Retrovirus-mediated insertional mutagenesis in zebrafish and identification of a molecular marker for embryonic germ cells.
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Animals↗
Biomedical subjects
C Yoon
Publications and source records attributed to C Yoon.
LET-23-mediated signal transduction during Caenorhabditis elegans development.
We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming alpha-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the "vulvaless" phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the "multivulva" phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development--larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways.
Animals↗
Cytokine secretion induced by superantigens in peripheral blood mononuclear cells, lamina propria lymphocytes, and intraepithelial lymphocytes.
Superantigens are potent inducers of T-cell proliferation and induce a broad range of cytokines, including tumor necrosis factor (TNF), gamma interferon, and interleukin 2 (IL-2). In the present study, we compared the abilities of different staphylococcal superantigens (staphylococcal enterotoxin B [SEB], staphylococcal enterotoxin E [SEE], and toxic shock syndrome toxin 1 [TSST-1]) to stimulate distinct cytokine profiles in peripheral blood mononuclear cells (PBMC), lamina propria lymphocytes (LPL), and intraepithelial lymphocytes (IEL). One million PBMC, LPL, and IEL were stimulated with various concentrations of superantigen (10 to 0.001 ng/ml) for 24, 48, and 72 h. Maximum cytokine production by PBMC, LPL, and IEL was observed for all three superantigens at 48 h at a concentration of 1 ng/ml. In PBMC, SEE and TSST-1 stimulated more IL-2 and gamma interferon than SEB. SEE and TSST-1 also stimulated more TNF and IL-4 production than SEB. In contrast, SEB stimulated more IL-6 than either SEE or TSST-1. In LPL, there was no SEE-induced IL-2 or IL-4 production, but IL-6, TNF, and gamma interferon were induced. SEB similarly induced no IL-2 or gamma interferon from the LPL, but IL-4, IL-6, and TNF were detected. TSST-1 stimulation of LPL resulted in IL-2 and TNF production but no IL-4, IL-6, or gamma interferon. In IEL, SEE induced no IL-2, IL-4, or gamma interferon but produced IL-6 and TNF, while SEB stimulation resulted in no IL-2 or gamma interferon but did result in detectable IL-4, IL-6, and TNF. Taken together, these data indicate that there are significant differences in the cytokine profiles induced by superantigens in LPL and IEL compared with those in PBMC, and these differences may relate to differences in activation requirements.
Bacterial Toxins↗
Primary non-Hodgkin's malignant lymphoma of the vulva--a case report.
A case of primary non-Hodgkin's malignant lymphoma of the vulva which occurred in a 68-year-old woman is presented. Non-Hodgkin's malignant lymphoma is infrequently involved in the female genital tract. Moreover, primary vulvar involvement of this tumor is very rare. To date only 6 cases have been reported in the literature. To our knowledge this is the first reported case of a non-Hodgkin's malignant lymphoma of the vulva in Korea.
Aged↗
Sequence specificity of the deoxyribonuclease activity of 1,10-phenanthroline-copper ion.
A statistical analysis of a data set composed of over 1600 scission events of DNA produced by the 2:1 1,10-phenanthroline-copper complex (OP-Cu) has demonstrated that the nucleotide 5' to the site of phosphodiester bond scission is a primary influence in the kinetics of cleavage at any sequence position. The scission was less affected by the 3' neighbor. For each of the sixteen possible dinucleotides, a kinetic parameter can be computed reflecting scission at the 3' nucleotide. When used to predict the scission pattern of a DNA sequence not part of the present data set, correlation coefficients of about 0.6 between predicted and observed patterns were obtained.
Base Sequence↗
Sequence-dependent variability of DNA structure. Influence of flanking sequences and fragment length on digestion by conformationally sensitive nucleases.
DNase I and 1,10-phenanthroline-copper are two nucleolytic activities which are sequence-dependent in their scission reaction yet are not nucleotide-specific at their site of cutting. When these two nucleases are used to digest identical sequences in 18-base pair oligonucleotides and in restriction fragments 10-fold longer, the digestion patterns are similar at sequence positions in the interior of the fragment. Changes in reactivity to 1,10-phenanthroline-copper associated with mutational changes in the lac promoter in biochemically functional restriction fragments are duplicated in 18-base pair oligonucleotides. The structural variability of a given DNA sequence detected by these conformationally sensitive nucleolytic activities is therefore encoded in local sequence and not sensitive to fragment length. Digestion patterns of a repeated 7-base pair sequence within a longer sequence have the same characteristic except for the two nucleotides at the 5' periphery of the direct repeat. This conclusion is based on the digestion pattern of a restriction fragment which contains the polyadenylation site of the mouse immunoglobulin mu heavy chain gene. Two pairs of different 7-base pair sequences repeated in this fragment retain their distinctive digestion patterns. DNA sequences which comprise the binding sites of regulatory proteins, retain a characteristic structure only influenced at their peripheries by two to three bases of the flanking sequence.
