Biomedical subjects

K Song

Publications and source records attributed to K Song.

At least 37 records · Page 2Linked to original sources

PubMed2001

Relationship between gene polymorphism of the PAI-1 promoter and myocardial infarction.

OBJECTIVE: To investigate the association between gene polymorphism of the plasminogen activator inhibitor-1 (PAI-1) and myocardial infarction (MI) in Chinese. METHODS: PAI-1 genotyping with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele specific polymerase chain reaction (ASPCR) was performed in 87 myocardial infarction patients and 92 unrelated healthy controls. All subjects' clinical features and PAI-1 activity were tested. RESULTS: There were two polymorphisms within the promoter, a G/A single base substitution polymorphism upstream at -844 bp, and a single guanosine deletion/insertion 4G/5G polymorphism -675 bp upstream from the start of transcription. Significant differences between the patients and the controls were observed neither for the frequencies of the GG, GA and AA genotypes nor for the PAI-1 activities of these three types. But for the 4G/5G polymorphism, there were significant differences between patients and controls for the frequencies of the 4G/4G, 4G/5G and 5G/5G genotypes (P < 0.05). In the MI group, the PAI-1 activity of the 4G/4G type was significantly higher than that of the 5G/5G type (P < 0.05). Further more, a positive correlation between the glucose level and PAI-1 activity was found (r = 0.34, P = 0.02). CONCLUSION: This study indicates that the 4G/5G gene polymorphism of PAI-1 is associated with myocardial infarction, that 4G/4G type is probably an important hereditary risk factor, and that glucose has functional importance in regulating PAI-1 activity.

Adult↗

PubMed2001

[Genomic structure and proteins sequence analysis of full-length of segment A of three infections bursal disease viruses].

The full-length of segment A of three infectious bursal disease viruses (IBDV), including an attenuated strain HZ2, an attenuated vaccine strain JD1 and a virulent field isolate ZJ2000, were cloned by long RT-PCR and sequenced respectively. All the three results revealed the identity of IB-DV with two overlapping open reading frames (ORF) flanked by 5'- and 3'-noncoding regions(NCR) in 3259 bp long. The strains shared high identity with each other at nucleotide or deduced amino acid level, and also had four unique sites H253, N279, T284, R330 which are common in other attenuated and some classic or highly virulent strains. The virulent strain ZJ2000 had several key amino acid mutations located in hypervariant region of VP2 and near the VP2-VP4 cleavage site of polypeptide, which is probably related to the virulence. Sequence comparison supported that VP2 is not the sole determinant of the virulence. The highly conservation in 5'- and 3'-NCR of different strains indicated the NCR may be not responsible for the virulence. But the same conservation appeared in VP5 revealed another complex relationship between VP5 and the virulence.

Amino Acid Sequence↗

PubMed2000

Human Ku70 interacts with heterochromatin protein 1alpha.

Ku is involved in the metabolism of DNA ends, DNA repair, and the maintenance of telomeres. It consists of a heterodimer of 70- and 80-kDa subunits. Recently we have demonstrated that Ku70 interacted with TRF2, a mammalian telomere-binding protein. Using the same yeast two-hybrid screening system, we now show that Ku70 also interacts with heterochromatin protein 1alpha (HP1alpha), a protein known to be associated with telomeres as well as heterochromatin. HP1 is a suppressor of the position effect variegation in Drosophila and acts as a transcriptional suppressor in mammalian cells. The interaction with Ku70 in the two-hybrid system was confirmed by a glutathione S-transferase pull-down study using bacterial recombinant proteins in vitro. The interaction was also reproduced in vivo in HeLa cells, where endogenous Ku70 coimmunoprecipitated with HP1alpha. This interaction was more effective in acidic pH and weakened considerably as the pH of the reaction buffer was elevated up to 7.5. Ku80 did not interact with HP1alpha directly. The interaction domains of Ku70 and HP1alpha included the Leu-Ser repeat (amino acids 200-385) and the chromo shadow domain, respectively. Ku70 was largely colocalized with transfected HP1alpha but not with a C-terminal deletion mutant, HP1alpha(Delta)C. In contrast to HP1alpha, Ku70 did not repress transcriptional activity of the reporter gene when tethered to DNA after transfection to mammalian cells. The implication of this interaction is discussed.

