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Y Kim

Publications and source records attributed to Y Kim.

At least 631 records · Page 35Linked to original sources
PubMed1994

Genome organization of a biologically active molecular clone of the lymphoproliferative disease virus of turkeys.

The lymphoproliferative disease retrovirus (LPDV) induces an acute, horizontally transmitted disease of turkeys that is often fatal. Although LPDV cannot be grown in cultured cells, it was possible to isolate molecular clones of biologically active integrated proviral genomes from spleens of infected turkeys. Based upon molecular hybridization and nucleotide sequence comparisons of its pol gene, LPDV was shown to represent a distinct group of avian retroviruses most closely related to avian sarcoma-leukemia viruses. Here we report the complete nucleotide sequence of the LPDV genome as well as amino acid sequence analysis of its gag gene products. The genetic organization of LPDV is characteristic of members of the oncovirus subfamily. Further sequence comparisons of the gag gene confirmed that LPDV is most closely related to Rous sarcoma virus (RSV). However, the gag, pro, and pol open reading frames (ORFs) were in different translational phases so that the expression of their mature gene products would require the double frame-shifting mechanism utilized by simian retroviruses, mouse mammary tumor virus, and human T-cell leukemia virus. In contrast, the RSV proteinase is synthesized as part of the gag precursor. The LPDV gag gene differs from that of RSV as well as from all other retroviruses in that it encodes a unique 31,000-Da (p31) protein, located between the MA and the CA coding sequences. FOur short ORFs of unknown function were present, Whether the putative products of these ORFs account for the acute nature of LPDV-induced disease remains to be determined.

Amino Acid Sequence↗
PubMed1994

Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI.

Cleavage specificity of RNase HI was examined on model Okazaki fragments, to determine the likely role of this nuclease in lagging strand DNA replication. Each substrate was prepared by annealing a short RNA primer, made by transcription in vitro, to a single-stranded synthetic DNA template, and subsequently extending the primer by DNA polymerization. The calf thymus RNase HI makes a structure-specific endonucleolytic cleavage in the RNA primer, releasing it intact, and leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. This specific cleavage, one nucleotide upstream of the RNA-DNA junction, is RNA primer sequence- and length-independent. Cleavage specificity is lost if the RNA primer is not extended with DNA, or if the substrate has a nick at the RNA-DNA junction. In addition, the cleavage at a single site requires Mg2+. Cleavage in the presence of Mn2+ is less specific. Neither human immunodeficiency virus reverse transcriptase nor Escherichia coli RNases H perform such a structure-specific cleavage before an RNA-DNA junction. Our work indicates that calf RNase HI is designed to recognize Okazaki fragments. It has the specificity to remove their initiator RNA segments, except for one ribonucleotide, by a single endonucleolytic cleavage in vivo.

Animals↗
PubMed1994

Enzymatic completion of mammalian lagging-strand DNA replication.

Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis from an upstream fragment to generate a nick, and then ligation. With our substrate, RNase H type I (RNase HI) makes a single cut in the initiator RNA, one nucleotide 5' of the RNA-DNA junction. The double strand specific 5' to 3' exonuclease removes the remaining monoribonucleotide. After dissociation of cleaved RNA, synthesis by DNA polymerase generates a nick, which is then sealed by DNA ligase I. The unique specificities of the two nucleases for primers with initiator RNA strongly suggest that they perform the same reactions in vivo.

Animals↗
PubMed1994

Pulsed field capillary electrophoresis of multikilobase length nucleic acids in dilute methyl cellulose solutions.

Pulsed field capillary gel electrophoresis in dilute methyl cellulose solutions is used to separate nucleic acid fragments in the size range 75-23,000 base pairs. Field inversion is shown to increase resolution for fragments longer than about 500 base pairs. Methyl cellulose solutions as dilute as 0.01% can be used. Intermolecular hydrogen bonding is suggested as the cause of apparent cellulose fiber entanglement at concentrations below the calculated entanglement limit. The 1-kb DNA ladder and the lambda DNA/HindIII restriction fragment mixtures are each baseline resolved in a 28-cm capillary in less than 9 min at 180 V/cm (dc component).

Bacteriophage lambda↗
PubMed1994

Effect of high-fat diet on gene expression of GLUT4 and insulin receptor in soleus muscle.

