Biomedical subjects
Publications and source records attributed to M Kim.
Multiple reliable and valid disease activities indices exist and have been used successfully in longitudinal studies. However, the definition of flare, using these intruments, has not been universally decided or accepted. Because flare is one of the three major patterns of lupus activity, it will remain an important outcome measure in both longitudinal and clinical trial studies.
The intestinal permeability to hesperidin glycosides was investigated by using a cultured monolayer of Caco-2 as a model for the small intestinal epithelium. Hesperidin glycosides were added to the apical side of the monolayer, and the substances that permeated to the basolateral side were determined by HPLC. Whereas hesperidin did not permeate across the Caco-2 monolayer, probably owing to its low solubility, the hesperidin glycosides did permeate. The transepithelial transport of hesperidin glycosides occurred in time- and dose-dependent manners. The transport was observed to be energy-independent, and was inversely correlated with the transepithelial electrical resistance (TEER) of the monolayer. These results suggest that hesperidin glycosides permeate across the Caco-2 cell monolayer via the paracellular pathway.
The effects of superoxide dismutase (SOD) and catalase on the autoxidation rate of L-ascorbic acid (ASA) in the absence of metal ion catalysts were examined. The stabilization of ASA by SOD was confirmed, and the enzyme activity of SOD, which scavenges the superoxide anion formed during the autoxidation of ASA, contributed strongly to this stabilization. The stabilization of ASA by catalase was observed for the first time; however, the specific enzyme ability of catalase would not have been involved in the stabilization of ASA. Such proteins as bovine serum albumin (BSA) and ovalbumin also inhibited the autoxidation of ASA, therefore it seems that non-specific interaction between ASA and such proteins as catalase and BSA might stabilize ASA and that the non-enzymatic superoxide anion scavenging ability of proteins might be involved.
Methemoglobin (MetHb) is an oxidation product of hemoglobin in which the sixth coordination position of ferric iron is bound to a water molecule or to a hydroxyl group. The most common cause of acquired MetHb-emia is accidental poisoning which usually is the result of ingestion of water containing nitrates or food containing nitrite, and sometimes the inhalation or ingestion of butyl or amyl nitrite used as an aphrodisiac. We herein report a case of MetHb-emia after ingestion of an aphrodisiac, later identified as dapsone by gas chromatograph/mass selective detector (GC/MSD). A 24-year old male was admitted due to cyanosis after ingestion of a drug purchased as an aphrodisiac. On arterial blood gas analysis, pH was 7.32, PaCO2 26.8 mmHg, PaO2 75.6 mmHg, and bicarbonate 13.9 mmol/L. Initial pulse oxymetry was 89%. With 3 liter of nasal oxygen supplement, oxygen saturation was increased to 90-92%, but cyanosis did not disappear. Despite continuous supplement of oxygen, cyanosis was not improved. On the fifth hospital day, MetHb was 24.9%. Methylene blue was administered (2 mg/kg intravenously) and the patient rapidly improved. We proved the composition of aphrodisiac as dapsone by the method of GC/MSD.
UNLABELLED: [18F]Fluorodeoxyglucose ([18F]FDG), a glucose analogue, has been widely used for tumor imaging. To investigate the mechanisms related to [18F]FDG uptake by tumors, an experiment involving nude mice was performed. METHODS: Human colon cancer cell lines SNU-C2A, SNU-C4 and SNU-C5 were transplanted to nude mice. Using immunohistochemical staining and Western blot, the expression of glucose transporter (Glut) isoforms (Glut-1 through -5) in xenografted tumors was analyzed. For the analysis of messenger ribonucleic acid (mRNA) expression, reverse-transcription polymerase chain reaction and Northern blot were used and the enzyme activity of hexokinase in cancer tissues was measured by continuous spectrophotometric rate determination. RESULTS: [18F]FDG uptake in SNU-C4 and SNU-C5 cells was higher than in normal colon cells. Among these cells and xenografted tumors, SNU-C5 showed the highest level of [18F]FDG uptake, followed by SNU-C4 and SNU-C2A. An immunostaining experiment showed intense staining of Glut-1 in SNU-C5 tumors but somewhat faint staining in SNU-C4. SNU-C5 tumors also showed positive staining with Glut-3, although this was not the case with SNU-C2A and SNU-C4. Western blot analysis showed the expression of Glut-1 and Glut-3 in all tumors. Experiments involving Northern blot analysis and reverse-transcription polymerase chain reaction confirmed the overexpression of Glut-1 mRNA in all tumors, with the highest level in SNU-C5. The level of Glut-3 mRNA was also elevated in SNU-C5 tumors but not in SNU-C2A and SNU-C4. The enzyme activity of hexokinase did not vary among different tumors. CONCLUSION: Gluts, especially Glut-1, are responsible for [18F]FDG uptake in a nude mouse model of colon cancer rather than hexokinase activity. Increased numbers of glucose transporters at the plasma membrane of cancer cells is attributed to an increased level of transcripts of glucose transporter genes and may be a cause of increased [18F]FDG uptake, at least in colon cancer tumors.
