Heterogeneous loss of the Y chromosome in leukocyte lineages of donor origin after stem cell transplantation.
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Adult↗
Biomedical subjects
M Kwon
Publications and source records attributed to M Kwon.
Molecular characterization of the porcine endogenous retrovirus subclass A and B envelope gene from pigs.
Xenotransplantation of porcine organs has the potential to overcome the current critical shortage of allogenic organs for transplantation in humans. However, the existence of porcine endogenous retroviruses (PERVs) presents a problem for the clinical use of xenografts from pigs. In an attempt to understand the molecular characteristics of PERVs, we cloned the PERV env gene from six pig breeds (ie, Berkshire, Duroc, Landrace, Yorkshire, and two types of miniature pigs) in Korea. A total of 141 env clones were isolated and their sequences were analyzed. Phylogenetic analyses of these genes revealed the presence of PERVs, from both classes A and B, in 54% and 46% of the env clones, respectively. Among these clones, 37 isolates had the correct open reading frame (ORF; 27 clones in subclass A and 10 clones in subclass B), while the others had premature termination. These PERV nucleotide sequences can be used in a database for comparisons of PERV distribution among different pig breeds and for monitoring PERV infection using isolates with functional ORFs. Recombinant envelope of subclass A and B with functional ORF was expressed by vaccinia virus systems. Additionally isolated env clones can be used for various experiments, such as PERV control and infectivity tests, and may enhance the understanding of molecular mechanisms through pseudotyped PERV viruses.
Animals↗
Clinical, hematological and cytogenetical data of 11 patients with myelodysplastic features and idiopathic thrombocytosis.
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Anemia, Refractory↗
Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation.
It has been proposed that the suppression of poleward flux within interpolar microtubule (ipMT) bundles of Drosophila embryonic spindles couples outward forces generated by a sliding filament mechanism to anaphase spindle elongation. Here, we (i) propose a molecular mechanism in which the bipolar kinesin KLP61F persistently slides dynamically unstable ipMTs outward, the MT depolymerase KLP10A acts at the poles to convert ipMT sliding to flux, and the chromokinesin KLP3A inhibits the depolymerase to suppress flux, thereby coupling ipMT sliding to spindle elongation; (ii) used KLP3A inhibitors to interfere with the coupling process, which revealed an inverse linear relation between the rates of flux and elongation, supporting the proposed mechanism and demonstrating that the suppression of flux controls both the rate and onset of spindle elongation; and (iii) developed a mathematical model using force balance and rate equations to describe how motors sliding the highly dynamic ipMTs apart can drive spindle elongation at a steady rate determined by the extent of suppression of flux.
Anaphase↗
Identification of a common mutation (Gly194Cys) in both Arab Moslem and Ashkenazi Jewish patients with dihydrolipoamide dehydrogenase (E3) deficiency: possible beneficial effect of vitamin therapy.
Dihydrolipoamide dehydrogenase (E3) deficiency with a clinical phenotype and genotype (Gly194Cys homozygous) previously identified only in Ashkenazi Jewish patients, was diagnosed in two Palestinian Arab siblings and two unrelated Ashkenazi Jewish patients. While three of the four patients died in childhood without specific treatment, the surviving patient at age 18 years may have benefited from long-term daily supplementation with a cocktail of riboflavin, biotin, coenzyme Q and carnitine.
Child↗
p22 is a novel plasminogen fragment with antiangiogenic activity.
