Biomedical subjects

C Kang

Publications and source records attributed to C Kang.

At least 73 records · Page 4Linked to original sources

PubMed1996

The number of identical kringle IV repeats in apolipoprotein(a) affects its processing and secretion by HepG2 cells.

A variable number of 5.6-kilobase kringle IV repeats in the human apolipoprotein(a) (apo(a)) gene results in a size polymorphism of the protein and correlates inversely with the plasma levels of the atherogenic lipoprotein(a) (Lp(a)). In order to analyze whether this association reflects a direct effect of kringle IV repeat number on Lp(a) plasma concentration, we have studied the expression of recombinant apo(a) (r-apo(a)) isoforms in the human hepatocarcinoma cell line HepG2. Following transient transfection of apo(a) cDNA expression plasmids that differed only in the number of kringle IV repeats, we observed a gradual decrease of Lp(a) in the medium of the cells with an increasing number of kringle IV repeats, mimicking the relationship present in humans in vivo. The analysis of apo(a) protein in the lysate and in the medium of cells that were transfected with a plasmid encoding an apo(a) isoform with 22 kringles revealed a predominant intracellular precursor with little secretion of the mature apo(a) protein. In contrast, transfection of a plasmid encoding an isoform with 11 kringles led to effective secretion of the mature peptide into the medium, indicating differential processing rates of apo(a) isoforms in the secretory path way. The intracellular accumulation of an apo(a) precursor in the endoplasmic reticulum was demonstrated by cell fractionation and [35S]Met metabolic labeling/temperature block experiments using HepG2 cells stably transfected with recombinant apo(a). The direct and causal effect of kringle IV repeat number on the expression of recombinant apo(a) in HepG2 cells, and presumably liver cells, provides a novel mechanism for the genetic regulation of the concentration of a protein.

Apolipoproteins A↗

PubMed1996

Subunit complementation of Escherichia coli adenylosuccinate synthetase.

Data are presented, based upon subunit complementation experiments, that suggest that Escherichia coli adenylosuccinate synthetase contains two shared active sites between its dimeric interface. This conclusion was alluded to by use of mutant forms of adenylosuccinate synthetase previously prepared by site-directed mutagenesis. The experiments indicate that, although the R143L and D13A mutants have low or no activity independently, when they are mixed, a significant amount of activity was obtained. These results indicate that the subunits exchange with each other to form heterodimers with a single viable wild-type active site. The kcat value for the active hybrid active site in the R143L-D13A heterodimer is virtually identical to that observed with the wild-type enzyme, and the other kinetic parameters are very similar to those found for the wild-type enzyme. An analysis of the restoration of the activity in the presence of substrates suggests that GTP and IMP stabilize the dimeric structure of the protein. A comparison of the restoration of the activity using different combinations of mutants provides evidence indicating that some of the GTP binding elements, including the P-loop, in the protein are important for subunit integrity. Also, for the first time, a comprehensive analysis of subunit complementation is performed for the two inactive mutants (R143L and D13A) where the dissociation constants for the R143L-D13A heterodimer and the D13A homodimer were determined to be 21 and 2.9 microM, respectively. A concentration dependence of the specific activity of the wild-type protein in this study shows that the Kd for dimer dissociation is approximately 1 microM.

Adenylosuccinate Synthase↗

PubMed1996

Involvement of arginine 143 in nucleotide substrate binding at the active site of adenylosuccinate synthetase from Escherichia coli.

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thio]phosphate (GMPSBDB) at pH 7.1 and 25 degrees C. Reaction of the enzyme with [8-3H]GMPSBDB results in the incorporation of 2 mol of the reagent/mol of subunit; in the presence of active site ligands the incorporation is reduced to 1 mol of reagent/mol of subunit. GMPSBDB reacts with Cys-291 in the initial rapid reaction which is accompanied by loss of 50% of the enzymatic activity; this reaction is not affected by the presence of active site ligands. In the slower reaction, GMPSBDB inactivates the enzyme by reacting with Arg-143. The inactivation kinetics of the slower phase are consistent with the formation of an enzyme--GMPSBDB complex having a Kd of 42 microM. Active site nucleotides, either adenylosuccinate or IMP + GTP, prevent both slower phase inactivation and labeling of Arg-143. Replacement of Arg-143 with a Leu by site-directed mutagenesis does not change the catalytic constant or the K(m) for aspartate but does significantly impair nucleotide binding: the Michaelis constants for IMP and GTP increase by 60-fold and 10-fold, respectively, in the R143L mutant. The crystal structure of the E. coli enzyme [Poland, B.W., Silva, M.M., Serra, M.A., Cho, Y., Kim, K. H., Harris, E.M.S., & Honzatko, R.B. (1993) J. Biol. Chem. 268, 25334--25342] shows that Arg-143 from one subunit projects into the putative active site of the other subunit. These results indicate that both subunits of dimeric adenylosuccinate synthetase contribute to each active site and that Arg-143 plays an important role in nucleotide binding.