Antigens, Nuclear↗

PubMed2000

An intra-Peyer's patch gene transfer model for studying mucosal tolerance: distinct roles of B7 and IL-12 in mucosal T cell tolerance.

Development of mucosal immunity and tolerance requires coordinated expression of a number of genes within the mucosa-associated lymphoid tissue (MALT). To study the roles of these genes in the MALT, we have established a MALT-specific gene transfer model using replication-defective adenovirus as vector. In this model, the target gene of interest is directly delivered into the Peyer's patch by intra-Peyer's patch injection of the recombinant virus. Using this gene transfer model, we investigated the roles of B7-1 and IL-12 in the development of mucosal tolerance. We found that intra-Peyer's patch injection of OVA induced Ag-specific T cell hyporesponsiveness, as manifested by decreased T cell proliferation and IL-2/IFN-gamma production upon subsequent immune challenge. Intra-Peyer's patch B7-1 gene transfer at the time of OVA administration partially reversed the inhibition of T cell proliferation and IL-2 secretion, but had no effect on IFN-gamma production. By contrast, intra-Peyer's patch IL-12 gene transfer completely restored T cell proliferation and IFN-gamma secretion and partially reversed IL-2 inhibition. Using an adoptive TCR transgenic model, we further demonstrated that B7 and IL-12 played distinct roles during the inductive phase of mucosal tolerance. B7 selectively increased T cell proliferation and IL-2 secretion without affecting IFN-gamma production, whereas IL-12 increased both IL-2 and IFN-gamma production. These results indicate that B7 alone may not be sufficient to abrogate mucosal tolerance, and that cytokines such as IL-12 may also be required. Based on these findings, we propose a new model to explain the paradoxical roles of B7 in mucosal immunity and tolerance.

Adenoviruses, Human↗

PubMed2000

Interaction of human Ku70 with TRF2.

Ku, a heterodimer of 70- and 80-kDa subunits, plays a general role in the metabolism of DNA ends in eukaryotic cells, including double-strand DNA break repair, V(D)J recombination, and maintenance of telomeres. We have utilized the yeast two-hybrid system to identify Ku70-interacting proteins other than Ku80. Two reactive clones were found to encode the dimerization domain of TRF2, a mammalian telomeric protein that binds to duplex TTAGGG repeats at chromosome ends. This interaction was confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells overexpressing TRF2. The transfected TFR2 colocalized with Ku70.

Animals↗

PubMed2000

Characteristics of levan fructotransferase from Arthrobacter ureafaciens K2032 and difructose anhydride IV formation from levan.

A microorganism producing levan fructotransferase was isolated from sugar-disclosed soil and it was identified as Arthrobacter ureafaciens. The major product from levan by enzyme reaction was identified as di-D-fructofuranose 2,6':6,2' dianhydride by mass spectrometry, nuclear magnetic resonance, and chemical analyses. Small amounts of several oligosaccharides and free fructose were also formed by enzyme reaction. An extracellular enzyme that produces di-D-fructofuranose 2,6':6,2' dianhydride from levan was purified from the culture broth of A. ureafaciens K2032. The enzyme had optimum activity around pH 5.8 and 45 degrees C and had a dimeric form in solution. The N-terminal amino acid residues of the purified enzyme were SAPGSLRAVYHMTPPSGXLXDPQ. The enzyme has narrow substrate range and converts the levan to di-D-fructofuranose 2,6':6,2' dianhydride with around 62.5% conversion yield.

Journal Article↗

PubMed2000

Dynamic evolution of plant mitochondrial genomes: mobile genes and introns and highly variable mutation rates.