Tumor or tumor-associated cells cleave circulating plasminogen into three or four kringle-containing antiangiogenic fragments, collectively referred to as angiostatin. Angiostatin blocks tumor growth and metastasis by preventing the growth of endothelial cells that are critical for tumor vascularization. Here, we show that cancer and normal cells convert plasminogen into a novel 22 kDa fragment (p22). Production of this plasminogen fragment in a cell-free system has allowed characterization of the structure and activity of the protein. p22 consists of amino acid residues 78-180 of plasminogen and therefore embodies the first plasminogen kringle (residues 84-162) as well as additional N- and C-terminal residues. Circular dichroism and intrinsic fluorescence spectrum analysis have defined structural differences between p22 and recombinant plasminogen kringle 1 (rK1), therefore suggesting a unique conformation for kringle 1 within p22. Proliferation of capillary endothelial cells but not cells of other lineages was selectively inhibited by p22 in vitro. In addition, p22 prevented vascular growth of chick chorioallantoic membranes (CAMs) in vivo. Furthermore, administration of p22 at low dose suppressed the growth of murine Lewis lung carcinoma (LLC) metastatic foci in vivo. This is the first identification of a single kringle-containing antiangiogenic plasminogen fragment produced under physiological conditions.
Allantois↗
In vitro selection of RNA against kanamycin B.
Aminoglycosides are well-known antibiotics that function by interacting with ribosomal RNA in bacteria. In order to understand the molecular details between RNA and the drug, RNA aptamer was selected against kanamycin B. After 12 cycles of selection, RNA was cloned and sequenced. Among 9 clones, sequences of three clones were identical, suggesting the selected RNA was enriched. Among the cloned RNA molecules, the triplicated RNA was the maximum binding RNA. It showed a 180 nM affinity (KD) to the cognate aminoglycoside, as measured by a surface plasmon resonance, and a competition assay using a fluorescence anisotropy technique. The affinity of the maximum binding RNA to a similar aminoglycoside, tobramycin, was much stronger than 12 nM of KD. The binding site of the aminoglycoside in the maximum binding RNA was a stem loop located at the end of the 5' region. A stem loop structural motif, found in this study, was similar to those previously reported, even though the sequences of the RNA were totally different from the known sequences of the aminoglycoside binding site of other aptamers. The present study suggests that the aminoglycoside-binding region in RNA does not have a sequence specificity, but has a shape-specific bulged stem loop, even though it has a nanomolar affinity.
Anti-Bacterial Agents↗
Oil components modulate physical characteristics and function of the natural oil emulsions as drug or gene delivery system.
Oil-in-water (o/w) type lipid emulsions were formulated by using 18 different natural oils and egg phosphatidylcholine (egg PC) to investigate how emulsion particle size and stability change with different oils. Cottonseed, linseed and evening primrose oils formed emulsions with very large and unstable particles. Squalene, light mineral oil and jojoba bean oil formed stable emulsions with small particles. The remaining natural oils formed moderately stable emulsions. Emulsions with smaller initial particle size were more stable than those with larger particles. The correlation between emulsion size made with different oils and two physical properties of the oils was also investigated. The o/w interfacial tension and particle size of the emulsion were inversely proportional. The effect of viscosity was less pronounced. To study how the oil component in the emulsion modulates the in vitro release characteristics of lipophilic drugs, three different emulsions loaded with two different drugs were prepared. Squalene, soybean oil and linseed oil emulsions represented the most, medium and the least stable systems, respectively. For the lipophilic drugs, release was the slowest from the most stable squalene emulsion, followed by soybean oil and then by linseed oil emulsions. Cationic emulsions were also prepared with the above three different oils as gene carriers. In vitro transfection activity was the highest for the most stable squalene emulsion followed by soybean oil and then by linseed oil emulsions. Even though the in vitro transfection activity of emulsions were lower than the liposome in the absence of serum, the activity of squalene emulsion, for instance, was ca. 30 times higher than that of liposome in the presence of 80% (v/v) serum. In conclusion, the choice of oil component in o/w emulsion is important in formulating emulsion-based drug or gene delivery systems.
Animals↗
In situ hybridization and immunolocalization of lignan reductases in woody tissues: implications for heartwood formation and other forms of vascular tissue preservation.