Adenylosuccinate Synthase↗

PubMed1996

Raman signature of the four-stranded intercalated cytosine motif in crystal and solution structures of DNA deoxycytidylates d(CCCT) and d(C8).

The Raman spectral signature of the four-stranded cytosine structure formed by intercalation of two hemiprotonated and parallel-stranded oligodeoxycytidylate duplexes (so-called i motif) has been obtained from the crystal structure of d(CCCT) [Kang, C.H., Berger, I., Lockshin, C., Ratliff, R., Moyzis, R., & Rich, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11636-11640]. Identification of Raman markers diagnostic of the cytosine quadruplex is complemented by results obtained in a pH titration of 2'-deoxycytosine-5'-monophosphate (5'-dCMP) to show that the spectral fingerprint associated with N3 protonation of cytosine is distinct from that of quadruplex formation. The Raman spectrum thus provides a definitive basis for evaluating quantitatively both the extent of cytosine quadruplex formation and the degree of cytosine N3 protonation in DNA. Application to aqueous d(CCCT) and d(C8) demonstrates that the four-stranded intercalated structure is formed by both of these oligodeoxycytidylates in aqueous solution. Whereas both 5'-dCMP and the d(CCCT) quadruplex exhibit a midpoint of titration (apparent pKc) of 4.5 +/- 0.2 at 10 degrees C, cytosine protonation in d(C8) is shifted significantly toward the physiological range, with pKc = 5.8 +/- 0.2. The difference in pKc between the two quadruplexes is equivalent to a free energy difference of 1.7 kcal/mol at 10 degrees C. The present findings extend the library of Raman conformation markers to deoxycytidylate residues in the novel i quadruplex. The significance of these results for probing solution conformations of telomeric DNA sequences is also considered.

Base Sequence↗

PubMed1996

Detection of hammerhead ribozyme-mediated cleavage and reduced expression of LacZ' mRNA in E. coli.

Hammerhead ribozymes have been shown to specifically suppress the expression of target genes in various cells, but their in vivo cleavage products have seldom been directly detected. A hammerhead ribozyme sequence was designed to cleave the phosphodiester bond just 3' to the GUC of SalI site of M13mp 18. The ribozyme was inserted some base pairs upstream of the target region without disrupting the reading frame of the lacZ' gene and without introducing any translational stop codons. More than 90% RNAs synthesized in vitro were cleaved at the expected site after 1-h incubation in the presence of 10 mM magnesium ion at 37 and 50 degrees C. Inclusion of the designed ribozyme sequence was also shown to suppress the expression of the fused lacZ' gene in E. coli cells. When the cells were infected by the ribozyme-containing phage, they remained colourless in the presence of X-gal, and the cellular beta-galactosidase activity was reduced by more than 90%. Insertion of the same ribozyme sequence in reverse orientation showed little effect on beta-galactosidase activity. Furthermore, a primer extension by reverse transcriptase revealed a cleavage product that resulted from cleavage of LacZ' mRNA at the targeted site as designed. Thus, our data demonstrated that the designed hammerhead ribozyme cleaves and reduces the expression of a fused LacZ' mRNA in E. coli cells.

Amino Acid Sequence↗

PubMed1996

In situ hybridization studies of cytomegalovirus and Epstein-Barr virus in reactive histiocytic hyperplasia with hemophagocytosis.