Gene expression of GLUT4 and insulin receptor in soleus muscle of high-fat and high-carbohydrate diet fed rats was studied by measuring mRNA. High-fat diet feeding increased plasma glucose but decreased plasma insulin level. Glucose uptake in soleus muscle measured by 2-deoxy-D-glucose technique was lower in high-fat than high-carbohydrate diet fed rats. GLUT4 mRNA level in soleus muscle was significantly decreased but insulin receptor mRNA was similar in high-fat compared with high-carbohydrate diet fed rats. Insulin receptor exists in two forms generated by alternative splicing of a primary gene transcript. There was no difference in the relative expression of insulin receptor isoform mRNAs between high-fat and high-carbohydrate diet fed rats. These results suggest that high-fat diet impairs glucose metabolism in muscle by reducing transcription of GLUT4 without affecting gene expression of insulin receptor.

Alternative Splicing↗
PubMed1994

Optical spectrum of myeloperoxidase. Origin of the red shift.

The optical spectrum of reduced myeloperoxidase (EC 1.11.1.7) displays an unusual red shift of the Soret band which is at 472 nm and the alpha-band which is at 636 nm. The spectral properties of myeloperoxidase can be modified by means of acid treatment. Upon short exposure to acid (pH 1.7) the red-shifted optical absorption spectrum of the reduced enzyme (lambda max at 472 nm) was blue-shifted (lambda max at 448 nm) but the spectrum of the reduced state could be restored by increasing the pH. By contrast, the resonance Raman spectra of both the oxidized and reduced enzyme are essentially the same at both pH 1.7 and pH 7.0. This shows that the optical spectrum and the resonance Raman spectrum are not directly correlated, which we interpret to indicate that the reversible effects of lower pH primarily affect the excited-state energy levels of the macrocycle. The EPR spectrum of the oxidized enzyme showed a reversible conversion from a high-spin rhombic spectrum (gx = 6.7, gy = 5.2) at neutral pH into a more axial high-spin spectrum (gx = gy = 5.8) at low pH. Upon prolonged exposure to acid (20 min) optical absorbance spectra, EPR spectra, resonance Raman spectra and the chlorinating activity were irreversibly affected. We propose that a negatively charged protonatable residue in the proximity of a pyrrole nucleus of the haem group is present that imposes the red shift in the optical absorption spectrum. This is consistent with the available X-ray structure data.

Electron Spin Resonance Spectroscopy↗
PubMed1994

Separation of nucleic acids by capillary electrophoresis in cellulose solutions with mono- and bis-intercalating dyes.

The effects of bis- and mono-intercalating dyes on the capillary electrophoretic separation of double-stranded DNA have been investigated in buffers containing (hydroxypropylmethyl)-cellulose. Broad bands and incomplete separations of phi X 174 DNA HaeIII digest were obtained with the bis-intercalators ethidium homodimer 1 (EthD-1) and ethidium homodimer 2 (EthD-2), under a wide range of dye and polymer concentrations. The above results were attributed to the presence of several intramolecular dye/DNA complexes for each fragment. For EthD-2/DNA complexes, but not for EthD-1/DNA complexes, additional bands beyond the expected number of fragments were observed. The extra bands were attributed to formation of intermolecular dimers. Use of the monomeric intercalating dyes, ethidium and propidium, allowed complete separation of all fragments of phi X 174 DNA HaeIII digest. A low-power green He-Ne laser (543.6 nm) was shown to be satisfactory for laser-induced fluorescence detection with any of these dyes.

Cellulose↗
PubMed1994

A loop between transmembrane helices IX and X of subunit I of cytochrome c oxidase caps the heme a-heme a3-CuB center.

Site-directed mutants were prepared of four consecutive and highly conserved residues (His-411, Asp-412, Thr-413, Tyr-414) of an extramembrane loop that connects putative transmembrane helices IX and X of subunit I of Rhodobacter sphaeroides cytochrome c oxidase. The modified enzymes were purified and analyzed by optical, resonance Raman, FTIR, and EPR spectroscopies. Consistent with our recent model in which both hemes are ligated to histidines of helix X [Hosler, J. P., et al. (1993) J. Bioenerg. Biomembr. 25, 121-136], substitutions for three of these four residues cause perturbations of either heme a or heme a3. Resonance Raman spectra of the mutant Y414F demonstrate that Tyr-414 does not participate in a hydrogen bond with the heme a formyl group, but its alteration does result in a 5-nm red-shift of the alpha-band of the visible spectrum, indicating proximity to heme a. The mutant D412N shows changes in resonance Raman and FTIR difference spectra indicative of an effect on the proximal ligation of heme a3. Changing His-411 to alanine has relatively minor effects on the spectral and functional properties of the oxidase; however, FTIR spectra reveal alterations in the environment of CuB. Conversion of this residue to asparagine strongly disrupts the environment of heme a3 and CuB and inactivates the enzyme. These results suggest that His-411 is very near the heme a3-CuB pocket. We propose that these residues form part of a cap over the heme a-heme a3-CuB center and thus are important in the structure of the active site.