DNA↗
Structure of an alternating-B DNA helix and its relationship to A-tract DNA.
The crystal structure of the synthetic DNA dodecamer CGCATATATGCG has been solved at 2.2-A resolution. Its central 6 base pairs adopt the alternating-B-DNA helix structure proposed nearly a decade ago. This alternating poly(AT) structure contrasts with the four known examples of what can be termed a poly(A) subfamily of B-DNA structures: CGCAAAAAAGCG, CGCAAATTTGCG, CGCGAATTCGCG, and CGCGAATTbrCGCG, their defining characteristic being a succession of two or more adenines along one strand, in a region of 4 or more A.T base pairs. All five helices show a characteristically narrow minor groove in their AT centers, but the mean propeller twist at A.T base pairs is lower in the alternating poly(AT) helix than in the poly(A) subfamily of helices. Three general principles emerge from x-ray analyses of B-DNA oligonucleotides: (i) GC and mixed-sequence B-DNA have a wide minor groove, whereas the minor groove is narrow in heteropolymer or homopolymer AT sequences. (ii) G.C base pairs have low propeller twist; A.T pairs can adopt a high propeller twist but need not do so. A high propeller twist can be stabilized by cross-strand hydrogen bonds in the major or minor groove, examples being the minor groove bonds seen in CCAAGATTGG and the major groove bonds that can accompany AA sequences in the poly(A) family. (iii) Homopolymer poly(A) tracts may be stiffer than are alternating AT or general-sequence DNA because of these cross-strand major groove hydrogen bonds. Poly(A) tracts appear internally unbent, but bends may occur at junctions with mixed-sequence DNA because of differences in propeller twist, base pair inclination, and base stacking on the two sides of the junction. Bending occurs most easily via base roll, favoring compression of the broad major groove.
Base Composition↗
The effect of acid perfusion on mucosal blood flow and intramural pH of rabbit duodenum.
To evaluate the role of blood flow for acid tolerance of the duodenal mucosa, we perfused the duodenums of anesthetized rabbits with different concentrations of hydrochloric acid (HCl). Acid perfusion stimulated blood flow to the duodenal wall in a concentration-dependent fashion up to 80 mmol/L HCl (0 mmol/L; 0.44 +/- 0.05, 10 mmol/L; 0.84 +/- 0.14, 50 mmol/L; 1.44 +/- 0.11, 80 mmol/L; 2.03 +/- 0.12, 100 mmol/L; 1.82 +/- 0.07 ml/gm/min X +/- SEM). The pH in the lamina propria of the mucosa, which was measured with antimony microelectrodes was not changed in experiments during perfusion with 50 and with 80 mmol/L HCl in normotension. Acidosis in the lamina propria could be demonstrated only when the duodenum was perfused with 100 and with 80 mmol/L HCl combined with hemorrhagic hypotension. Damage to the mucosa, which developed after 30 and 60 minutes of acid perfusion, also showed a H+-dependent pattern. Reduction of blood flow by hemorrhagic hypotension aggravated the morphologic damage. We conclude that luminal acid stimulates blood flow in the duodenum. The decrease in blood flow induced by hypotension results in a greater susceptibility to mucosal damage.
Animals↗
Nuclease activity of 1,10-phenanthroline-copper ion: reaction with CGCGAATTCGCG and its complexes with netropsin and EcoRI.
The self-complementing dodecamer 5'-CGCGAATTCGCG-3' and its complexes with the antibiotic netropsin and the restriction endonuclease EcoRI provide substrates of known three-dimensional structure to study the stereochemistry and mechanism of the artificial nuclease of 1,10-phenanthroline-copper ion [(OP)2Cu+]. Analysis of the reaction products with the 5'-32P dodecamer on 20% sequencing gels has demonstrated the presence of 3'-phosphoglycolate ends in addition to 3'-phosphomonoester ends expected from previous studies. A reaction intermediate, which is a precursor to 3'-phosphomonoester termini, has been trapped; in contrast, no comparable species for the 5'-phosphomonoester termini can be detected when 3'-labeled DNAs are utilized as substrates. The reactive oxidative species formed by the coreactants (OP)2Cu+ and hydrogen peroxide is distinguishable in its chemistry from the hydroxyl radicals produced by cobalt-60 gamma-irradiation. The freely diffusible hydroxyl radicals generated by cobalt-60 irradiation produce equivalent amounts of 3'-phosphomonoester and 3'-phosphoglycolate termini whereas the 3'-phosphomonoesters are the preferred product of (OP)2Cu+ and H2O2. On the basis of the structures of the products obtained, the principal site of attack of the coordination complex is on the C-1 of the deoxyribose within the minor groove. This conclusion is supported by the footprinting of netropsin binding to the dodecamer. Crystallographic results have demonstrated that netropsin binds to the minor groove at the central AATT residue. A clear protection of attack by the coordination complex at the deoxyriboses associated with A-5, T-6, T-7, and C-9 is fully consistent with attack from the minor groove without intercalation during the course of the cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Rates of nucleotide substitution in Drosophila mitochondrial DNA and nuclear DNA are similar.
