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J Yim

Publications and source records attributed to J Yim.

33 records · Page 2Linked to original sources
PubMed1998

Molecular characterization of the Drosophila melanogaster gene encoding the pterin 4alpha-carbinolamine dehydratase.

We have isolated and characterized the cDNA and the genomic DNA encoding Drosophila melanogaster pterin 4alpha-carbinolamine dehydratase (PCD). The amino acid sequence deduced from the cDNA sequence was very similar to those of PCDs previously reported in other species (19-57% identity). The protein coding region of the cDNA was expressed in E. coli as a histidine fusion protein, and the expressed protein proved to have PCD activity. The characterization of the Drosophila genomic clone revealed that the Drosophila PCD gene is interrupted by two introns. The potential promoter region, deduced from the determination of the transcription start point (tsp), lacks the distinct TATAAA box consensus sequence.

Amino Acid Sequence↗
PubMed1998

Cloning and structure of a rabbit protein inhibitor of neuronal nitric oxide synthase (PIN) gene and its pseudogene.

Neuronal nitric oxide synthase (nNOS), the biosynthetic enzyme for the free radical neurotransmitter nitric oxide, was found to be inhibited by the protein inhibitor of nNOS, designated PIN (Jaffrey, S.R. and Snyder, S.H., (1996) Science 274, 774-777). The cDNA clone encoding a rabbit PIN was isolated, and the translated PIN protein was89 amino acid long sharing 100% of its deduced amino acid sequence with rat PIN. Using a radiolabelled riboprobe derived from the rabbit PIN cDNA, the rabbit PIN gene was isolated from a rabbit genomic library and the structural organization of the gene was determined. The gene contains three exons separated by two introns spanning approx. 2.3kb of genomic DNA. 5' RACE analysis mapped the transcriptional initiation site 98bp upstream of the initiator methionine codon. Characterization of the 5' flanking genomic region revealed that the rabbit PIN promoter is TATA-less, but contains a CCAAT box as well as various putative transcription factor-binding elements. The 3' untranslated region contains consensus polyadenylation signal (AATAAA). We also isolated an intronless gene with 93% nucleotide sequence similarity to the rabbit PIN cDNA. Sequence analysis indicates that the open reading frame was interrupted by a premature stop codon and frameshift which resulted in a processed pseudogene of the rabbit PIN.

Amino Acid Sequence↗
PubMed1997

Characterization of citrate synthase purified from Drosophila melanogaster.

Citrate synthase which condenses acetyl-CoA and oxaloacetate to citrate was purified from Drosophila melanogaster. Some physicochemical as well as enzymatical properties were investigated. The optimum pH and temperature were pH 8.0-9.0 and 45 degrees C, respectively. The molecular weight of the enzyme was determined as 81,000 Da by gel filtration and the purified active enzyme consisted of two identical subunits which had a molecular mass of 48,700 on SDS-PAGE. Homogeneity of the purified enzyme was confirmed by SDS-PAGE and also by N-terminal amino acid sequence analysis. The Michaelis constants (K(m)) of the enzyme for acetyl-CoA and oxaloacetate were 6.7 microM and 3.1 microM, respectively. Kinetic studies showed that citrate synthase follows the concerted mechanism which forms a ternary complex. Propionyl-CoA, ATP, and intermediates of the TCA cycle, succinyl-CoA and alpha-ketoglutarate, behaved as inhibitors in vitro. Using pig and chicken heart enzymes for comparison, we found similarities at the N-terminal region. However, in the Ouchterlony immunodiffusion test, the polyclonal antibody raised against Drosophila citrate synthase did not show any crossreaction with pig, chicken or pigeon enzymes.

Animals↗
PubMed1997

The binding of two dimers of IciA protein to the dnaA promoter 1P element enhances the binding of RNA polymerase to the dnaA promoter 1P.

Transcription of the dnaA gene from the promoter 1P has been shown to be activated in vitro and in vivo by the binding of IciA protein to two sites on the dnaA promoter region [Lee, Y. S., Kim, H., and Hwang, D. S. (1996) Mol. Microbiol . 19, 389-396; Lee, Y. S., and Hwang, D. S. (1997) J. Biol. Chem. 272, 83-88]. In vitro transcription assays using DNA fragments carrying variable combinations of two IciA binding sites revealed that IciA binding site I (IciA I site), which is located upstream of the promoter 1P, is responsible for the transcriptional activation. Binding of one dimeric IciA protein to the IciA I site is followed by binding of the second dimer. Two dimers of IciA protein, rather than one dimer, on the IciA I site appeared to enhance the binding of RNA polymerase to the promoter 1P, resulting in the activation of transcription from the promoter 1P.

Bacterial Proteins↗
PubMed1997

Molecular characterization of a nonsuppressible allele (prC4) of the Drosophila purple gene.

