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Biomedical subjects

Y S Kim

Publications and source records attributed to Y S Kim.

At least 19 recordsLinked to original sources
PubMed1995

Cloning and characterization of rat pancreatic beta-cell/liver type glucose transporter gene: a unique exon/intron organization.

Four overlapping lambda genomic clones encoding rat pancreatic beta-cell/liver type glucose transporter (GLUT2) have been isolated and characterized. The gene is about 35 kb long and contains 14 exons and 13 introns. Contrary to the exon 1 of the human or mouse counterpart, the rat GLUT2 gene has three additional noncoding exons which were identified by 5'-RACE and all four were designated exon 1a, 1b, 1c, and 1d. The intron sequences bordering the splice site junctions generally follow the GT/AG rule except for one intron which begins with GC. The exon sequences determined from genomic DNA sequencing showed some differences when compared to the published rat GLUT2 cDNA. Transcription initiation site was determined by primer extension and located 661 bp upstream of the ATG translation initiation codon. Several potential binding sites for transcription factors such as C/EBP, Sp1, AP1, HNF-5, and UPE were observed and they may be responsible for the regulation of GLUT2 gene expression. The promoter region of rat GLUT2 showed little homology when compared with those of human or mouse. However, striking sequence identity (84%) was found when the adjacent intron regions flanking exon 1c were compared with the -970/-721 region of the mouse GLUT2 promoter. A series of deleted mutant constructs of the putative promoter region linked to the CAT reporter gene showed promoter activity in the primary hepatocyte culture. The region containing -4542/+240 showed the highest CAT activity and further deletion of the region showed gradual decrease in CAT activity.

Alternative Splicing↗
PubMed1995

Regional expression and dietary regulation of rat small intestinal peptide and amino acid transporter mRNAs.

RT-PCR was used to obtain rat small intestinal cDNAs for two peptide transporters, showing conclusively for the first time that both are present in normal intestinal mucosa. Sequencing of these cDNAs showed them to be highly homologous and similar to two different types of peptide transport proteins from either colorectal carcinoma cells (Caco-2) or human and rabbit intestine. An even distribution profile of steady state levels of mRNA for both peptide transporters was observed along the longitudinal axis of small intestine. Both were upregulated in the distal regions of intestine by a high protein diet. Also, high levels of the rat high affinity glutamate transporter EAAC1 were observed in the distal intestine. These results suggest that the distal regions of small intestine play an important role in the absorption of some amino acids and peptides. Furthermore this area appears to be a primary site where dietary-induced changes in peptide and amino acid transport occurs.

Amino Acid Sequence↗
PubMed1995

Human dipeptidyl peptidase IV gene promoter: tissue-specific regulation from a TATA-less GC-rich sequence characteristic of a housekeeping gene promoter.

The dipeptidyl peptidase IV gene encodes a plasma-membrane exopeptidase that is highly expressed in small intestine, lung and kidney. In order to better understand the mechanisms responsible for this tissue-specific expression we cloned, sequenced and functionally characterized the 5'-flanking region of the human dipeptidyl peptidase IV gene. The first 500 bases of the 5'-flanking sequence constituted an unmethylated CpG island, contained several Sp1-binding sites and lacked a consensus TATA box, all characteristics of gene promoters lacking tissue-specific expression. RNase-protection analysis using both small intestinal and Caco2 cell RNA indicated that the dipeptidyl peptidase IV transcript was initiated from no fewer than six major and 12 minor start sites. The 5'-flanking sequence also exhibited functional promoter activity in transient transfection experiments. Here, various lengths of the sequence were cloned upstream of a luciferase gene and introduced into cultured cells using lipofectin. A region located between bases -150 and -109 relative to the start of translation was found to be important for high-level promoter activity in both Caco2 and HepG2 cells. Moreover, Caco2 cells and HepG2 cells, which express high levels of dipeptidyl peptidase IV activity, exhibited much higher normalized luciferase activity after transfection than did 3T3, Jurkat or COS-7 cells, which have low enzyme levels. Sodium butyrate was found to increase both enzyme activity and normalized luciferase in HepG2 cells. Thus the dipeptidyl peptidase IV promoter possesses the ability to initiate transcription in a tissue-specific fashion in spite of having the sequence characteristics of a housekeeping gene promoter.