We studied 14 adult patients presenting with fever and cytopenia of the peripheral blood and histiocytic hyperplasia with hemophagocytosis (HHH) in the bone marrow regarding an association of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) by using in situ hybridization (ISH) and also evaluated the clinical and laboratory findings according to the encountered organisms. ISH using a CMV RNA probe demonstrated infected cells in 6 out of 14 cases (43%), and ISH using an EBV EBER RNA probe demonstrated infected nuclei in 5 out of the same 14 cases (36%) of HHH. No cases showed a positive reaction with both probes. Three cases showed a negative reaction with both probes. The mean age of all patients was 29 years; and that of the CMV-positive patients was 27 years and that of the EBV-positive patients was 36 years. Organomegaly was found in 3 out of 6 CMV-positive patients (1 hepatomegaly, 1 splenomegaly, 1 hepatosplenomegaly), and 4 out of 5 EBV-positive patients (lymphadenopathy in all 4 cases, hepatosplenomegaly in 2 cases). One of the CMV-positive case had acute myeloblastic leukemia, and 2 EBV-positive cases had underlying malignancy (1 Hodgkin's disease, 1 non-Hodgkin's lymphoma). Seven out of the 14 HHH cases (50%) died within several months after diagnosis. Nucleic acid hybridization methods can be used for the routine examination of the association of CMV or EBV.

Adult↗

PubMed1996

[Anti-reflux ureterocysto-reimplantation in treatment of ectopic ureters: report of 51 cases].

From 1974 to 1994, anti-reflux ureterocysto-reimplantation was performed on 51 children (1 boy, 50 Girls) aged from 10 months to 13 years. The follow-up duration of 45 cases was 3 months to 14 years. The clinical symptoms of frequency of urination, lumbago and repeatedly fever, etc, were not observed except urinary incontinence in 2 cases. The results showed that ureterocysto-reimplantation is simple and traumatic. Some important aspects such as preoperative location and intraoperative identification of the unilateral or bilateral ectopic ureters were analyzed.

Adolescent↗

PubMed1995

Molecular structure of the halogenated anti-cancer drug iododoxorubicin complexed with d(TGTACA) and d(CGATCG).

4'-Deoxy-4'-iododoxorubicin, a halogenated anthracycline derivative, is an anticancer agent currently under Phase II clinical trials. In preclinical studies, it has demonstrated significantly reduced levels of cardiotoxicity compared to currently employed anthracyclines. It also has modified pharmacological properties resulting in an altered spectrum of experimental antitumor activity. The iodine atom at the 4' position of the sugar ring reduces the basicity and enhances the lipophilicity of this compound as compared to related anthracycline drugs. We report here single crystal X-ray diffraction studies of the complexes of 4'-deoxy-4'-iododoxorubicin with the hexanucleotide duplex sequences d(TGTACA) and d(CGATCG) at 1.6 and 1.5 A, respectively. The iodine substituent does not alter the geometry of intercalation as compared to previously solved anthracycline complexes, but appears to markedly affect the solvent environment of the structures. This could have consequences for the interaction of this drug with DNA and DNA binding proteins in cells.

Antibiotics, Antineoplastic↗

PubMed1995

Identification of an essential second metal ion in the reaction mechanism of Escherichia coli adenylosuccinate synthetase.

This study reports that two Mg2+ ions are required for Escherichia coli adenylosuccinate synthetase activity. The first metal ion is presumably coordinated with beta- and gamma-phosphoryl groups of GTP to provide an electron sink, and the second one seems to interact with aspartate in the enzyme active site. Regarding the latter metal ion, kinetic studies show that aspartate and the second Mg2+ ion bind to the enzyme active site randomly with a kcat value of 1.47 s-1 and with Km values for aspartate and Mg2+ of 225 and 114 microM, respectively. The dissociation constants for aspartate and Mg2+ of the enzyme.GTP.IMP.(aspartate or Mg2+) complex are 79.2 and 40.0 microM, respectively. However, variable amounts of aspartate or Mg2+ did not show any significant changes in the Km values for GTP and IMP. Kinetic studies using Mn2+ and Ca2+ ions indicate that the kcat values (0.930 and 0.235 s-1, respectively) were slightly decreased compared with the value obtained using Mg2+; however, the Km values for aspartate and GTP in the presence of Mn2+ and Ca2+ were significantly decreased compared with those obtained using Mg2+ ion (4.5 and 4.6 times for Mn2+ ion and 5.6 and 5.8 times for Ca2+ ion, respectively). On the other hand, the Km values for IMP were not significantly changed (1.9 and 1.8 times for Mn2+ and Ca2+ ions, respectively). Taken together, these kinetic results imply that aspartate may interact with Mg2+ to form a Mg.aspartate complex in the enzyme active site. An inhibition study of the enzyme with ZnCl2 (its Ki value is 29 nM) also suggested that Zn2+ competes with aspartate as well as Mg2+, implying that Zn2+ might form a complex with aspartate in the active site. On the basis of these results, it is suggested that Mg.aspartate complex formation in the active site of adenylosuccinate synthetase may be important in activation of the protonated amino group of aspartate, enhancement of the enzyme's binding affinity, and its specificity for aspartate.