While the majority of DNA in eukaryotes is in the nucleus, a small but functionally significant amount is found in organelles such as chloroplasts and mitochondria. A recent, rather remarkable, finding has been that in vertebrates the DNA in the mitochondria (mtDNA) is evolving 5-10 times faster than the DNA in the nucleus. No similar studies have been done with invertebrates. Using the technique of DNA X DNA hybridization, we have measured the degree of nucleotide substitution between Drosophila melanogaster and Drosophila yakuba for both single-copy nuclear DNA (scnDNA) and mtDNA. The change in melting temperature is the same in both types of DNA hybrids. Thus we conclude that mtDNA and scnDNA are evolving at similar rates in these Drosophila. Considerable DNA sequence data are available for the mtDNAs studied, allowing us to estimate that a 1 degree C change in melting temperature corresponds to a 1.5-2% base-pair mismatch.
Animals↗
Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G.
The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.
Base Sequence↗
The molecular origin of DNA-drug specificity in netropsin and distamycin.
X-ray analysis of the complex of netropsin with the B-DNA dodecamer of sequence C-G-C-G-A-A-T-T-BrC-G-C-G reveals that the antitumor antibiotic binds within the minor groove by displacing the water molecules of the spine of hydration. Netropsin amide NH furnish hydrogen bonds to bridge DNA adenine N-3 and thymine O-2 atoms occurring on adjacent base pairs and opposite helix strands, exactly as with the spine of hydration. The narrowness of the groove forces the netropsin molecule to sit symmetrically in the center, with its two pyrrole rings slightly non-coplanar so that each ring is parallel to the walls of its respective region of the groove. Drug binding neither unwinds nor elongates the double helix, but it does force open the minor groove by 0.5-2.0 A, and it bends back the helix axis by 8 degrees across the region of attachment. The netropsin molecule has an intrinsic twist that favors insertion into the minor groove of B-DNA, and it is given a small additional twist upon binding. The base specificity that makes netropsin bind preferentially to runs of four or more A X T base pairs is provided not by hydrogen bonding but by close van der Waals contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the drug molecule. Substitution of one or more pyrroles by imidazole could permit recognition of G X C base pairs as well, and it could lead to a class of synthetic "lexitropsins," capable of reading any desired short sequence of DNA base pairs.
Base Sequence↗
Helix geometry and hydration in an A-DNA tetramer: IC-C-G-G.
The DNA oligomer of sequence IC-C-G-G has been synthesized, and its X-ray crystal structure solved at a resolution of 2.0 A, using anomalous scattering from iodines in phase analysis: 48 cycles of Jack-Levitt restrained least-squares refinement resulted in a residual error of 19.9% over all data, or 16.5% for two-sigma data. Two double-helical tetramers stack in the crystal to form a continuous octamer, except for the two missing phosphate connections across the center. The octamer has a mean helix rotation of 33.7 degrees (10.7 base-pairs per turn), rise of 2.87 A, mean inclination angle of base-pairs of 14 degrees, and mean base-pair propeller twist of +16.3 degrees. Local variations in both helix rotation and base plane roll angles, including those across the center of the octamer, are as predicted from base sequence by sum functions sigma 1 and sigma 2. The three known DNA octamers: IC-C-G-G/IC-C-G-G, G-G-T-A-T-A-C-C and G-G-C-C-G-G-C-C, make up a graded series in this order, with monotonically changing structural parameters. An exhaustive comparison of torsion angle correlations among the known A helices confirms some structural expectations and reveals some new features. 86 water molecules have been located per double-helical IC-C-G-G tetramer (the asymmetric unit), of which 451/2 per tetramer lie within a first hydrogen-bonded shell of hydration. No ordered water structure is observed comparable to the minor groove spine of hydration in B-DNA.
Base Sequence↗
[The protective effect of systemic HCO3- administration on the gastric mucosa in shock].