3T3 Cells↗
PubMed1995

Genomic aberrations in human hepatoblastomas detected by 2-dimensional gel analysis.

Hepatoblastoma is a frequent embryonic tumor found in children. The mechanism of its formation and development remain unknown. We analyzed genomic DNA aberrations associated with three hepatoblastomas by means of restriction landmark genomic scanning (I. Hatada et al., Proc. Natl. Acad. Sci. USA, 88:9523-9527, 1991). About 2000 NotI restriction landmarks were monitored in each of the hepatoblastomas, which revealed that the intensity of eight spots decreased in the tumor genome, suggesting allelic loss. Three of these spots were identical to those that decreased in human hepatocellular carcinomas. These results suggested that common, early events are involved in the genesis of the two types of liver tumor.

Carcinoma, Hepatocellular↗
PubMed1995

Association of sialyl-Lewis(a) and sialyl-Lewis(x) with MUC-1 apomucin ina pancreatic cancer cell line.

We have shown previously that the mucins of the human pancreatic cancer cell line, SW1990, have both sialyl-Lewis(a) and sialyl-Lewis(x) carbohydrate ligands that are implicated in tumor cell metastasis. In the present study, we undertook to identify the protein core of these mucins. SW1990 mucins that carry sialyl-Lewis(a) and sialyl-Lewis(x) bound to the MUC1 peptide-specific mAb 139H2. Removal of most of the sialic acids from SW1990 mucins by neuraminidase greatly enhanced binding of two other MUC1 peptide specific antibodies, HMFG-2 and SM-3. After removal of sialic acids, most of the mucins rich in sialyl-Lewis(a) and sialyl-Lewis(x) oligosaccharides no longer bound to a DEAE-cellulose column at pH 8.0. These results indicate that at least part of the sialyl-Lewis(a) and sialyl-Lewis(x) in SW1990 cells is associated with the MUC1 polypeptide. Moreover, sialic acids play an important role in determining the net negative charge of sialyl-Lewis(a) and sialyl-Lewis(x) rich mucins and in obscuring MUC1 peptide regions.

Amino Acid Sequence↗
PubMed1995

Cloning of no tI-cleaved genomic DNA fragments appearing as spots in 2D gel electrophoresis.

RLGS (Restriction Landmark Genomic Scanning) is a simple and rapid scanning of genomic DNA in two-dimensional electrophoresis. Human genomic DNA is first cleaved by NotI, and the cleaved ends are radio-labeled and cleaved further by EcoRV, followed by size-fractionation by first dimensional electrophoresis. The sample is then cleaved in situ by the second enzyme HinfI and resolved by the second dimensional electrophoresis. Nearly 2,000 spots emerge with spot intensities reflecting the copy number in the genome. Because of the resolving power and capacity to scan the entire genome, RLGS has been used to monitor genomic aberrations and imprinting. Here, we report a means of cloning the DNA in spots. The DNA was eluted and ligated to biotinylated NotI and HinfI likers followed by affinity separation using streptavidin. The ligated fragment was recovered by EcoRV cleavage, the target sequence of which was located in the NotI linker and amplified by PCR using a primer pair, the sequences of which lie in the linkers. The products were then cloned into a vector for further tests. An amplified spot in stomach cancer genomic DNA and a dwindling spot in liver cancer genomic DNA were taken as examples for cloning.

Base Sequence↗
PubMed1995

Mucin gene expression in normal, preneoplastic, and neoplastic human gastric epithelium.

