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S K Rhee

Publications and source records attributed to S K Rhee.

At least 37 records · Page 2Linked to original sources
PubMed1999

Transcriptional analysis of levU operon encoding saccharolytic enzymes and two apparent genes involved in amino acid biosynthesis in Zymomonas mobilis.

Extracellular levansucrase (LevU) and sucrase (InvB) are two of the three saccharolytic enzymes involved in the sucrose metabolism of Zymomonas mobilis. The levU and invB genes were clustered with a 155bp interval on the chromosome. Both genes were transcribed constitutively at the basal level and the transcription of both genes was induced significantly when sucrose was added to the medium. These genes were transcribed as a bicistronic mRNA and the expression was modulated by a single promoter, which is located upstream of the levU gene. The transcriptional initiation site was mapped to -64bp from the translation start site of levU gene. These results indicated that two genes are most likely to constitute an operon. The glk operon, which encodes four glycolytic enzymes, was located close to the levU operon on the chromosome. Two apparent ORFs (ORF3 and 4) were found at the intervening sequence located between the glk and levU operons. These ORFs were transcribed divergently and showed high homology at the amino acid level with the bacterial global regulatory protein (Lrp) and aspartate racemase.

Amino Acid Isomerases↗
PubMed1999

Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1.

Plasmids with different selectable markers were constructed and used to transform the Hansenula polymorpha strain DL1. It was shown that, depending on the host mutant strain, the use of these plasmids enables rapid selection of transformants with plasmids integrated in low (1-2), moderate (6-9) or high (up to 100) copy numbers. The vectors and mutant described are potentially useful for the construction of efficient producers of heterologous proteins in H. polymorpha.

Blotting, Southern↗
PubMed1999

A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1.

To facilitate the selection of multiple gene integrants in Hansenula polymorpha, a rapid and copy-number-controlled selection system was developed using a vector containing a telomeric autonomous replication sequence and the bacterial aminoglycoside 3-phosphotransferase (APH) gene. Direct use of the unmodified APH gene as a dominant selectable marker resulted in the extremely slow growth of transformants and the frequent selection of spontaneous resistance. For the proper performance of the APH gene, a set of deleted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters of H. polymorpha were fused to the APH gene. The fusion construct with the 578-bp GAPDH promoter conferred G418 resistance sufficient to allow rapid growth of transformants, and thus facilitated the selection of transformants with up to 15 tandem copies of the vector. To increase further the integration copy number within the gene-dose-dependent range, the GAPDH promoter was serially deleted down to the -61 nucleotide. With this weak expression cassette, the integration copy number could easily be controlled between 1 and 50. Tandemly integrated copies of plasmids near the end of the chromosome were mitotically stable over 150 generations. The dosage-dependent selection system of this study would provide a powerful tool for the development of H. polymorpha as an industrial strain to produce recombinant proteins.

Bacterial Proteins↗
PubMed1999

Phenylacetyl-CoA:acceptor oxidoreductase, a membrane-bound molybdenum-iron-sulfur enzyme involved in anaerobic metabolism of phenylalanine in the denitrifying bacterium Thauera aromatica.

Phenylacetic acids are common intermediates in the microbial metabolism of various aromatic substrates including phenylalanine. In the denitrifying bacterium Thauera aromatica phenylacetate is oxidized, under anoxic conditions, to the common intermediate benzoyl-CoA via the intermediates phenylacetyl-CoA and phenylglyoxylate (benzoylformate). The enzyme that catalyzes the four-electron oxidation of phenylacetyl-CoA has been purified from this bacterium and studied. The enzyme preparation catalyzes the reaction phenylacetyl-CoA + 2 quinone + 2 H2O --> phenylglyoxylate + 2 quinone H2 + CoASH. Phenylacetyl-CoA:acceptor oxidoreductase is a membrane-bound molybdenum-iron-sulfur protein. The purest preparations contained three subunits of 93, 27, and 26 kDa. Ubiquinone is most likely to act as the electron acceptor, and the oxygen atom introduced into the product is derived from water. The protein preparations contained 0.66 mol Mo, 30 mol Fe, and 25 mol acid-labile sulfur per mol of native enzyme, assuming a native molecular mass of 280 kDa. Phenylglyoxylyl-CoA, but not mandelyl-CoA, was observed as a free intermediate. All enzyme preparations also catalyzed the subsequent hydrolytic release of coenzyme A from phenylglyoxylyl-CoA but not from phenylacetyl-CoA. The enzyme is reversibly inactivated by a low concentration of cyanide, but is remarkably stable with respect to oxygen. This new member of the molybdoproteins represents the first example of an enzyme which catalyzes the alpha-oxidation of a CoA-activated carboxylic acid without utilizing molecular oxygen.