BACKGROUND: The prognosis for gastric cancer remains dismal; novel agents that target specific molecular pathways are needed as adjuvant therapy. Camptothecin (CPT), on inhibitor of topoisomerase I, is effective in the treatment of certain solid tumors; its effects on gastric cancer are largely undefined. The purpose of this study was to (1) characterize the effects of CPT on the growth of a human gastric cancer and (2) assess potential cellular mechanisms responsible for CPT-mediated growth inhibition. METHODS: The human gastric cancer SIIA was transplanted subcutaneously into athymic nude mice. After tumors reached approximately 100 mm2, mice were randomized into 3 groups to receive either CPT (5 or 10 mg/kg) or vehicle (control) intraperitoneally 3 days per week for 3 weeks; tumor size was measured biweekly. To assess potential mechanisms of CPT-mediated inhibition, SIIA cells were treated with CPT (20 mumol/L) and cells were counted over a time course; apoptosis was assessed by Hoechst stain and DNA laddering. Expression of p53 (a tumor suppressor), p21Waf1 and p27Kip1 (cell cycle inhibitors), and Bcl-2 and Bcl-XL (antiapoptotic proteins) was determined. RESULTS: CPT (5 and 10 mg/kg) significantly inhibited tumor growth of SIIA gastric cancers compared with controls. CPT-mediated inhibition of SIIA cell proliferation was associated with an increase in apoptosis. Moreover, CPT treatment resulted in induction of p53, p21Waf1, and p27Kip1 and a decrease in Bcl-2 and Bcl-XL RNA and protein levels. CONCLUSIONS: Treatment with CPT effectively inhibited the growth of the human gastric cancer SIIA; the mechanism involved was induction of apoptosis mediated by up-regulation of p53, p21Waf1/Cip1, and p27Kip1 and the down-regulation of Bcl-2 and Bcl-XL. Novel agents such as CPT, which target specific molecular pathways, may prove clinically useful in the adjuvant treatment of gastric cancers.
OBJECTIVES: To determine (a) the long-term and short-term effectiveness and safety of pharmacological and nonpharmacological interventions for attention-deficit/hyperactivity disorder (ADHD) in children and adults and (b) whether combined interventions are more effective than individual interventions. SEARCH STRATEGY: MEDLINE (from 1966), CINAHL (from 1982), HEALTHStar (from 1975), PsycINFO (from 1984), EMBASE (from 1984), and the Cochrane Library searches were completed in November 1997. Reference lists of eligible studies and files of members of the research team and partner organizations were also searched. SELECTION CRITERIA: Studies were selected if they focused on the treatment of ADHD in humans and were published in any language as a full report in peer-reviewed journals. Studies including conditions other than ADHD were reported if separate subgroup analyses for patients with ADHD were provided. DATA COLLECTION AND ANALYSIS: Two reviewers independently extracted data for 41 variables on general characteristics, along with detailed information on interventions, outcomes, and tests. Differences were resolved by consensus or by a third researcher. Studies were not combined quantitatively because the quality of reporting was low and heterogeneity existed across outcome measures and tests. MAIN RESULTS: Seventy-eight studies (77 randomized controlled trials) met the inclusion criteria. Twenty-three studies compared drugs and showed few, if any, differences among methylphenidate (MPH), dextroamphetamine (DEX), and pemoline; studies comparing stimulants with tricyclic antidepressants (2) were inconclusive. Six studies compared drugs with nondrug interventions and showed consistently that stimulants, particularly MPH, may be more effective than nonpharmacological interventions. Twenty studies compared combination therapies with a stimulant or a nondrug intervention alone; no additional beneficial effects for combination therapies were shown. Nine studies compared tricyclic antidepressants with placebo and showed that desipramine may be more effective than placebo; no consistent effect was shown for imipramine. Fourteen studies (13 in school children and 1 in adults) evaluated long-term therapy (> or = 12 weeks) and showed a trend to general improvement regardless of treatment, but the length of followup was inadequate. MPH may reduce behavioral disturbance in children with ADHD while it is taken. Academic performance does not appear to be improved with stimulants. Twelve studies evaluated treatment in adults with ADHD. For MPH vs. placebo, the results were contradictory. Antidepressants may be effective in adults, but no beneficial effect was seen with pemoline, nicotine, or phenylalanine compared with placebo. Thirty-two reports (29 studies) evaluated adverse effects of drug therapy; many of the side effects associated with stimulant use appear to be relatively mild and of short duration and to respond to dosing or timing adjustments. Data are inadequate on the long-term effects and severity of adverse effects of most interventions. CONCLUSIONS: This report describes rigorous systematic reviews on the treatment of ADHD, ready for incorporation into evidence-based clinical practice guidelines or performance measures. The report also provides a detailed description of the many limitations of the evidence available and provides recommendations to fill existing knowledge gaps. Studies on ADHD have low reporting quality, methodological flaws, and heterogeneity across outcome measures and tests. A detailed description is included of the many limitations of the available evidence plus recommendations to fill existing knowledge gaps. Fulfilling such knowledge gaps will not be easy and will require genuine collaboration among decisionmakers.
A recombinant adenovirus containing the Von Hippel-Lindau (VHL) cDNA was constructed (AdVHL) and used to investigate the function of this tumor suppressor gene. Exposure of renal and breast cancer cell lines to AdVHL resulted in high levels of VHL mRNA and protein. AdVHL infection resulted in G1 cell cycle arrest and growth inhibition of renal and breast cancer cell lines. AdVHL-mediated cell cycle arrest was associated with induction of the cyclin-dependent kinase inhibitor (CDKI) p27Kip1 and inhibition of CDK2 and cyclinB1-dependent cdc2 activities. Nuclear run-on analyses and actinomycin D inhibition studies indicate that the induction of p27Kip1 RNA by VHL is mediated at least in part through an increase in p27Kip1 mRNA synthesis. Furthermore, [35S]methionine pulse-chase studies indicate that the increase in p27Kip expression is also regulated through posttranscriptional control mechanisms. These studies support a novel concept that the tumor suppressor gene VHL controls cell cycle progression by regulation of p27Kip1 at both the mRNA and protein levels.
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Vascular endothelial cells (ECs), forming a boundary between the circulating blood and the vessel wall, are constantly subjected to fluid shear stress due to blood flow. The aim of this study was to determine the role of the recently identified IkappaB kinases (IKKs) in shear stress activation of NF-kappaB and to elucidate the upstream signaling mechanism that mediates IKK activation. Our results demonstrate that IKKs in ECs are activated by shear stress in a rapid and transient manner. This IKK activation is followed by IkappaB degradation and NF-kappaB translocation into the nucleus. Transfection of plasmids encoding catalytic inactive mutants of IKKs, i.e. hemagglutinin (HA)-IKKalpha(K44M) and HA-IKKbeta(K44A), inhibits shear stress-induced NF-kappaB translocation. In addition, constructs encoding antisense IKKs, i.e. HA-IKKalpha(AS) and HA-IKKbeta(AS), attenuate shear stress induction of a promoter driven by the kappaB enhancer element. Preincubation of the EC monolayer with a monoclonal anti-alphavbeta3 integrin antibody (clone LM609) attenuates shear stress induction of IKK. Inhibition of tyrosine kinases by genistein causes a similar down-regulating effect. These results suggest that the integrin-mediated signaling pathway regulates NF-kappaB through IKKs in ECs in response to shear stress.
