trifluoromethyl-5-hydroxyprazole (PTMHP), PTMHP-sulfate and PTMHP-glucuronide conjugates were identified as the metabolites of flupyrazofos. However, O,O-diethyl O-(1-phenyl-3-trifluoromethylpyrazol-5-yl) phosphate (flupyrazofos oxon) was not detected. A HPLC method with UV detection was used to investigate the hepatic disposition of flupyrazofos and its metabolite PTMHP. The concentrations of flupyrazofos, PTMHP and PTMHP conjugates in outflow perfusate reached steady-state levels within 20 min after commencing perfusion of 7.3 microM flupyrazofos. At steady state, the mean extraction ratio of flupyrazofos was 0.93 (+/- 0.01) and clearance was 26.1 (+/- 0.2) ml min-1 which nearly approached perfusate flow rate (28 ml min-1). PTMHP accounted for 55.7 (+/- 5.8)% of eliminated flupyrazofos and was recovered as unchanged PTMHP, PTMHP-sulfate and PTMHP-glucuronide in the bile as well as the outflow perfusate.

Animals↗

PubMed2001

Tyrosine phosphorylation translocates beta-catenin from cell-->cell interface to the cytoplasm, but does not significantly enhance the LEF-1-dependent transactivating function.

beta-catenin plays an essential role in cells, not only as a cadherin-associated complex, but also as a signaling molecule in the nucleus. Tyrosine phosphorylation of beta-catenin has been shown to correlate with tumorigenesis, cell migration, and developmental processes. However, its exact effects on downstream targets in the nucleus are not yet clear. In this study, we used HCT-15 colon carcinoma and NIH 3T3 fibroblasts as models to investigate the effects of a phosphotyrosine phosphatase (PTPase) inhibitor on the localization of beta-catenin, the binding affinity to LEF-1 (Lymphoid Enhancer Factor), and on LEF-1-dependent transactivation function. Treatment with a PTPase inhibitor, pervanadate, increased the tyrosine phosphorylation of beta-catenin in a time-dependent manner and led to its relocation from cell-cell interfaces to the cytoplasm. This phosphorylation/dephosphorylation of beta-catenin does not require its presence at cell-cell interfaces. However, tyrosine phosphorylation of beta-catenin does not change its binding affinity to LEF-1 nor enhance cyclin D1 transactivation, a nuclear target of beta-catenin/LEF-1. This result suggests that tyrosine phosphorylation of beta-catenin has effects on the binding to cadherins in the cytoplasm but not on its LEF-1-dependent transactivating function in the nucleus.

3T3 Cells↗

PubMed2001

New evidence that nuclear import of endogenous beta-catenin is LEF-1 dependent, while LEF-1 independent import of exogenous beta-catenin leads to nuclear abnormalities.

The once accepted idea that LEF-1 transports beta-catenin into nuclei has recently been challenged by experiments using exogenous beta-catenin. Here, we investigated the effects of beta-catenin and LEF-1 on nuclear import of beta-catenin using different combinations of exogenous and endogenous molecules over longer lengths of time than previously studied. Nuclear beta-catenin is not detectable in corneal fibroblasts and epithelia or NIH 3T3 and MDCK cells. In LEF-1 transfections, we show that the B-box of LEF-1 is required to move cytoplasmic endogenous beta-catenin into the nuclei of such cells, proving that LEF-1 does transport endogenous beta-catenin into nuclei. Moreover, transfection of uveal melanoma cells with B-box deficient LEF-1 inhibits nuclear import of beta-catenin by endogenous LEF-1. However, the movement of overexpressed exogenous beta-catenin into nuclei is unaffected by the presence or absence of LEF-1 and forms abnormal nuclear aggregates that are a prelude to subsequent apoptosis. We conclude that nuclear transport of exogenous beta-catenin independently of LEF-1 has questionable physiological significance.

Active Transport, Cell Nucleus↗

PubMed2001

One micrometer resolution NMR microscopy.

We obtained a magnetic resonance image of 1 microm resolution and 75 microm(3) voxel volume for a phantom filled with hydrocarbon oil within an hour at 14.1 T. For this work, a specially designed probe with a high sensitivity RF coil and gradient coils generating over 1000 G/cm was built. The optimal pulse sequence was analyzed in consideration of the bandwidth, diffusion coefficients, and T(1) and T(2) relaxations of the medium. The system was applied to the in vivo imaging of a geranium leaf stem to get the images of 2 microm resolution and 200 microm(3) voxel volume.