We summarize our recent studies showing that angiosperm mitochondrial (mt) genomes have experienced remarkably high rates of gene loss and concomitant transfer to the nucleus and of intron acquisition by horizontal transfer. Moreover, we find substantial lineage-specific variation in rates of these structural mutations and also point mutations. These findings mostly arise from a Southern blot survey of gene and intron distribution in 281 diverse angiosperms. These blots reveal numerous losses of mt ribosomal protein genes but, with one exception, only rare loss of respiratory genes. Some lineages of angiosperms have kept all of their mt ribosomal protein genes whereas others have lost most of them. These many losses appear to reflect remarkably high (and variable) rates of functional transfer of mt ribosomal protein genes to the nucleus in angiosperms. The recent transfer of cox2 to the nucleus in legumes provides both an example of interorganellar gene transfer in action and a starting point for discussion of the roles of mechanistic and selective forces in determining the distribution of genetic labor between organellar and nuclear genomes. Plant mt genomes also acquire sequences by horizontal transfer. A striking example of this is a homing group I intron in the mt cox1 gene. This extraordinarily invasive mobile element has probably been acquired over 1,000 times separately during angiosperm evolution via a recent wave of cross-species horizontal transfers. Finally, whereas all previously examined angiosperm mtDNAs have low rates of synonymous substitutions, mtDNAs of two distantly related angiosperms have highly accelerated substitution rates.

Biological Evolution↗

PubMed2000

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of tumor cells but not normal cells; its role in normal nontransformed tissues is unknown. We report here that chronic blockade of TRAIL in mice exacerbated autoimmune arthritis, and that intraarticular TRAIL gene transfer ameliorated the disease. In vivo, TRAIL blockade led to profound hyperproliferation of synovial cells and arthritogenic lymphocytes and heightened the production of cytokines and autoantibodies. In vitro, TRAIL inhibited DNA synthesis and prevented cell cycle progression of lymphocytes. Interestingly, TRAIL had no effect on apoptosis of inflammatory cells either in vivo or in vitro. Thus, unlike other members of the tumor necrosis factor superfamily, TRAIL is a prototype inhibitor protein that inhibits autoimmune inflammation by blocking cell cycle progression.

Animals↗

PubMed2000

Identification of a putative DEAD-box RNA helicase and a zinc-finger protein in Candida albicans by functional complementation of the S. cerevisiae rok1 mutation.

We identified two novel genes, CHR1 and CSR1, of the fungal pathogen Candida albicans, by functional complementation of the Saccharomyces cerevisiae rok1 mutation. The Rok1 protein is a member of the DEAD protein family of ATP-dependent RNA helicases. ROK1 is required for cell cycle progression and also for rRNA processing. The CHR1 gene product of 578 amino acids is highly homologous to the Rok1 protein (54% identity) and is considered to be a putative DEAD-box RNA helicase. We predict that the CSR1 gene encodes a 73 kDa protein of 612 amino acids with five zinc-finger motifs at the C-terminal region. CHR1 or CSR1 on a high-copy number plasmid showed a slow-growth phenotype in a condition where the ROK1 expression is turned on from the GAL1 promoter. This result is consistent with the lethality caused by the ROK1 overexpression. We conclude that CHR1 encodes a functional homologue of Rok1 protein and CSR1 is a heterologous suppressor of the rok1 mutation.

Amino Acid Sequence↗

PubMed2000

Identification and characterization of a bidirectional promoter from the intergenic region between the human DDX13 and RD genes.

The human DDX13 gene encodes a putative RNA helicase of the DExH-box family. In an earlier report we showed that the human DDX13 and RD genes were arranged head-to-head in the class III MHC complex and their ATG start codons were separated by 745 base pairs. We have now analyzed the common 745 bp intergenic region in detail and characterized their promoters. Northern blot analysis revealed that DDX13 and RD exhibit distinct patterns of steady-state expression among multiple human tissues. The promoter regions for DDX13 and RD genes were identified by deletion analysis from 740 bp to 176 bp of the intergenic region fused to a chloramphenicol acetyltransferase (CAT) reporter gene using transient transfection assays. Results indicated that a promoter sequence as small as 176 bp is sufficient for basal expression of both genes in HeLa and HepG2 cells. Functional analysis using a bidirectional reporter system demonstrates that the sequence 262 bp proximal to the DDX13 gene is sufficient for concurrent expression in both directions. However, the common 740 bp intergenic region showed promoter activity in DDX13 only, suggesting the presence of a negatively acting region for the RD gene within the region -267 to -744. It appears that RD expression is controlled by a complex system of positively and negatively acting elements present on distant portions of both genes.