Mucins synthesized by malignant cells may contribute (via decreased cellular adhesion and immune recognition) to cancer invasion and metastases. Human mucins are derived from a heterogeneous family of genes, labeled MUC1-6. Our aim was to determine the pattern of mucin gene expression in normal, preneoplastic (intestinal metaplasia), and malignant gastric specimens. Probes and antibodies for specific mucin tandem repeat sequences were used for RNA and immunohistochemical analysis. Normal stomach mucosa was characterized by expression of MUC1, MUC5, and MUC6 mRNA and immunoreactive protein, without MUC2, MUC3, and MUC4 gene expression. In contrast, high levels of MUC2 and MUC3 mucin mRNA and immunoreactive protein were found in specimens with intestinal metaplasia. Gastric cancers exhibited markedly altered secretory mucin mRNA levels compared with adjacent normal mucosa, with decreased levels of MUC5 and MUC6 mRNA and increased levels of MUC3 and MUC4 mRNA. Overall, immunoreactive MUC1 mucin was detected in 72% of 33 gastric cancers, and secretory mucin core peptides were expressed in 34% (MUC2), 45% (MUC3), 19% (MUC5), and 57% (MUC6) of these specimens. Coexpression of multiple (three or more) mucin core proteins occurred in 15 of 25 (60%) advanced (stages III and IV) cancers compared with 1 of 8 (12.5%) early (stages I and II) cancers (P < 0.048). We conclude that human gastric epithelium has a unique mucin gene pattern, which becomes markedly altered in preneoplastic and neoplastic specimens. Increased mucin gene heterogeneity in gastric adenocarcinomas is associated with advanced cancer stage.

Adenocarcinoma↗
PubMed1995

The laminin alpha 1 chain Ile-Lys-Val-Ala-Val (IKVAV)-containing peptide promotes liver colonization by human colon cancer cells.

Laminin, a major basement membrane-specific glycoprotein, promotes the attachment, migration, and invasion of a variety of tumor cells. Since laminin is present in the perisinusoidal matrix of the liver, we studied its effects on liver colonization by human colon cancer cells (HM7, LiM6) previously shown to have liver-metastasizing ability in athymic mice. These malignant cells expressed high levels of a 32-kDa laminin-binding protein on Western blot analysis when compared to the low metastatic parental cell line. Coinjection of laminin alpha chain-derived peptides which contain the amino acid sequence Ile-Lys-Val-Ala-Val (IKVAV) significantly stimulated liver colonization as determined by liver weight (P < 0.005) and number of tumor nodules (P < 0.02) 3 weeks after splenic-portal inoculation into nude mice. No stimulation was seen with a control peptide containing the same amino acids but in a scrambled sequence. In contrast, the Tyr-Ile-Gly-Ser-Arg peptide from the laminin beta 1 chain significantly inhibited HM7 liver colonization. These differences were not due to alterations in the number of cells initially reaching the liver as determined by injection of [125I]iododeoxyuridine-labeled tumor cells, but retention in the liver was stimulated by the IKVAV-containing peptides. Flow analysis indicated that the IKVAV peptide may act, in part, by stimulating homotypic adhesion of tumor cells. These data suggest that interactions of colon cancer cells with the IKVAV site on laminin may play a role in the formation of metastatic foci in the liver through cell-cell or cell-substratum interactions which promote metastasis.

Adenocarcinoma↗
PubMed1995

Access to peptide regions of a surface mucin (MUC1) is reduced by sialic acids.

Mucinous glycoproteins are present on the surfaces of tumor cells. Knowledge of which parts of the mucin molecule are accessible targets for cells of the immune system is important in the development of successful therapeutic approaches. One breast (ZR-75-1), two colon (Colo 205 and SW1116), and three pancreas (Capan-2, HPAF and SW1990) cancer cell lines were examined. The reactivities of antibodies HMFG-2, specific for the tripeptide (DTR) in the 20 amino acid tandem repeat of MUC1, and SM-3 (PDTRP) were greatly enhanced by pre-treating cells with an inhibitor of O-glycosylation, benzyl-alpha-N-acetylgalactosamide. However, desialylation of cell surfaces with neuraminidase or pre-treatment with an inhibitor of carbohydrate processing, monensin, also greatly enhanced the reactivities of HMFG-2, SM-3 and HMFG-1 (PDTR). Thus, sialic acids on termini of neighboring oligosaccharides significantly limit access to the peptide region recognized by antibodies HMFG-1/2 and SM-3.