A simple and rapid assay method for three stimulant drugs (amphetamine, methamphetamine, and dimethamphetamine) in human urine using solid-phase microextraction was developed. In solid-phase microextraction, the drugs were equilibrated between the adsorbent coated-fiber and aqueous sample matrix. After adsorption of the analytes, the fiber was directly transferred to the injector of a gas chromatograph, where the analytes were thermally desorbed and subsequently separated by the gas chromatograph and detected by mass spectrometer. The solid-phase microextraction method, which did not require solvents, was found to be a fast and simple analytical method. We optimized the solid-phase microextraction technique, for factors such as the NaCl salt effect (30%), pH effect (pH=12.4), equilibration time (30 min), desorption time (1 min) and coated-fiber type (100 microm poly(dimethylsiloxane)) and detected the stimulants in human urine, obtained from human subjects. The detection limits of each drug were below 1-10 ng/ml. The developed method can be applied to the abused drug test.
The human insulin-like growth factor II gene is regulated in a development-dependent manner and is not expressed in most adult tissues. However, high levels of insulin-like growth factor II mRNA are detected in many human tumors including rhabdomyosarcoma, an embryonal tumor of skeletal muscle origin. In this study, we demonstrate that the developmentally regulated transcription factor AP-2 is expressed at higher levels in human fetal skeletal muscle and rhabdomyosarcoma cells compared to human adult skeletal muscle. Endogenous insulin-like growth factor II mRNA derived from the P3 as well as transfected P3 promoter activity were modestly and consistently increased to the same extent following treatment of the rhabdomyosarcoma cell line RD with forskolin, a compound implicated in AP-2 transactivation. This effect of AP-2 on increased transcriptional activity was confirmed by nuclear run-on assays. Expression of AP-2B, a dominant-negative inhibitor of AP-2, suppressed the P3 promoter activity in AP-2 expressing RD cells. Furthermore, five AP-2 protected regions corresponding to six AP-2 specific binding sites were detected in the insulin-like growth factor II P3 promoter. These data together suggest that AP-2 may contribute to the high expression of IGF-II in rhabdomyosarcoma cells.
Partial PTK6 (also known as Brk) cDNA was initially isolated by reverse transcription-PCR of normal human melanocyte mRNAs and the full-length cDNA encodes a non-receptor protein tyrosine kinase with an SH3 domain, an SH2 domain, and a kinase catalytic domain. We have cloned the human PTK6 gene by screening human genomic lambda libraries using the full-length PTK6 cDNA as probe. The human PTK6 gene consists of 8 exons encompassing 8.8 kb and all the splicing junctions followed the conserved GT/AG rule. Coding sequence of the PTK6 gene was identical to that of the cDNA cloned from T-47D, human breast tumor cell line. Although the amino acid sequence of the PTK6 polypeptide showed the strongest homology to those of the Src family members of protein tyrosine kinases, exon-intron boundaries of the PTK6 gene were quite different from those of the Src family genes, which are evolutionarily conserved. The 813-bp 5'-flanking sequence of the PTK6 gene upstream of a luciferase reporter gene conferred significant promoter activity, at approximately 60% level of the SV40 promoter, in transient expression assays into MCF-7, human breast tumor cell line. PTK6 mRNA was expressed at very high level in colon and at high levels in small intestine and prostate, and at low levels in some tested fetal tissues. These results suggest that PTK6 constitutes an evolutionarily distinct family of non-receptor protein tyrosine kinases and may function as an intracellular signal transducer in specific tissues.
We have utilized the differential display PCR method to isolate transcripts expressed during early embryogenesis of Xenopus laevis. Among many transcripts that have been found to be expressed differentially during the development, one transcript which was expressed predominantly in the unfertilized egg, was isolated as a full-length cDNA and the sequence was determined. This cDNA contained a predicted size of 198 amino acids. A search of the GenBank database revealed that the predicted amino acid sequence of the cDNA is highly homologous-87.8% identical-to the recently identified human protein, HsYKT6, a prenylated vesicle associated-SNARE ((soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor)). Thus we have named the gene as Xsnare1. RT-PCR analysis showed that the Xsnare1 mRNA expressed throughout the oogenesis, in egg and in the early phase of embryogenesis and the level of expression declined after gastrulation. These results suggest that the Xsnare1, a maternally active, putative Xenopus homologue of prenylated v-SNARE, is a developmentally regulatory gene and may be play a role in the process of the early development of Xenopus laevis.