Blotting, Northern↗

PubMed2000

Nuclear matrix of calreticulin in hepatocellular carcinoma.

Nuclear matrix protein profiles of malignant cells vary from their normal counterparts. By two-dimensional gel electrophoresis, we analyzed nuclear matrix proteins in 11 hepatocellular carcinomas and compared them with corresponding non-neoplastic liver tissue. Although the compositions were mostly similar, several peptides were noted predominantly in the former. The most prominent one was an acidic protein of apparent Mr 62,000, which was identified to be calreticulin upon NH2-terminal amino acid sequencing. By immunoblotting, calreticulin was confirmed to be present abundantly in the nuclear matrix fraction of carcinomas but not in that of the nonmalignant liver tissue. Interestingly, the total content of calreticulin was similar between them. By immunofluorescence microscopy, evident nuclear immunostaining was detected in carcinomas. Calreticulin was also found to be in the nuclear matrices of various carcinoma cell lines. We conclude that calreticulin is a component of the nuclear matrix. The formation and/or expansion of the calreticulin-nuclear matrix may be related to the activated cell growth.

Adult↗

PubMed2000

Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12).

IL-12 is a heterodimeric cytokine that is known to induce tumor regression and long-term antitumor immunity. Recombinant adeno-associated virus (rAAV) vectors are advantageous for gene therapy in that they lack pathogenicity in humans, infect dividing as well as nondividing cells, and show a broad range of infectivity. We constructed an rAAV vector expressing interleukin-12 (IL-12) for cancer immunotherapy studies in a mouse model by inserting murine IL-12 (mIL-12) p35 and p40 cDNAs into the plasmid pRep4 and inserting the encephalomyocarditis virus internal ribosomal entry site between the p35 and p40 cDNAs. The mIL-12 expression cassette containing the Rous sarcoma virus promoter and a simian virus 40 polyadenylation signal was subcloned into the AAV plasmid p008Sub/NeoR, which contains two AAV inverted terminal repeat sequences and the NeoR gene driven by the thymidine kinase promoter. rAAV virions (10(4) infectious particles/ml) were generated by cotransfection of rAAV-mIL-12 and a helper plasmid (pAAV/Ad) into 293 cells previously infected with adenovirus 5. After infection of D6 fibroblasts with rAAV-mIL-12, G418-resistant clones were isolated. Each of the 1D D6 clones isolated produced up to 5.2 ng/10(6) cells/48 hours of mIL-12 as determined by enzyme-linked immunosorbent assay. Induction of interferon-gamma, enhanced lymphocyte proliferation, and cytotoxicity assays confirmed biologically functional IL-12 production by the vector. This is the first report indicating that an rAAV vector expresses mIL-12, which can be used to model the effects of mIL-12 alone and/or in combination with other antitumor agents.

Animals↗

PubMed2000

Regulation of T-helper-1 versus T-helper-2 activity and enhancement of tumor immunity by combined DNA-based vaccination and nonviral cytokine gene transfer.

Intramuscular (i.m.) injections of a plasmid encoding human carcinoembryonic antigen (CEA) elicited both humoral and cellular immune responses in mice, but only partial inhibition of the growth of transplanted syngeneic CEA-positive P815 tumor cells (CEA/P815). Coinjection of the CEA vector with a vector encoding either interferon-gamma (IFN gamma) or IL-12 promoted IgG2a isotype anti-CEA antibody production, anti-CEA/P815 CTL activity and greater resistance to CEA/P815 tumor challenge. As well, CEA/P815-stimulated IFN gamma secretion in vitro was increased, but IL-4 diminished, consistent with a T-helper type 1 (Th1) response. In contrast, coinjection of the CEA vector with an IL-4 vector increased IgG1 production, but reduced CTL activity and resistance to tumor challenge. The latter treatment inhibited CEA/P815-dependent IFN gamma production but enhanced IL-4 secretion, consistent with a Th type 2 (Th2) response. Antitumor immunity was enhanced when the CEA and IL-12 plasmids were coinjected at the same muscle site, but not at separate sites despite increased serum IL-12 levels. Though the tumor cells expressed neomycin phosphotransferase, mice immunized with vectors encoding that protein (without CEA) were not protected against tumor growth, and produced no CTLs except for low levels when coinjected with an IL-12 vector. Thus, we show that immunity elicited by DNA vaccination against CEA can be biased to a protective type (high Th1 and CTL activity) or nonprotective type (high Th2 and low CTL activity) by i.m. coinjection of cytokine-expressing plasmids. IL-12 appears to act locally, but not systemically, through an adjuvant effect.