Adenylosuccinate Synthase↗

PubMed1995

Crystal structure of the T4 regA translational regulator protein at 1.9 A resolution.

The translational regulator protein regA is encoded by the T4 bacteriophage and binds to a region of messenger RNA (mRNA) that includes the initiator codon. RegA is unusual in that it represses the translation of about 35 early T4 mRNAs but does not affect nearly 200 other mRNAs. The crystal structure of regA was determined at 1.9 A resolution; the protein was shown to have an alpha-helical core and two regions with antiparallel beta sheets. One of these beta sheets has four antiparallel strands and has some sequence homology to RNP-1 and RNP-2, which are believed to be RNA-binding motifs and are found in a number of known RNA-binding proteins. Structurally guided mutants may help to uncover the basis for this variety of RNA interaction.

Amino Acid Sequence↗

PubMed1995

Stable loop in the crystal structure of the intercalated four-stranded cytosine-rich metazoan telomere.

In most metazoans, the telomeric cytosine-rich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-A resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C.C+ hydrogen bonds. The base-paired strands are parallel to each other, and the two duplexes are intercalated into each other in opposite orientations. One TAA end forms a highly stabilized loop with the 5' thymine Hoogsteen-base-paired to the third adenine. The 5' end of this loop is in close proximity to the 3' end of one of the other intercalated cytosine strands. Instead of being entirely in a DNA duplex, this structure suggests the possibility of an alternative conformation for the cytosine-rich telomere strands.

Animals↗

PubMed1995

Identification and characterization of a human cDNA homologous to yeast SKI2.

A monoclonal antibody, 170A1, which recognizes a nucleolar peptide of molecular weight 90,000, was raised. The protein was conserved among various vertebrates. To characterize the antigen, we screened a human fetal liver expression library using the monoclonal antibody as a probe. Molecular analyses of immunopositive clones suggested the presence of a novel cDNA. It appeared to be a single-copy gene and encoded about 4- and 5-kb mRNAs. The gene appeared to be expressed in every cell tested so far. Its deduced amino acid sequence revealed an overall homology to recently described yeast SKI2. The SKI2 gene of Saccharomyces cerevisiae encoded a nucleolar protein that is involved in the antiviral system. We report here the partial human cDNA sequence and the localization of the corresponding gene on chromosome 6p21.

Amino Acid Sequence↗

PubMed1995

Extension of the four-stranded intercalated cytosine motif by adenine.adenine base pairing in the crystal structure of d(CCCAAT).

The crystal structure of d(CCCAAT), refined at 2.0 A resolution, shows a four stranded molecule in which two parallel duplexes intercalate with opposite polarity, using cytosine.protonated cytosine base pairs. The intercalation motif in this structure is extended by adenine.adenine base pairs. Two topologically distinct broad grooves are found in the lath-like central part of the molecule with the phosphate groups on one side bent over towards each other, stabilized by bridging water molecules. At the 3' ends, two arrangements of intermolecular A.A.T base triplets are found, involving both asymmetric and symmetric A.A base pairs joined to thymine residues by Watson-Crick and reverse Hoogsteen base pairing, respectively.

Adenine↗

PubMed1995

Antisense RNA-mediated inhibition of mouse hepatitis virus replication in L2 cells.

We have successfully used antisense RNA to inhibit replication of the mouse hepatitis virus (MHV) in a cell culture system. MHV is a single-stranded RNA virus of positive polarity. Mouse L2 cells were stably transfected with an antisense construct that targets regions of genes 5 and 6 of the virus. High levels of expression from this construct, which is under control of the human elongation factor 1 alpha promoter, were found. After infection of the antisense cell lines with MHV, replication of the virus was significantly reduced compared with control cells. In a viral plaque assay, smaller plaques were found in the antisense cell lines. In addition, up to a 92% inhibition in the number of viral particles produced in one antisense cell line could be seen. This inhibitory effect decreased at longer (> 16 hour) infection times. It was possible to both increase the amount of inhibition and prolong the inhibitory effect by reducing the multiplicity of infection. Our results suggest that antisense RNA may be an effective tool to slow down progression of MHV infection in mice.