Low-intensity fluorescence of rhodamine-123 (Rh-123) discriminates a quiescent hematopoietic stem cell (HSC) population in mouse bone marrow, which provides stable, long-term hematopoiesis after transplantation. Rh-123 labels mitochondria with increasing intensity proportional to cellular activation, however the intensity of staining also correlates with the multidrug resistance (MDR) phenotype, as Rh-123 is a substrate for P-glycoprotein (P-gp). To address the mechanisms of long-term repopulating HSC discrimination by Rh-123, mouse bone marrow stem and progenitor cells were isolated based on surface antigen expression and subsequently separated into subsets using various fluorescent probes sensitive to mitochondrial characteristics and/or MDR function. We determined the cell cycle status of the separated populations and tested for HSC function using transplantation assays. Based on blocking studies using MDR modulators, we observed little efflux of Rh-123 from HSC obtained from young (3- to 4-week-old) mice, but significant efflux from HSC derived from older animals. A fluorescent MDR substrate (Bodipy-verapamil, BodVer) and Rh-123 both segregated quiescent cells into a dim-staining population, however Rh-123-based separations resulted in better enrichment of HSC function. Similar experiments using two other fluorescent probes with specificity for either mitochondrial mass or membrane potential indicated that mitochondrial activation is more important than either mitochondrial mass or MDR function in defining HSC in young mice. This conclusion was supported by morphologic studies of cell subsets separated by Rh-123 staining.
The accurate function of C5 protein, the protein component of Escherichia coli RNase P, is uncertain in vivo. A controllable expression system for C5 protein was constructed which can be used to investigate effects of C5 protein on various cellular functions including biosynthesis of RNase P in vivo. The semisynthetic rnpA gene encoding C5 protein was fused to the tac promoter of the pKK223-3 expression vector. This tac promoter expression system produced a high level of C5 protein upon induction with isopropyl-beta-D-thiogalacto-pyranoside. When the overexpressed C5 protein was purified and used for reconstitution of RNase P, the reconstituted enzyme was active. The N-terminal amino acid of the overexpressed C5 protein was leucine specified by the second codon of the rnpA gene. The more controllable expression system was constructed by introducing the lacIq gene into the vector sequence itself.
We constructed an adenoviral vector containing human p16 cDNA in order to evaluate the cytotoxic effects of exogenous p16 expression on cancer cell proliferation and to explore the potential use of p16 in cancer gene therapy. Following infection of human breast (MCF-7, MDA-MB-231, and BT549), osteosarcoma (U-2 OS and Saos-2), cervical (C33a), and lung cancer (H358) cell lines with the recombinant adenovirus Adp16, high levels of p16 expression were observed in all cell lines. Cancer cell lines which were mutant or null for p16 but wild-type for the retinoblastoma gene product (pRb) (MCF-7, MDA-MB-231, BT549 and U-2 OS) were 7-22-fold more sensitive to the cytotoxic effects of Adp16 than to a control virus. In contrast, cancer cell lines which were wild-type for p16 but mutant or null for pRb (Saos-2, C33a and H358) were <threefold more sensitive to Adp16 when compared to a control virus. Analysis of 5-bromodeoxyuridine incorporation into DNA following infection with Adp16 showed a loss of S phase in those cell lines which were null or mutant for p16 but expressed a functional pRb. This cell cycle arrest was associated with binding of the p16 protein to cyclin-dependent kinase 4 and dephosphorylation of pRb. In contrast, human cancer cell lines expressing a wild-type p16 and a mutant pRb or no pRb showed no substantial loss of S phase following Adp16 infection. Based on these studies, we conclude that p16-mediated cytotoxicity is tightly associated with the presence of functional pRb in human cancer cells, and that tumor cells which are mutant or null for p16 are candidates for Adp16 mediated cancer gene therapy.
Formation cyclic boronates for beta-blockers, by use of triethylamine (TEA) and pyridine as catalysts, gives more effective product yield. Eleven beta-blockers, formed by cyclic boronation with n-butylboronic acid and TEA, produced higher yields than by on-column thermal reaction and seven beta-blockers were shown to give the highest yields in the phenyl cyclic boronation with pyridine or TEA by on-column thermal and general reaction. The phenyl cyclic boronate of nadolol produced one peak in the chromatogram and the n-butyl cyclic boronate showed two peaks. On-column derivatization and n-butylboronic acid and pyridine was effective in saving analysis time and for convenience, even though some n-butyl cyclic boronates by cyclic boronation with TEA gave a better yield. In n-butyl cyclic boronation with pyridine by on-column thermal reaction the detection limits were 0.1 to 4 ng/microL in urine with a signal-to-noise ratio of 10:1.