Purple gene encodes 6-pyruvoyl tetrahydropterin synthase (PTP synthase) in Drosophila. The enzyme PTP synthase catalyzes the conversion of dihydroneopterin triphosphate (H2-NTP) to 6-pyruvoyl tetrahydropterin (PTP), an important intermediate for pterin compounds. The extreme purple mutant, prC4, shows a very low activity of PTP synthase. The mutant purple gene has been cloned by screening with the subgenomic library of prC4. The size and expression level of PTP synthase gene transcripts in prC4 were almost the same as those of the wild type. The genomic DNA was also examined in the purple region by Southern blot analysis, but no changes in restriction pattern could be detected. Compared with the wild type PTP synthase sequence, the mutant PTP synthase of prC4 showed three missense mutations: the replacement of alanine 7 by serine (A7S), leucine 9 by phenylalanine (L9P), and aspartic acid 168 by glycine (D168G). Significance of these mutations was discussed in relation to the formation of the oligomeric structure of PTP synthase.

Alleles↗
PubMed1997

Purification, cloning, and functional expression of dihydroneopterin triphosphate 2'-epimerase from Escherichia coli.

Dihydroneopterin triphosphate (H2NTP) 2'-epimerase from Escherichia coli catalyzes the epimerization of H2NTP to dihydromonapterin triphosphate (H2MTP). The enzyme was purified 954-fold to apparent homogeneity by a combination of ammonium sulfate fractionation and column chromatography of Cibacron blue 3GA dye ligand, phenyl-Sepharose CL-4B, methotrexate-agarose, and Superdex 200 HR 10/30 FPLC column. The molecular mass of the epimerase determined on a Superdex column was 82.6 kDa, while the subunit molecular mass determined on SDS-polyacrylamide gel electrophoresis was 13.7 kDa. This implies that the epimerase most probably exists as homohexamer. The 20-amino acid sequence from the N terminus was determined (AQPAAIIRIKNLRLRTFIGI). Based on this sequence, the gene encoding the epimerase was cloned using a simple polymerase chain reaction approach. Translation of the nucleotide sequence of the cloned gene revealed the presence of an open reading frame containing 120 amino acids with a predicted molecular mass of 13,993 Da. The epimerase gene located in a 2.3-kilobase BamHI-EcoRI fragment from Kohara's clone 406 was overexpressed 300-fold, which was confirmed by the prominent increase in the 14-kDa protein band on SDS-polyacrylamide electrophoresis gels. It showed no homology with the sequences of isomerases or other enzymes in GenBank/EMBL data bases.

Amino Acid Sequence↗
PubMed1997

Clinical characteristics and management of acute stroke in patients with atrial fibrillation admitted to US university hospitals.

UNLABELLED: The optimal evaluation and management of patients with atrial fibrillation who suffer an acute ischemic stroke remains controversial. METHODS: Medical records of 171 consecutive patients with atrial fibrillation and acute stroke at six U.S. university hospitals were reviewed. Data collected included the use of antithrombotic therapy, brain and cardiac imaging, bleeding complications, stroke risk factors, and contraindications to anticoagulation. RESULTS: Mean age was 75.4 years. Cardiovascular risk factors associated with increased stroke risk were present in 87%; 35% had at least one contraindication to anticoagulation. Half of the patients with stroke risk factors and no contraindications to anticoagulation were not receiving any antithrombotic therapy at the time of admission. Of the 22 patients who were treated with warfarin, and had INR values on admission, 16 had levels of < 2.0; only six had INR values between 2.0 and 3.0. Transthoracic echocardiography was performed in 107 patients (63%); intracardiac thrombi were visualized in only 5%. Initial brain imaging revealed hemorrhagic transformation in nine. Heparin was used in 93 patients (54%), usually within 48 hours of stroke onset. Patients who received delayed heparin typically did not have repeat brain imaging prior to starting heparin. One patient had a delayed symptomatic cerebral hemorrhage. Of the survivors, 47% were discharged and treated with warfarin (or warfarin plus aspirin), 28% with ASA, 7% with other antithrombotic therapies, and 18% with no antithrombotic therapy. CONCLUSION: Antithrombotic therapy was underutilized and inadequately monitored in atrial fibrillation patients prior to stroke onset. After hospital admission, a wide range of diagnostic and management strategies, which often did not follow current recommendations, were employed.

Acute Disease↗
PubMed1996

Isolation by preparative free-flow electrophoresis and aqueous two-phase partition from rat adipocytes of an insulin-responsive small vesicle fraction with glucose transport activity.

Preparative free-flow electrophoresis and aqueous two-phase polymer partition were used to obtain a plasma membrane-enriched fraction of adipocytes isolated from epididymal fat pads of the rat together with a fraction enriched in small vesicles with plasma membrane characteristics (thick membranes, clear dark-light-dark pattern). The electrophoretic mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged molecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fat cells treated or not treated with 100 microU/ml insulin and the resident proteins of the two fractions analyzed by SDS-PAGE, the two fractions exhibited characteristic responses involving specific protein bands. Insulin treatment for 2 min resulted in the loss of a 90 kDa band from the plasma membrane. At the same time, a ca. 55-kDa peptide band that was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 glucose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation by treatment of adipocytes with insulin.