Magnetic Resonance Spectroscopy↗

PubMed2001

Evaluation of lymph node metastases in squamous cell carcinoma of the esophagus with positron emission tomography.

BACKGROUND: Previous studies suggest positron emission tomography (PET) may improve staging accuracy of esophageal cancer compared with conventional methods, especially in detecting occult distant metastases. We evaluated the accuracy of PET in the detection of lymph node metastasis prospectively with pathologic findings. METHODS: Fifty-three patients with squamous cell carcinoma underwent whole-body PET scan and chest computed tomography (CT). The findings of PET and chest CT of 50 patients who underwent curative esophagectomy with radical lymph node dissection were compared with the pathologic findings. RESULTS: Among 53 primary esophageal tumors, PET detected 51 (96.2%) and CT detected 49 (92.5%) tumors correctly. Nodal metastases were present in 108 of 436 dissected lymph node groups. PET detected 56 metastatic node groups (51.9% sensitivity, 94.2% specificity, 83.7% accuracy), compared with CT, which detected 16 (14.8% sensitivity, 96.7% specificity, 76.6% accuracy; sensitivity: p < 0.005). CONCLUSIONS: PET was more sensitive than CT in the detection of nodal metastases and may improve staging of squamous cell carcinoma of the esophagus.

Adult↗

PubMed2001

Mechanisms of female urinary continence under stress: frequency spectrum analysis.

Intravesical and urethral pressure signals during cough and Valsalva maneuvers for 15 continent women were analyzed with frequency spectrum analysis. Clear modulation of the urethral pressure changes by the intravesical pressure rise during stress maneuvers was demonstrated in the frequency bands of 14 and 7 Hz for cough and Valsalva, respectively. The linearity between the urethral and intravesical pressure signals was strong for cough, but relatively weaker for Valsalva. The observed linearity lead to the formulation of a modified continence equation to mathematically quantify stress leak point pressure (sLPP): sLPP=MUCP/(1-alpha1)+RBP. This algebraic equation demonstrated that sLPP depends on pressure transmission, resting bladder pressure, and maximum urethral closure pressure. The equation was validated with excellent theoretical predictions for the 15 continent subjects (R(2)=0.98 and 0.97 for cough and Valsalva leak point pressure, respectively) and good but somewhat weaker predictions for 46 stress incontinent women (R(2)=0.79 and 0.48, respectively). It has been shown that pressure transmission plays the most important role in female continence function, while it may be attributable to passive structural origin as evidenced by the minimal time delay between the two pressure signals, in the order of a few milliseconds. It can be concluded that coughing seems to have a more mechanical, rather than neuromuscular basis for its signal dynamics. This study suggests that a complete assessment of female stress continence function requires comprehensive urodynamic information in terms of pressure transmission, maximum urethral closure pressure, and resting bladder pressure.

Adult↗

PubMed2001

Microbial reduction of perchlorate in pure and mixed culture packed-bed bioreactors.

Perchlorate (ClO4-) has been detected in a large number of surface and ground waters in the US. Due to health concerns of perchlorate in drinking water, the California Department of Health Services has established a provisional action level of 18 microg/L. Several microbial isolates have been obtained capable of microbiological perchlorate reduction through cell respiration, but few of these have been tested for perchlorate removals to these low levels. The feasibility of using one isolate (KJ) for water treatment was tested in a packed-bed bioreactor by comparing minimum detention times necessary to achieve complete removal of perchlorate. Perchlorate was reduced approximately from 20 mg/L to non-detectable (< 4 microg/L) levels in acetate-fed columns inoculated with KJ or mixed cultures. The complete conversion of perchlorate to chloride was demonstrated by a stoichiometric ratio of perchlorate to chloride of 1.0 +/- 0.14. Perchlorate removal to non-detectable levels required a minimum empty bed contact time (EBCT) of only 2.1 min for the column inoculated with KJ, vs. 31 min for the mixed culture column. Acetate was used at a molar ratio of C2H3O2-/ClO4- of 2.9 (n = 6) for the mixed culture, while more than twice as much acetate was consumed on average (6.6 +/- 2.0, n = 156) by the pure culture. These results demonstrate that detention times of packed-bed bioreactors can be substantially reduced using isolate KJ, but that larger concentrations of acetate will be necessary to reduce perchlorate to low levels necessary for drinking water.