Amino Acid Sequence↗
PubMed1999

A family of telomere-associated autonomously replicating sequences and their functions in targeted recombination in Hansenula polymorpha DL-1.

A family of multiple autonomously replicating sequences (ARSs) which are located at several chromosomal ends of Hansenula polymorpha DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a chromosome, as a probe showed several homologues in the genome of H. polymorpha. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical to that of HARS36. All three fragments harbored an ARS motif and ended with 18 to 23 identical repetitions of 5'-GGGTGGCG-3' which resemble the telomeric repeat sequence in other eukaryotes. Transformation of H. polymorpha with nonlinearized plasmids containing the newly obtained telomeric ARSs almost exclusively resulted in the targeted integration of a single copy or multiple tandem copies of the plasmid into the chromosomes. The sensitivity to exonuclease Bal31 digestion of the common DNA fragment in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36. Even though the frequencies of targeted recombination were varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through homologous recombination at the telomeric repeats, which serve as high-frequency recombination targets.

Base Sequence↗
PubMed1998

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae PMR1 gene.

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPase. The cloned gene product exhibits 60.3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevesiae PMR1 gene product, a P-type Ca(2+)-ATPase.

ATP-Binding Cassette Transporters↗
PubMed1998

Heteromeric gap junction channels in rat hepatocytes in which the expression of connexin26 is induced.

More than one isotype of the gap junction channel-forming protein subunit, known as connexin, are synthesized in most mammalian cells. Using a modified primary cell culture of rat hepatocytes, in which both connexin32 and connexin26 were expressed in a comparable degree, the molecular composition of connexin subtypes in a gap junction channel (i.e., homomeric or heteromeric) was studied. A fluorescent dye Lucifer Yellow-coupling among hepatocytes was blocked in the presence of 20 microM beta-glycyrrhetinic acid, and antagonist for the gap junction channel. Similarly, either one of the antibodies raised against connexin32 or connexin26 completely inhibited dye-coupling activity among hepatocytes. In addition, we studied the dye-coupling properties at different extracellular pHs in two primary rat hepatocytes: one in which connexin26 was induced, and the other which expresses connexin32 as a major gap junction channel protein. The dye-coupling among the connexin26-induced hepatocytes was more sensitive to lowering pHs than among the normal hepatocytes, in which less than 5% of gap junction protein is connexin26. Taken together, data obtained in our study strongly suggest that the connexins (32 & 26) are assembled to form heteromeric, rather than homomeric, gap junction channels in the connexin226-induced rat hepatocytes.

Administration, Topical↗
PubMed1998

Amyloid beta protein-(1-42) forms calcium-permeable, Zn2+-sensitive channel.