Adipocytes↗
PubMed1996

Structure and expression of wild-type and suppressible alleles of the Drosophila purple gene.

Viable mutant alleles of purple (pr), such as prbw, exhibit mutant eye colors. This reflects low 6-pyruvoyl tetrahydropterin (PTP) synthase activity required for pigment synthesis. PTP synthase is also required for synthesis of the enzyme cofactor biopterin; presumably this is why some pr alleles are lethal. The prbw eye color phenotype is suppressed by suppressor of sable [su(s)] mutations. The pr gene was cloned to explore the mechanism of this suppression. pr produces two PTP synthase mRNAs: one constitutively from a distal promoter and one in late pupae and young adult heads from a proximal promoter. The latter presumably supports eye pigment synthesis. The prbw allele has a 412 retrotransposon in an intron spliced from both mRNAs. However, the head-specific mRNA is reduced > 10-fold in prbw and is restored by a su(s) mutation, while the constitutive transcript is barely affected. The Su(s) protein probably alters processing of RNA containing 412. Because the intron containing 412 is the first in the head-specific mRNA and the second in the constitutive mRNA, binding of splicing machinery to nascent transcripts before the 412 insertion is transcribed may preclude the effects of Su(s) protein.

Alcohol Oxidoreductases↗
PubMed1996

Analysis of the relative level of gene expression from different retroviral vectors used for gene therapy.

We have analyzed the relative level of gene expression and viral titer from different types of retroviral vectors used for gene therapy, the LTR-based MFG vector and the internal promoter-containing vectors, LNCX, LNSX and LXSN. The CAT gene was used for comparison of retroviral vector gene expression in both transfected and transduced cells, while the neo gene was used to evaluate viral liter. In transfected cells, MFG-CAT expressed higher levels of CAT then the other vectors, LNC-CAT was next, while L-CAT-SN and LNS-CAT produced much lower levels. CAT expression from MFG-CAT was particularly high in the human T lymphoid cell lines CEM-SS and H9. In nonselected transduced cells. CAT expression from MFG was 10- to 50-fold higher than with the other vectors. Similar observations were made with retroviral constructs expressing human EPO and murine GM-CSF. In transient transfection assays, the titer of MFG was at least five-fold higher than the other vectors as determined by Southern analysis and G418 resistance. Analysis of the steady-state RNAs produced after transfection of the packaging cell lines showed that MFG expressed a significantly higher level of genomic RNA, which contains the packaging signal, than the other vectors while still expressing a high level of the subgenomic RNA encoding CAT. The high level of genomic RNA most likely contributes directly to the higher titer of MFG. We also compared viral titers from subcloned PA317 producer lines containing LNC-CAT and MFG-CAT-Neo, and confirmed that the titer of the MFG virus was higher than that of the LNCX. In selected subcloned transduced NIH3T3 cells, average levels of CAT activity were nine-fold higher from MFG-based vector. Our results suggest that there are significant differences in both the titer and the level of gene expression between retroviral vectors which are currently being used in gene therapy clinical trials.

Animals↗
PubMed1996

A simple and rapid method for the determination of recombinant retrovirus titer by G418 selection.

We have developed a simple and rapid method for the determination of retroviral titer by G418 selection which is both accurate and reproducible. NIH3T3 cells were transduced with recombinant retroviral vectors harboring the neo gene and selected in the presence of the antibiotic G418 for 3 days. Cells were then trypsinized, harvested, and counted on a hemacytometer. The control NIH3T3 cells were completely dead 3 days after treatment with G418, while cells transduced with retroviral vectors containing the neo gene produced an average of 15 progeny cells per colony. To estimate viral titer, therefore, the total number of trypsinized G418-resistant cells was divided by 15. Retroviral titers measured by this method did not differ significantly from those determined by the conventional method based on counts of G418-resistant colonies 10-14 days after G418 selection. The method worked well with various retroviral constructs and its efficacy was not influenced by cDNA insert. This method not only reduces the amount of work but also shortens the time needed for determining viral titer.

3T3 Cells↗
PubMed1990

GTP hydrolysis by transitional endoplasmic reticulum from rat liver inhibited by all-trans-retinol.

GTP hydrolysis by an endoplasmic reticulum fraction from rat liver enriched in part-rough, part-smooth transition elements was inhibited by all-trans-retinol half maximally at a concentration of about 10 micrograms/ml. Similar results were obtained with GTPase activity partially purified by ion-exchange (DE-52) chromatography. The inhibition was non-competitive and given by both retinol and retinaldehyde but not by retinoic acid or alpha-tocopheryl acetate. The hydrolysis of other nucleoside di- and triphosphates was much less affected by retinol. The activity was inhibited by detergents but at much higher concentrations than by retinol. The results suggest that enhancement of cell-free transfer from endoplasmic reticulum to Golgi apparatus by retinol observed previously at low concentrations of cytosol may be mediated through an interaction with GTP.

Animals↗