Vascular plants have evolved with remarkable ways to form and protect the vasculature apparatus. In certain woody shrubs, the secondary xylem can have within its center a hollowed pith surrounded by secondary xylem, whereas in most trees there is a solid core of heartwood. Both types of woody systems have, however, the commonality of accumulating so-called 'secondary' metabolites, albeit to different extents, whose roles are to protect and preserve the vascular (lignified) tissues. This investigation had as its purpose establishing the nature of the cells involved in the biosynthesis of these specialized 'secondary' metabolites in plants forming heartwood and hollow piths, respectively. This was achieved by determining the tissue-specific expression of two lignan biosynthetic pathway enzymes: pinoresinol-lariciresinol reductase (PLR) and phenylcoumaran benzylic ether reductase (PCBER), soluble enzymes which catalyze analogous benzylic ether reductions of 8-8' and 8-5' linked lignans, respectively. Using Forsythia intermedia, reverse transcription-PCR (RT-PCR) and northern blots revealed that PLR mRNA accumulated mainly in young stems, as well as in young roots and petioles. Furthermore, PLR-specific DIG-labeled riboprobes established that in the stems its mRNA accumulated in the radial parenchyma cells [and to a lesser extent in the developing vessels], as well as in the cambial cells of developing secondary xylem. In addition, immunocytochemical localization of PCBER in Pinus taeda established that it was in the axial and radial parenchyma cells of secondary xylem of stems. That is, irrespective of whether the woody plants formed hollowed piths or heartwood, the 'secondary' metabolite pathways leading to the protective lignans predominantly involved axial and radial parenchyma cells. This is in contrast to monolignol coupling (i.e. the entry point to both the lignans and lignins), which appears to be more restricted to the vascular cambial regions.
Asteraceae↗
Dirigent proteins and dirigent sites in lignifying tissues.
Tissue-specific dirigent protein gene expression and associated dirigent (site) localization were examined in various organs of Forsythia intermedia using tissue printing, in situ mRNA hybridization and immunolabeling techniques, respectively. Dirigent protein gene expression was primarily noted in the undifferentiated cambial regions of stem sections, whereas dirigent protein sites were detected mainly in the vascular cambium and ray parenchyma cell initials. Immunolocalization also revealed cross-reactivity with particular regions of the lignified cell walls, these being coincident with the known sites of initiation of lignin deposition. These latter regions are considered to harbor contiguous arrays of dirigent (monomer binding) sites for initiation of lignin biopolymer assembly. Dirigent protein mRNA expression was also localized in the vascular regions of roots and petioles, whereas in leaves the dirigent sites were primarily associated with the palisade layers and the vascular bundle. That is, dirigent protein mediated lignan biosynthesis was initiated primarily in the cambium and ray cell initial regions of stems as well as in the leaf palisade layers, this being in accordance with the occurrence of the lignans for defense purposes. Within lignified secondary xylem cell walls, however, dirigent sites were primarily localized in the S(1) sublayer and compound middle lamella, these being coincident with previously established sites for initiation of macromolecular lignin biosynthesis. Once initiation occurs, lignification is proposed to continue through template polymerization.
Asteraceae↗
Induced compression wood formation in Douglas fir (Pseudotsuga menziesii) in microgravity.
In the microgravity environment of the Space Shuttle Columbia (Life and Microgravity Mission STS-78), were grown 1-year-old Douglas fir and loblolly pine plants in a NASA plant growth facility. Several plants were harnessed (at 45 degrees ) to establish if compression wood biosynthesis, involving altered cellulose and lignin deposition and cell wall structure would occur under those conditions of induced mechanical stress. Selected plants were harnessed at day 2 in orbit, with stem sections of specific plants harvested and fixed for subsequent microscopic analyses on days 8, 10 and 15. At the end of the total space mission period (17 days), the remaining healthy harnessed plants and their vertical (upright) controls were harvested and fixed on earth. All harnessed (at 45 degrees ) plant specimens, whether grown at 1 g or in microgravity, formed compression wood. Moreover, not only the cambial cells but also the developing tracheid cells underwent significant morphological changes. This indicated that the developing tracheids from the primary cell wall expansion stage to the fully lignified maturation stage are involved in the perception and transduction of the stimuli stipulating the need for alteration of cell wall architecture. It is thus apparent that, even in a microgravity environment, woody plants can make appropriate corrections to compensate for stress gradients introduced by mechanical bending, thereby enabling compression wood to be formed. The evolutionary implications of these findings are discussed in terms of "variability" in cell wall biosynthesis.