Amyloid beta protein (AbetaP) forms senile plaques in the brain of the patients with Alzheimer's disease. The early-onset AD has been correlated with an increased level of 42-residue AbetaP (AbetaP1-42). However, very little is known about the role of AbetaP1-42 in such pathology. We have examined the activity of AbetaP1-42 reconstituted in phospholipid vesicles. Vesicles reconstituted with AbetaP show strong immunofluorescence labeling with an antibody raised against an extracellular domain of AbetaP suggesting the incorporation of AbetaP peptide in the vesicular membrane. Vesicles reconstituted with AbetaP showed a significant level of 45Ca2+ uptake. The 45Ca2+ uptake was inhibited by (i) a monoclonal antibody raised against the N-terminal region of AbetaP, (ii) Tris, and (iii) Zn2+. However, reducing agents Trolox and dithiothreitol did not inhibit the 45Ca2+ uptake, indicating that the oxidation of AbetaP or its surrounding lipid molecules is not directly involved in the AbetaP-mediated Ca2+ uptake. An atomic force microscope was used to image the structure and physical properties of these vesicles. Vesicles ranged from 0.5 to 1 microm in diameter. The stiffness of the AbetaP-containing vesicles was significantly higher in the presence of calcium. The stiffness change was prevented in the presence of zinc, Tris, and anti-AbetaP antibody but not in the presence of Trolox and dithiothreitol. Thus the stiffness change is consistent with the vesicular uptake of Ca2+. These findings provide biochemical and structural evidence that AbetaP1-42 forms calcium-permeable channels and thus may induce cellular toxicity by regulating the calcium homeostasis in Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed1998

Glycosylation of human alpha 1-antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts.

Human alpha 1-antitrypsin (alpha 1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human alpha 1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant alpha 1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant alpha 1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted alpha 1-AT. The human alpha 1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of alpha 1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of alpha 1-AT in S. cerevisiae. The alpha 1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of alpha 1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein.

Amino Acid Sequence↗
PubMed1998

Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules.

Intercellular connexin channels (gap junction channels) have long been thought to mediate molecular signaling between cells, but the nature of the signaling has been unclear. This study shows that connexin channels from native tissue have selective permeabilities, partially based on pore diameter, that discriminate among cytoplasmic second messenger molecules. Permeability was assessed by measurement of selective loss/retention of tracers from liposomes containing reconstituted connexin channels. The tracers employed were tritiated cyclic nucleotides and a series of oligomaltosaccharides derivatized with a small uncharged fluorescent moiety. The data define different size cut-off limits for permeability through homomeric connexin-32 channels and through heteromeric connexin-32/connexin-26 channels. Connexin-26 contributes to a narrowed pore. Both cAMP and cGMP were permeable through the homomeric connexin-32 channels. cAMP was permeable through only a fraction of the heteromeric channels. Surprisingly, cGMP was permeable through a substantially greater fraction of the heteromeric channels than was cAMP. The data suggest that isoform stoichiometry and/or arrangement within a connexin channel determines whether cyclic nucleotides can permeate, and which ones. This is the first evidence for connexin-specific selectivity among biological signaling molecules.

Animals↗
PubMed1998

Efficient production of intact human parathyroid hormone in a Saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3).

When human parathyroid hormone (hPTH) is expressed as a secretory product in yeast, the main problem is the aberrant proteolytic cleavage that reduces the yield of intact protein. To overcome this problem, we developed an hPTH expression system using a host strain in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 48 h of culture, most of the hPTH secreted by the yap3 disruptant remained intact, whereas more than 90% of the hPTH secreted by the wild-type strain was cleaved. When the authentic hPTH was incubated in each of the culture supernatants of untransformed yap3 disruptant and wild-type strain, the proteolysis proceeded much more slowly in the culture supernatant of yap3 disruptant than in that of the wild type. The extent of hPTH proteolysis was also significantly reduced by the addition of pepstatin A, a specific aspartic protease inhibitor. The results suggest that YAP3 is involved in the internal cleavage of hPTH expressed in yeast. The correct processing of the intact hPTH secreted in the yap3 disruptant demonstrates that the yeast mutant lacking the YAP3 activity is a suitable host for the high-level expression of intact hPTH.

Aspartic Acid Endopeptidases↗
PubMed1998

Cloning of the ribosomal protein L41 gene of Phaffia rhodozyma and its use a drug resistance marker for transformation.