Amino Acid Sequence↗
PubMed1995

Cloning and expression of a cDNA for mu-class glutathione S-transferase from rabbit liver.

A mu-class glutathione S-transferase (GST) cDNA clone, pHMB1, from rabbit liver has been constructed, using a 748-base-pair fragment of GST Yb1 cDNA as a probe. The nucleotide sequence of pHMB1 has been determined, and the complete amino acid sequence has been deduced. Recombinant clone pHMB1 contains a cDNA insert of 1443 base pairs with 654 nucleotides of open reading frame, 33 nucleotides of 5'-untranslated region, and 756 nucleotides of 3'-untranslated region. The open reading frame encodes a polypeptide (rbGST mu I) comprising 218 amino acids with molecular weight of 25,417. Compared to published mu-class GST sequences, rbGST mu I is 73 and 77% identical to rat Yb1 and human GST4 in amino acid sequence, respectively. The pHMB1 was expressed in Escherichia coli using expression vector pIH821 and the expressed GST was purified as a single band on polyacrylamide gel electrophoresis by maltose- and glutathione-affinity column chromatography. Rabbit liver GST protein expressed by this system was catalytically active. The functional characterization was done on the expressed protein. The rabbit liver GST expressed in E. coli showed greater activity toward 1,2-dichloro-4-nitrobenzene than mu-class isozymes in rabbit hepatic tissue (T. Primiano and R.F. Novak (1993) Arch. Biochem. Biophys. 301, 404-410). Enzymatic activity of expressed protein toward the substrate 1-chloro-2,4-dinitrobenzene was inhibited by triethyltin bromide, Cibacron blue, triphenyltin chloride, bromosulfophthalein, and hematin. RNA blot hybridization demonstrated that the pHMB1 mRNA was well expressed in rabbit liver, brain, and kidney.

Amino Acid Sequence↗
PubMed1995

Characterization and biodistribution of a mouse/human chimeric antibody directed against pancreatic cancer mucin.

BACKGROUND: Nd2 is a murine monoclonal antibody (MoAb) directed against purified mucins of the human pancreatic cancer cell line SW1990. The authors previously reported promising results with Nd2 for immunotargeting pancreatic cancer. However, murine MoAbs induce human anti-mouse antibodies (HAMAs), a serious problem for clinical use. Mouse/human chimeric antibodies may be less immunogenic and therefore reduce the incidence of HAMAs. In this study, the binding affinity, tumor specificity, biodistribution, and immunoimaging of chimeric Nd2 were evaluated. METHODS: The affinity of chimeric Nd2 was evaluated by competition radioimmunoassay and Scatchard analysis using 125I-chimeric Nd2, 125I-murine Nd2, and SW1990 mucin. Immunoreactivity against pancreatic cancer tissues was examined histochemically by the avidin-biotin peroxidase complex method. The biodistribution of the MoAbs was examined in athymic nude mice bearing SW1990 xenografts that were administered intravenous 125I-labeled chimeric or murine Nd2. 111In-chimeric Nd2 was injected into the same xenograft models, and scintigrams were obtained on day 3. RESULTS: Affinity analysis and immunohistochemical studies showed that chimeric Nd2 had the same affinity to SW1990 mucin and the same specificity for pancreas cancer tissues as murine Nd2. Intravenous administration of 125I-chimeric Nd2 resulted in a maximum tumor accumulation of 43% of the initial dose/gram of tumor, which was almost identical to the accumulation of 125I-murine Nd2. Distinct immunoscintigrams of tumors in nude mice were obtained with 111In-chimeric Nd2. CONCLUSION: Chimeric Nd2 may have clinical potential in the radioimmunodetection and immunotherapy of pancreatic cancer.