Absorption↗

PubMed2001

Induction of CTL responses and identification of a novel epitope of hepatitis B virus surface antigens in C57BL/6 mice immunized with recombinant vaccinia viruses.

MHC class I-restricted cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV) surface antigens (HBsAg) has been suggested to play essential roles in viral clearance and pathogenesis of HBV-induced hepatitis. In the present study, we analyzed CTL responses to endogenously synthesized or exogenously introduced HBsAg in C57BL/6 mice (H-2(b)). We show that the endogenously synthesized surface antigens of adr-type HBV encoded by recombinant vaccinia virus efficiently elicit CTL responses in C57BL/6 mice previously defined as non-responders to vaccinia-HBV immunization. We also show that two peptides, S(179-186) (FVQWFVGL) and S(208-216) (ILSPFLPLL), serve as effective motifs for CTL response in H-2(b) system after in vitro restimulation of the primed T cells with either of the two synthetic peptides. S(208-215) has recently been identified as a CTL epitope which could be produced by exogenous pathway only, in contrast to the current result, while S(179-186) appeared a novel epitope for CTL response. In addition, we show that soluble HBsAg also elicits CTL responses in H-2(b) mice upon in vitro restimulation with the two peptides, although less efficiently compared with the recombinant vaccinia viruses. These findings may provide an efficient experimental system for studying H-2(b)-restricted immune responses against endogenously synthesized and exogenously introduced HBsAg.

Animals↗

PubMed2001

Malignant esophagogastric junction obstruction: palliative treatment with an antireflux valve stent.

The authors assessed the efficacy of an antireflux valve stent in the palliation of malignant esophagogastric junction (EGJ) obstruction after in vitro testing of the stent. Seventeen patients with inoperable malignant EGJ obstruction were treated. Antireflux valves, made of three polyurethane leaflets, were attached to the distal part of the stent to prevent reflux. When the flow rate of normal saline was 100 mL/sec in the forward direction, the valve fully opened at a pressure of 10 mm Hg. When the flow rate of normal saline was 0.35 mL/sec in the backward direction, the valve nearly completely closed at a pressure of 10 mm Hg. Stent placement was successful in all patients without complications. The median dysphagia score decreased significantly, from 3.0 (dysphagia to liquids) to 1.0 (dysphagia to normal solid food) (P < .0005). No patients experienced reflux symptoms. There was one case of stent migration. A valve stent that can prevent major reflux is an effective device for the palliation of malignant EGJ obstruction.

Aged↗

PubMed2001

A case of disseminated extrahepatic hepatocellular carcinoma after US-guided biopsy and percutaneous ethanol injection therapy.

A case of disseminated extrahepatic hepatocellular carcinoma (HCC) occurring after ultrasound (US)-guided biopsy and percutaneous ethanol injection therapy is presented. A 72-year-old man with hepatitis-C-virus-related cirrhosis underwent percutanous ethanol injection therapy (PEIT) two times with complete remission: the first for moderately-differentiated HCC in segment six (S6), and the second for well-differentiated HCC in another part of S6. Imaging studies including carbon dioxide (CO(2))-US angiography, incremental computed tomography, and dynamic magnet resonance imaging showed that both HCCs were hypovascular. Twenty-one months after the first PEIT and 7 months after the second, a 5.5x4.5 cm extrahepatic mass interfaced with S6 of the liver was detected by imaging studies. The patient underwent surgery for extrahepatic HCC. Grossly, the main tumor was 5.5x4.5 cm with capsule and septum; the disseminated tumors were detected on the surface of the liver, including the right diaphragm and the falx ligamentosa. Histologically, it was moderately- to poorly-differentiated HCC, which, although not attributed to direct track seeding, was suspected of being induced by the percutaneous US-guided biopsy procedure or by PEIT, irrespective of a hypovascular tumor. Further studies may provide insight into the risk factor engendered by these procedures.

Journal Article↗