Cycadopsida↗
Human dihydrolipoamide dehydrogenase gene transcription is mediated by cAMP-response element-like site and TACGAC direct repeat.
Dihydrolipoamide dehydrogenase is a common component of four multienzyme complexes which are involved in oxidation of carbohydrates, lipids and amino acids. To better understand the regulation of human DLD gene expression, we have analyzed the proximal promoter region of this gene. DNase I footprinting analysis of the promoter region (-322 to +47 bp) revealed four major protein-binding domains (termed P1-P4). Nested deletions and site-specific mutations of approximately 100 bp proximal promoter region identified two elements, TACGAC direct repeat sequence and cAMP-response element (CRE)-like site, which are localized in the P2 and P1 domains, respectively, and mediate basal transcription of the DLD gene. Electrophoretic mobility supershift assays showed that the CRE-like site is associated with CRE binding protein. Interestingly, when DLD promoter constructs (-1.8 kb to +47 bp and -78 to +47 bp) fused with the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into human HepG2 cells either in the presence or absence of 0.5 mM 8-Br-cAMP, the levels of CAT expression remained unaffected. In addition, endogenous DLD mRNA levels in HepG2 cells also remained unaffected by treatment with 0.5 mM 8-Br-cAMP. These results indicate that the CRE binding protein is essential for basal transcription of the human DLD promoter, but does not confer cAMP-dependent gene regulation.
5' Untranslated Regions↗
Purification and characterization of A61. An angiostatin-like plasminogen fragment produced by plasmin autodigestion in the absence of sulfhydryl donors.
Plasmin, a broad spectrum proteinase, is inactivated by an autoproteolytic reaction that results in the destruction of the heavy and light chains of the protein. Recently we demonstrated that a 61-kDa plasmin fragment was one of the major products of this autoproteolytic reaction (Fitzpatrick, S. L., Kassam, G., Choi, K. S., Kang, H. M., Fogg, D. K., and Waisman, D. M. (2000)Biochemistry 39, 1021-1028). In the present communication we have identified the 61-kDa plasmin fragment as a novel four kringle-containing protein consisting of the amino acid sequence Lys(78)-Lys(468). To avoid confusion with the plasmin(ogen) fragment, angiostatin(R) (Lys(78)-Ala(440)), we have named this protein A(61). Unlike angiostatin, A(61) was produced in vitro from plasmin autodigestion in the absence of sulfhydryl donors. A(61) bound to lysine-Sepharose and also underwent a large increase in fluorescence yield upon binding of the lysine analogue, trans-4-aminomethylcyclohexanecarboxylic acid. Circular dichroism suggested that A(61) was composed of 21% beta-strand, 14% beta-turn, 18% 3(1)-helix and 8% 3(10)-helix. A(61) was an anti-angiogenic protein as indicated by the inhibition of bovine capillary endothelial cell proliferation. Plasminogen was converted to A(61) by HT1080 cells and bovine capillary endothelial cells. Furthermore, a plasminogen fragment similar to A(61) was present in the serum of humans as well as normal and tumor-bearing mice. These results establish that plasmin turnover can generate anti-angiogenic plasmin fragments in a nonpathological setting.
Angiostatins↗
Functional coordination of three mitotic motors in Drosophila embryos.