Animals↗
PubMed1995

Substrate specificity and detailed characterization of a bifunctional amylase-pullulanase enzyme from Bacillus circulans F-2 having two different active sites on one polypeptide.

Bacillus circulans F-2 amylase-pullulanase enzyme (APE) displayed dual activity with respect to glycosidic bond cleavage. The enzyme was active on alpha-1,6 bonds in pullulan, amylopectin, and glycogen, while it showed alpha-1,4 activity against malto-oligosaccharides, amylose, amylopectin, and soluble starch, but not pullulan. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as two competing substrates was used to distinguish the dual specificity of the enzyme from the single-substrate specificity known for pullulanases and alpha-amylases. Enzyme activities were inhibited by some metal ions, and by metal-chelating agents with a different mode. The enzyme-inhibitory results of amylase and pullulanase with Hg2+ and Co2+ ions were different, indicating that the activation mechanisms of both enzyme activities are different. Cyclomaltoheptaose inhibited both alpha-amylase and pullulanase activities with inhibition constants (Ki) of 0.029 and 0.06 mg/ml, respectively. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide confirmed a carboxy group at the active sites of both enzymes. The N-terminal sequence of the enzyme was: Ala-Asp-Ala-Lys-Lys-Thr-Pro- Gln-Gln-Gln-Phe- Asp-Ala-Leu-Trp-Ala-Ala-Gly-Ile-Val-Thr-Gly-Thr-Pro-Asp-Gly-Phe. The purified enzyme displayed Michaelis constant (Km) values of 0.55 mg/ml for amylose, and 0.71 mg/ml for pullulan. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which the two substrates are hydrolyzed at different active sites. These results suggest that amylopullulanases, which possess both alpha-1,6 and alpha-1,4 cleavage activities at the same active site, should be distinguished from APEs, which contain both activities at different active sites on the same polypeptide. Also, it is proposed that the Enzyme Commission use the term 'amylase-pullulanase enzyme' to refer to enzymes which act on starch and cleave both alpha-1,6-bonds in pullulan and alpha-1,4 bonds in amylose at different active sites.

Amino Acid Sequence↗
PubMed1995

Down-regulation of phospholipase C-gamma 1 during the differentiation of U937 cells.

Phospholipase C (PLC)-gamma 1 and -gamma 2 play a pivotal role in signal transduction for cell proliferation and differentiation. The enzyme activity and protein level of PLC-gamma 1 were markedly decreased in the human histiocytic leukemia U937 cell line during the differentiation process which is induced by phorbol 12-myristate 13-acetate (PMA) but those of PLC-gamma 2 were not altered. Northern blot analysis showed that the levels of PLC-gamma 1 and -gamma 2 transcripts were not changed. These results suggest that the expression of PLC-gamma 1 during the PMA-induced differentiation may be down-regulated by post-transcriptional processing.

Cell Differentiation↗
PubMed1995

Transcriptional regulation of phospholipase C-gamma 1 gene during muscle differentiation.

Phospholipase C (PLC) has been known to be a key effector protein in signal transduction pathway for cell proliferation and differentiation. Here, we report the transcriptional regulation of PLC-gamma 1 during myogenic differentiation. Both PLC-gamma 1 mRNA and protein levels were increased during myogenesis. The activity of PLC-gamma 1 was also comparable to the amount estimated by Western blot. Comparing promoter activities of various PLC-gamma 1 gene upstream regions in myoblast and myotube, the responsible region was mapped between -551 and -480. From these results, we conclude that the transcription of PLC-gamma 1 is increased by the control of GPE1 region of PLC-gamma 1 gene during myogenesis.