Recent research has shown adequate mucosal blood flow and blood bicarbonate availability to be crucial in the prevention of gastric ulceration, yet the relative importance of these two factors is unknown. We studied the incidence of ulceration in rats subjected to hypovolaemic shock under varying conditions of blood flow and acid-base balance. At the start of the experiment 2 ml of 0.1 M HCl were instilled into the stomach. In the control series, severe systemic acidosis had developed after 2 hours of shock (mean arterial blood pressure 40 mm Hg) and 6 out of 6 stomachs were ulcerated. Mucosal blood flow, measured with radioactive microspheres, had drastically decreased. Prostacyclin, dissolved in 0.01 M phosphate, improved gastric blood flow, but did not help to correct acidosis, and 6 out of 8 stomachs were ulcerated. A higher degree of protection was observed when prostaglandin was dissolved in 0.16 M HCO-3. Intraarterial infusion of high concentrations of bicarbonate completely eliminated acidosis, with ulceration occurring in only 1 out of 6 rats infused with 0.5 M bicarbonate and in 1 out of 10 with 1 M bicarbonate. Blocking carbonic anhydrase with acetazolamide (100 mg/kg) completely prevented the protective effect of a 1 M HCO-3 infusion, and 6 out of 8 stomachs were ulcerated. Substituting TRIS buffer for bicarbonate in the infusion also eliminated acidosis, but neither concentrations of 1 M or 5 M buffer prevented the stomachs from ulceration (6 out of 6 with 1 M buffer and 5 out of 6 with 5 M buffer). We conclude that bicarbonate is the essential factor in prevention of ulceration in our shock model and that blood flow is also important as one component of a bicarbonate-dependent protection system involving carbonic anhydrase.
Acetazolamide↗
The relative importance of HCO3- and blood flow in the protection of rat gastric mucosa during shock.
Recent research has shown adequate mucosal blood flow and blood bicarbonate availability to be crucial in the prevention of gastric ulceration, yet the relative importance of these two factors is unknown. We studied the incidence of ulceration in rats subjected to hypovolemic shock under varying conditions of blood flow and acid-base balance. At the start of the experiment 2 ml of 0.1 M HCl were instilled into the stomach. In the control series, severe systemic acidosis had developed after 2 h of shock (B.P. 40 mmHg) and 6 of 6 stomachs ulcerated. Mucosal blood flow, measured with radioactive microspheres, had drastically decreased. Prostacyclin dissolved in 0.01 M phosphate improved gastric blood flow but did not help to correct acidosis, and 6 of 8 stomachs ulcerated. A higher degree of protection was observed when the prostaglandin was dissolved in 0.16 M HCO3-. Intraarterial infusion of high concentrations of bicarbonate completely eliminated acidosis with ulceration occurring in 1 of 6 rats infused with 0.5 M and in 1 of 10 with 1 M bicarbonate. Blocking the carbonic anhydrase with acetazolamide (100 mg/kg) completely prevented the protective effect of a 1 M HCO3- infusion, and 6 out of 8 stomachs ulcerated. Substituting TRIS buffer for bicarbonate in the infusion also eliminated acidosis, but neither concentrations of 1 M or 5 M buffers prevented the stomachs from ulceration (6 out of 6 with 1 M buffer and 5 of 6 with 5 M buffer). We conclude that bicarbonate is the essential factor in prevention of ulceration in our shock model and that blood flow is also important as one component of a bicarbonate-dependent protection system involving carbonic anhydrase.
Acid-Base Equilibrium↗
1978 anesthesia morbidity and mortality survey: Southern California Society of Oral and Maxillofacial Surgeons.
The morbidity and mortality experience with outpatient general anesthesia of the Southern California Society of Oral and Maxillofacial Surgeons was surveyed for a second five-year period. One hundred percent of the active members responded with data on educational background, time in practice, and equipment and drugs in use, as well as with morbidity and mortality data. No deaths resulting from anesthetics were reported during this survey period. The decreasing occurrence of problems is attributed to improved training, careful monitoring of patients, and preparation for anesthesia emergencies through mandatory in-office peer evaluation.
Adult↗
Dentist-physician relationship in extended care facilities.
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Aged↗
Dialysis access graft thrombolysis: randomized study of pulse-spray versus continuous urokinase infusion.
PURPOSE: To compare pulse-spray to continuous-infusion thrombolysis with high-dose urokinase in thrombosed dialysis access grafts. METHODS: A prospective randomized controlled trial was performed. From August 1992 to September 1993, 30 thrombosed polytetrafluoroethylene (PTFE) grafts in 24 patients were included, 15 grafts in each group. The success of thrombolysis, mean time to thrombolysis, mean urokinase dose, and 60-day patency rate were evaluated. RESULTS: In the pulse-spray group, the mean time to thrombolysis was 72 min with a mean urokinase dose of 560,000 U. The 60-day patency rate was 71%. In the continuous-infusion group, the mean infusion time to thrombolysis was 55 min with a mean dose of 479,000 U. The 60-day patency rate was 73%. CONCLUSION: No statistically significant difference was found between the two techniques in the mean time to thrombolysis, the mean urokinase dose used, or the 60-day patency rate.
Arm↗