Animals↗

PubMed1995

[Surgical treatment of penoscrotal transposition associated hypospadias].

Thirty-four patients with penoscrotal transposition associated with hypospadias were surgically treated from 1978 to 1992. Penoscrotal hypospadias was noted in 23, and perineal hypospadias in 11. Incomplete penoscrotal transposition was scen in 24 patients, and complete penoscrotal transposition in 10. 32 patients out of 34 obtained satisfactory results after the correction of penoscrotal transposition. Complication occurred in 5 patients (urethral fisula 3 and orifice structure 2). The operative procedures of penoscrotal transposition were discussed.

Abnormalities, Multiple↗

PubMed1994

Start site selection at lacUV5 promoter affected by the sequence context around the initiation sites.

The effects of single base pair substitutions at the initiation sites of lacUV5 promoter on the transcription start site selection by E. coli RNA polymerase were systematically studied. Transcription start sites were mapped by sizing the cytosine-specifically terminated transcripts produced in vitro by using a chain terminator 3'-deoxycytidine 5'-triphosphate (3'-dCTP) in transcription reactions. Transcription of a prototype lacUV5 promoter initiated with three purines (-1G, +1A and +2A; +1 representing the predominant start site) located 6-8 bp downstream from the Pribnow box. All the substitutions affected the start site selection, resulting in a change in the number of start sites (from 3 to 2 or 1) and/or a shift of the major start site (to -1 or +2). None of the variants started outside the 3-bp region and at the positions substituted by a pyrimidine. Purine-to-pyrimidine changes suppressed not only initiation at the substituted position but also, in some cases, at the other purine position. Purine-to-purine changes also shifted the major start site or suppressed the initiation at other sites. Changes at -2 and +5 also affected the start site selection. Thus, the sequence context around the initiation sites of lacUV5 promoter strongly influences the selection of initiating nucleotides by E. coli RNA polymerase.

Base Sequence↗

PubMed1994

Replacement of Asp333 with Asn by site-directed mutagenesis changes the substrate specificity of Escherichia coli adenylosuccinate synthetase from guanosine 5'-triphosphate to xanthosine 5'-triphosphate.

The aspartate residue of the (N/T)KXD concensus sequence for GTP-binding proteins is present in the eight available sequences of adenylosuccinate synthetase. Reported here is a comprehensive analysis of the substrate specificity of mutant enzymes, where the conserved Asp333 of the synthetase from Escherichia coli is changed to asparagine, glutamate, and glutamine by site-directed mutagenesis. The mutants D333N, D333E, and D333Q generally show decreased kcat values and increased Km values for GTP. The decreased values of kcat exhibited by the mutants indicate that the interactions between Asp333 and the guanine are relayed by some mechanism to the catalytic residues around the gamma-phosphate of GTP, and that the energy provided by the interaction between Asp333 and the guanine moiety of GTP is utilized for rearrangement of the catalytic residues. The three mutants each have higher affinity for xanthosine 5'-triphosphate (XTP) and ITP than does the wild-type enzyme. In fact, the D333N mutant uses XTP more effectively than the wild-type enzyme employs GTP as a substrate. The side-chain of Asp333 forms hydrogen bonds with the N-1 and the exocyclic amino group of the guanine base of GTP. In the D333N mutant, this interaction is probably replaced by hydrogen bonds between the amide side chain of Asn333 and N-1 and the 2-oxo group of XTP. The D333Q mutant can use UTP as a substrate more effectively than the wild-type enzyme. The longer side chain of glutamine at residue 333 favors pyrimidine nucleotides over the purine nucleotides, GTP, XTP, and ITP. These results demonstrate that Asp333 in the (N/T)KXD consensus sequence of adenylosuccinate synthetase from E. coli is a determinant for GTP-specificity.

Adenylosuccinate Synthase↗

PubMed1994

Cloning and sequence analysis of the gene encoding the low potential cytochrome c of Synechocystis PCC 6803.

The gene for the low potential cytochrome c (petK) was isolated from a genomic library of Synechocystis PCC 6803. The nucleotide sequence of this gene contains two regions with sequence similarity to two regions in the gene for the high potential cytochrome c6 of the organism. The sixth iron ligand can be identified with a conserved histidine. Experiments demonstrate the reduction of the low potential cytochrome by reduced ferredoxin II. The heme of the cytochrome is flanked by lysines which may be involved in orienting the ferredoxin near the site of electron donation.

Amino Acid Sequence↗