It is well established that multiple microtubule-based motors contribute to the formation and function of the mitotic spindle, but how the activities of these motors interrelate remains unclear. Here we visualize spindle formation in living Drosophila embryos to show that spindle pole movements are directed by a temporally coordinated balance of forces generated by three mitotic motors, cytoplasmic dynein, KLP61F, and Ncd. Specifically, our findings suggest that dynein acts to move the poles apart throughout mitosis and that this activity is augmented by KLP61F after the fenestration of the nuclear envelope, a process analogous to nuclear envelope breakdown, which occurs at the onset of prometaphase. Conversely, we find that Ncd generates forces that pull the poles together between interphase and metaphase, antagonizing the activity of both dynein and KLP61F and serving as a brake for spindle assembly. During anaphase, however, Ncd appears to have no effect on spindle pole movements, suggesting that its activity is down-regulated at this time, allowing dynein and KLP61F to drive spindle elongation during anaphase B.
Anaphase↗
Identification and characterization of the antimicrobial peptide corresponding to C-terminal beta-sheet domain of tenecin 1, an antibacterial protein of larvae of Tenebrio molitor.
An active fragment was identified from tenecin 1, an antibacterial protein belonging to the insect defensin family, by synthesizing the peptides corresponding to the three regions of tenecin 1. Only the fragment corresponding to the C-terminal beta-sheet domain showed activity against fungi as well as Gram-positive and Gram-negative bacteria, whereas tenecin 1, the native protein, showed activity only against Gram-positive bacteria. CD spectra indicated that each fragment in a membrane-mimetic environment might adopt a secondary structure corresponding to its region in the protein. The leakage of dye from liposomes induced by this fragment suggested that this fragment acts on the membrane of pathogens as a primary mode of action. A comparison between the structure and the activity of each fragment indicated that a net positive charge was a prerequisite factor for activity. To the best of our knowledge this is the first report in which the fragment corresponding to the beta-sheet region in antibacterial proteins, which consists of alpha-helical and beta-sheet regions, has been identified as a primary active fragment.
Amino Acid Sequence↗
Identification and characterization of novel antimicrobial decapeptides generated by combinatorial chemistry.
Novel combinatorial libraries consisting of simplified amino acid sequences were designed to screen for peptides active against the Candida albicans membrane. A novel decapeptide, KKVVFKVKFK, that had a unique primary amino acid sequence was identified in this work. This peptide irreversibly inhibited the growth of C. albicans and showed a broad range of antibacterial activity but no hemolytic activity. Circular dichroism spectra revealed that the predominant secondary structure of this peptide strongly depended on the membrane-mimetic environments; the peptide preferred to form an amphipathic alpha-helical structure in the presence of 50% trifluoroethanol, while it preferred to adopt a distorted alpha-helical structure in the presence of sodium dodecyl sulfate micelles. Experiments in which dye was released from vesicles indicated that this novel antimicrobial peptide killed microorganisms through the action on the membrane as its primary target. Replacement of amino acids in this active decapeptide on the basis of information from the libraries could provide unique information about factors affecting its antimicrobial activity such as its secondary structure, net positive charge, and hydrophobicity.
Anti-Bacterial Agents↗
Primary amino acid sequence and structure of human pyruvate carboxylase.
Pyruvate carboxylase (PC) (pyruvate:carbon dioxide ligase (ADP-forming), EC 6.4.1.1.), a nuclear-encoded mitochondrial enzyme, catalyzes the conversion of pyruvate to oxaloacetate. We have isolated and characterized cDNAs spanning the entire coding region of human PC. The sequence of human PC has an open reading frame of 3537 nucleotides which encodes for a polypeptide with a length of 1178 amino acids. The identity of the cDNA as PC is confirmed by comparison to PC cDNAs of other species and sequenced peptide fragments of mammalian PC. The M(r) of the full length precursor protein is 129,576 and that of the mature apoprotein is 127,370. RNA blot analysis from a variety of human tissues demonstrates that the highest level of PC mRNA is found in liver corresponding to this tissue's high level of PC activity. Based on homology with other biotin-containing proteins, the ATP, pyruvate, and biotin-binding sites can be identified. One of two patients with documented PC deficiency was found to be missing PC mRNA, further confirming the identity of this cDNA.
Amino Acid Sequence↗
[Three dimensional cholangiography with spiral CT for analysis of biliary tract: preliminary report].
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Cholangiography↗