Animals↗
PubMed1995

Single-dose pharmacokinetics, safety, and tolerance of linopirdine (DuP 996) in healthy young adults and elderly volunteers.

The pharmacokinetics, safety, and tolerance of linopiridine ([3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one]; DuP 996) a potential therapeutic agent for Alzheimer's disease, were assessed in double-blind, placebo-controlled, randomized studies in which single oral doses were given to 64 healthy young or elderly males. Young subjects received escalating doses of 0.5 to 55 mg, whereas elderly subjects were given doses of 20 to 45 mg. Linopirdine plasma and urine samples were quantified after liquid extraction by a specific HPLC method using UV detection. In both groups, linopirdine disposition was characterized by rapid absorption (mean Tmax, < 1 hr) and elimination (mean t1/2, 0.4-3.2 hr). Urinary excretion of unchanged drug was negligible. The pharmacokinetic parameters showed large inter- and intrasubject variability. Linopirdine was well-tolerated in both young and elderly volunteers. The most frequently reported adverse event was headache. The subjects who received linopirdine did not experience clinically important changes in vital signs, electrocardiograms (ECGs), electroencephalograms (EEGs), or clinical laboratory evaluations.

Administration, Oral↗
PubMed1995

Synthesis and characterization of fluoropolymeric substrata with immobilized minimal peptide sequences for cell adhesion studies. I.

In this work, poly(tetrafluoroethylene-co-hexafluoropropylene) (also known as fluorinated ethylene propylene; FEP) was functionalized at the surface using a radio frequency glow discharge plasma. This particular surface modification produced controlled densities of hydroxyl functionality on the FEP surface. These surface hydroxyl groups provided sites for the covalent attachment of minimal peptide sequences, that are specific for neuronal attachment. FSCA, ATR-FTIR, ToF-SIMS, and fluorescence spectroscopy were used to evaluate peptide reaction efficiencies and to verify that intact peptide sequences were covalently attached to the FEP surfaces. These modified substrata were then used to study the cell attachment and response to covalently bound minimal peptide sequences. Cell attachment and differentiation results using NG108-15 and PC12 neuronal cell lines are presented in the adjoining paper by Ranieri et al.

Amino Acid Sequence↗
PubMed1995

Effect of intermittent and stepwise administration of a beta-adrenergic agonist, L644,969, on rat growth performance and skeletal muscles.

The effects of intermittent and stepwise administration of a beta-adrenergic agonist, L644,969 on rat growth performance and skeletal muscle growth were investigated in two studies. In experiment 1, 34 juvenile male rats were randomly assigned to two treatment groups: (1) control, 0 ppm L644,969 for 21 days; (2) BL10, 10 ppm of L644,969 for 7 days, then 0 ppm L644,969 for another 7 days, plus 10 ppm of L644,969 for the final 7 days. L644,969 increased (P < 0.05) weight gain during the 7 day initial administration period. When L644,969 was withdrawn, no difference in weight gain was observed between the two groups. Re-administration of L644,969 after the withdrawal period increased (P < 0.01) weight gain up to 3 days of re-administration, but no effect was observed thereafter. In experiment 2, 18 juvenile female rats were divided into three groups: (1) control, 0 ppm L644,969 for 22 days; (2) BL10, 10 ppm L644,969 for 22 days; (3) BL10-60, 10 ppm L644,969 for 11 days, then 60 ppm L644,969 for another 11 days. Feeding 10 ppm L644,969 increased (P < 0.05) the rate of weight gain up to 11 days, but not thereafter. Raising the dose of L644,969 from 10 to 60 ppm increased body weight gain during the initial 8 day period, but no effect was observed thereafter. Weights of skeletal muscles and heart were significantly increased by 10 ppm L644,969. Raising the dose to 60 ppm increased the mean skeletal muscle weights without statistical significance. Muscular concentration of cAMP was significantly decreased by the long-term administration of L644,969.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