The ribosomal protein L41 gene of Phaffia rhodozyma was cloned and used as a dominant selectable marker for cycloheximide resistance in the transformation of P. rhodozyma. Electrotransformation with a plasmid containing a ribosomal DNA fragment as a targeting signal typically yielded 800 to 1,200 transformants/microgram of DNA with an integrated copy number of about seven per haploid genome.

Cloning, Molecular↗
PubMed1997

H+-pumping ATPase has little stimulatory effect on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles.

We studied the effect of a H+ electrochemical potential generated by F1F0 ATPase on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles. The F1F0 ATPase of V. alginolyticus catalyzed the pumping of H+ coupled to ATP hydrolysis as measured in fluorescence quenching experiments. Consequently, this enzyme leads to the generation of a H+ electrochemical potential. The H+ electrochemical potential generated by F1F0 ATPase was completely abolished by 30 microM N,N'-dicyclohexylcarbodiimide (DCCD) or 5 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) at 30 degrees C. The treatment of membrane vesicles with 30 microM DCCD at 30 degrees C had little influence on the translocation activity of uncleavable OmpF-Lpp, a model secretory protein, as compared to the intact membrane vesicles. On the other hand, the NADH:quinone oxidoreductase of V. alginolyticus is known to be a Na+ pump that leads to generation of a Na+ electrochemical potential. This Na+ electrochemical potential stimulates protein translocation into inside-out membrane vesicles prepared from V. alginolyticus in the presence of Escherichia coli SecA [Tokuda, H., Kim, Y. J., and Mizushima, S. (1990) FEBS Lett. 264, 10-12] From these results, it is evident that the stimulatory effect of the Na+ electrochemical potential on protein translocation in V. alginolyticus is not affected by the H+ electrochemical potential influence of F1F0 ATPase.

Adenosine Triphosphatases↗
PubMed1997

Fatty acid carbon isotope ratios in humans on controlled diets.

Carbon stable isotope ratios for six serum fatty acids (FA) are reported for human subjects on controlled fat diets to determine the range of natural isotope abundance and to demonstrate the leveling effect of a well-controlled diet. Twenty-nine subjects were randomly assigned to one of three controlled diets containing high, medium, or low fat. Diets were consumed for 8 wk. Serum samples were collected at baseline (0), 5, 6, 7, and 8 wk. FA were extracted and methylated. Isotope ratios were analyzed by high-precision gas chromatography combustion-isotope ratio mass spectrometry. At baseline, mean delta 13C for 16:0b, 16:1a, 18:0a, 18:1c,18:2n-6d and 20:4n-6bc were -24.1, -21.7, -21.6, -25.6, -29.6, and -25.0/1000, respectively, with an average standard deviation of 1.9/1000. Most delta 13C decreased during the diet period and appeared to have stabilized by week 5 at -25.3, -21.9, -22.3, -26.5, -30.1, and -24.5/1000, respectively. Between-subjected variability decreased from 1.74 to 1.20/1000 on the controlled diets. Measurement variability was 0.53/1000. The within-subject variability during weeks 5-8 was 0.57/1000 (range of 0.32-0.84/1000), showing a minimum biological fluctuation on controlled diets. There was no diet group effect on delta 13C of serum FA. Except for 18:2, the delta 13C of experimental diets was lower than that of serum FA, consistent with observations in animals. These data show that carbon isotope ratios stabilize in response to controlled diets within 5 wk, reflecting the isotope ratio of their dietary source, and establish isotope ratio fluctuations for endogenous compounds for future studies.

Adult↗
PubMed1997

Desaturation and interconversion of dietary stearic and palmitic acids in human plasma and lipoproteins.