Amino Acid Sequence↗
PubMed1994

FT-IR and near-infrared FT-Raman studies of the secondary structure of insulinotropin in the solid state: alpha-helix to beta-sheet conversion induced by phenol and/or by high shear force.

Insulinotropin (glucagon-like peptide I) is a peptide containing 31 amino acid residues. It stimulates the secretion of the hormone insulin. The solubility of this peptide is highly dependent on its environment and the treatment that it has undergone. For instance, synthetic insulinotropin is highly soluble in neutral phosphate-buffered saline (1 mg/mL). However, the application of shear force by stirring renders it extremely insoluble (1 micrograms/mL). This property may be explained in terms of a change in peptide secondary structure with no alteration in primary structure. In order to understand this phenomenon, FT-IR and near-IR FT-Raman were employed to examine four samples prepared under different experimental conditions. It was found that solubility decreases as the alpha-helix is converted to an antiparallel beta-sheet structure.

Amino Acid Sequence↗
PubMed1994

Correlation of the AUA symptom index with urodynamics in patients with suspected benign prostatic hyperplasia.

The AUA symptom index is widely used to access patients with suspected benign prostatic hyperplasia (BPH). In order to determine how well symptoms as assessed by this index correlate with urodynamic findings, we evaluated 83 patients referred to our urology clinics with symptoms of BPH. All patients completed the AUA symptom index and then underwent a multichannel urodynamic evaluation. Patients were classified as obstructed, unobstructed, or equivocal according to the Abrams Griffiths nomogram. The AUA symptom index was recorded as the total score and, for purposes of symptom classification, further subdivided into an obstructive score (questions 3, 5, and 6) and an irritative score (questions 1, 2, 4, and 7). The mean age of the 83 patients was 67 (45-84). The mean total AUA symptom score was 16.6 (6-34), mean obstructive score was 6.1 (0-15), and the mean irritative score 10.4 (3-20). Pressure flow analysis using the Abrams-Griffiths nomogram classified 28 patients (34%) as obstructed, 17 (20%) as unobstructed, and 38 (46%) as equivocal. Using the analysis of variance procedure (ANOVA) there was no statistically significant difference in the mean total (P = 0.446), obstructive (P = 0.979), or irritative (P = 0.136) scores. Detrusor instability was present in 45 patients (54%). While total and obstructive scores were not significantly different in patients with detrusor instability vs. those with stable bladders, irritative scores were higher in patients with instability (P = 0.028) using the T-test procedure. Using ANOVA, the difference in post void residual (PVR) between the groups was not quite statistically significant (P = 0.057).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

Constitutive activation of the collagenase promoter in recessive dystrophic epidermolysis bullosa fibroblasts: role of endogenously activated AP-1.

Recessive dystrophic epidermolysis bullosa (RDEB) is a mutilating disease of the skin characterized by recurrent blistering and erosions that result from compromised integrity of the basement membrane zone. In this study, fibroblasts derived from the skin of RDEB patients were characterized for expression of the major metalloproteinases, particularly interstitial collagenase. Consistent with previous reports on increased collagenase protein levels in fibroblasts from some RDEB patients, we found that steady-state levels of collagenase mRNA were significantly increased in fibroblast strains derived from three of five RDEB patients compared to fibroblasts obtained from normal donors. Stromelysin mRNA was elevated in the same three fibroblast strains, whereas expression of neither the 72- nor the 92-kDa type IV collagenases was different from that of controls. Tissue inhibitor of metalloproteinases was expressed in RDEB fibroblasts at levels similar to those observed in normal fibroblasts. To investigate the mechanism behind the steady-state elevation in collagenase and stromelysin expression, AP-1 expression and activation were studied. Although levels of Jun expression were not different from those seen in normal fibroblasts, AP-1 activity, as assessed by ability to bind to a TPA response element-containing oligonucleotide, was endogenously elevated in RDEB fibroblasts compared to normal fibroblasts. Transfection studies using a plasmid construct containing the collagenase promoter linked to a CAT reporter gene demonstrated that RDEB fibroblasts were able to support active transcription of the promoter compared to normal fibroblasts. These studies support the hypothesis that RDEB fibroblasts contain chronically activated AP-1, and perhaps other transactivating factors, that contribute to the cellular phenotype of collagenase and stromelysin overexpression.

Cells, Cultured↗