Dietary saturated fatty acids are implicated as a risk factor for atherosclerosis. The conversion of the major dietary saturated fatty acids stearic acid (18:0) and palmitic acid (16:0) to monounsaturated fatty acids in whole plasma and lipoprotein fractions is reported for seven healthy adult humans over 6 d using [U-13C]stearic acid (18:0*) and [U-13C]palmitic acid (16:0*) and high-precision mass spectrometry. A tracer dose (28-32 mg) of 18:0* or 16:0* was loaded into an emulsion and orally administered before breakfast. Serial blood samples were collected on day 1 and fasting blood was drawn daily until day 7. Overall conversion of 18:0 to 18:1 was approximately 14%, whereas that of 18:0 to 16:0 was approximately 2% in plasma up to 144 h. Conversion of 16:0 to 16:1 was < 2%, whereas conversion of 16:0 to 18:0 was approximately 6%. No other fatty acid metabolites were detected for 18:0* or 16:0*. The conversion products were observed mainly in chylomicrons and very-low-density lipoproteins, indicating that the intestine and liver have comparable roles in desaturating 18:0 and 16:0. Overall, these data indicate that dietary 18:0 desaturation is severalfold greater than 16:0 desaturation. The low level (14%) of 18:0 desaturation in omnivorous adults may have little influence on blood lipid profiles relevant to atherosclerosis risk.

Adolescent↗
PubMed1997

Nocardioides pyridinolyticus sp. nov., a pyridine-degrading bacterium isolated from the oxic zone of an oil shale column.

A bacterial strain which is able to degrade pyridine was previously isolated from the oxic zone of an oil shale column and described as Pimelobacter sp. strain OS4T. However, Pimelobacter species have been transferred to the genera Nocardioides and Terrabacter. Strain OS4T was identified as a member of the genus Nocardioides on the basis of chemotaxonomic analysis and phylogenetic inference based on 16S ribosomal DNA (rDNA) sequence analysis. The G+C content of strain OS4T is 72.5 mol%. The cell wall peptidoglycan contains LL-diaminopimelic acid as the diamino acid. The predominant menaquinone is MK-8(H4). The cellular fatty acid profile of strain OS4T is similar to that of the genus Nocardioides. The 16S rDNA similarity of strain OS4T with previously described Nocardioides species is 94.5% +/- 0.7%, and a phylogenetic tree based on 16S rDNA sequences revealed a distinct lineage for strain OS4T within the evolutionary radiation enclosed by the genus Nocardioides. Therefore, on the basis of our data, we propose that strain OS4T should be placed in the genus Nocardioides as a member of a new species, Nocardioides pyridinolyticus. The type strain of the new species is strain OS4 (= KCTC 0074BP).

Base Composition↗
PubMed1997

Anaerobic and aerobic degradation of pyridine by a newly isolated denitrifying bacterium.

New denitrifying bacteria that could degrade pyridine under both aerobic and anaerobic conditions were isolated from industrial wastewater. The successful enrichment and isolation of these strains required selenite as a trace element. These isolates appeared to be closely related to Azoarcus species according to the results of 16S rRNA sequence analysis. An isolated strain, pF6, metabolized pyridine through the same pathway under both aerobic and anaerobic conditions. Since pyridine induced NAD-linked glutarate-dialdehyde dehydrogenase and isocitratase activities, it is likely that the mechanism of pyridine degradation in strain pF6 involves N-C-2 ring cleavage. Strain pF6 could degrade pyridine in the presence of nitrate, nitrite, and nitrous oxide as electron acceptors. In a batch culture with 6 mM nitrate, degradation of pyridine and denitrification were not sensitively affected by the redox potential, which gradually decreased from 150 to -200 mV. In a batch culture with the nitrate concentration higher than 6 mM, nitrite transiently accumulated during denitrification significantly inhibited cell growth and pyridine degradation. Growth yield on pyridine decreased slightly under denitrifying conditions from that under aerobic conditions. Furthermore, when the pyridine concentration used was above 12 mM, the specific growth rate under denitrifying conditions was higher than that under aerobic conditions. Considering these characteristics, a newly isolated denitrifying bacterium, strain pF6, has advantages over strictly aerobic bacteria in field applications.

Aerobiosis↗