Analysis of Variance↗

PubMed2000

IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen (CEA) studied in immune-gene knockout mice.

Intramuscular (i.m.) injection of a plasmid encoding human carcinoembryonic antigen (CEA) elicited immunity against transplanted syngeneic (C57BL/6) CEA-positive Lewis lung carcinoma (CEA/LLC) cells, but tumors still appeared in all mice. In wild-type mice, coinjection of an IL-12 plasmid markedly enhanced anti-CEA humoral, T-helper-1 and cytotoxic T lymphocyte (CTL) responses, and resistance to a CEA/LLC tumor challenge such that 80% of mice remained tumor free. Injection of the IL- 12 plasmid alone was not protective. To analyze immune requirements, we immunized gene knockout (KO) mice of C57BL/6 background, deficient in either CD3, CD4, CD8, interferon gamma (IFNgamma), perforin or Fas ligand (FasL). Only CD3+ mice expressing both CD4 and CD8, which appear equally important, as well as IFNgamma and perforin, could fully resist a tumor challenge. IL-12 stimulated CTL activity, which was strictly CD3/CD8/perforin-dependent. FasL-KO mice had normal CTL activity and tumor resistance, indicating that only the perforin lytic pathway was involved. CD4-KO and IFNgamma-KO mice still generated CTLs. CEA-stimulated IFNgamma production occurred in both CD4- or CD8-KO mice and in both cases was augmented by IL-12. In IFNgamma-KO mice, IL-12 still enhanced anti-CEA antibody production but only moderately restored impaired DTH and tumor resistance. We conclude that the immune requirements for tumor rejection are stringent, involving multiple mechanisms which are all enhanced by IL-12.

Animals↗

PubMed2000

A comparison of remifentanil and fentanyl in patients undergoing surgery for intracranial mass lesions.

UNLABELLED: We compared the effects of remifentanil versus fentanyl during surgery for intracranial space-occupying lesions. Patients were randomly assigned to receive either remifentanil (0.5 microg. kg(-1). min(-1) IV during the induction of anesthesia reduced to 0.25 microg. kg(-1). min(-1) after endotracheal intubation; n = 49) or fentanyl (dose per usual practice of the anesthesiologist; n = 54). Anesthesia maintenance doses of isoflurane, nitrous oxide, and opioid were at the anesthesiologist's discretion for both groups. There were no differences between opioid groups for the frequency of responses (hemodynamic, movement, and tearing) to intubation, pinhead holder placement, skin incision, or closure of the surgical wound. Adverse event frequencies were similar between groups. Times to follow verbal commands (P < 0.001) and tracheal extubation (P = 0. 04) were more rapid for remifentanil. The percentage of patients with a normal recovery score (were alert or arousable to quiet voice, were oriented, were able to follow commands, had motor function unchanged from their preoperative evaluation, were not agitated, and had modified Aldrete Scores of 9-10) at 10 min after surgery was more for remifentanil (45% vs 18%; P = 0.005). By 20 min, no difference between groups existed (P = 0.27). Anesthesiologists used more isoflurane in the fentanyl group (4.22 vs 1.93 minimum alveolar anesthetic concentration hours). Neurosurgeons, blinded to treatment group, favored the use of remifentanil. Similar frequencies of light anesthesia responses and other adverse events suggest that intraoperative depths of anesthesia were similar in the two groups. Under these conditions, emergence was more rapid with remifentanil. This is consistent with the necessity for less isoflurane use in the remifentanil group and the intrinsic rapid clearance of this opioid. IMPLICATIONS: Patients given remifentanil-based anesthesia for craniotomy had faster recovery times from anesthesia than did those given fentanyl-based anesthesia.

Anesthesia Recovery Period↗

PubMed2000

Cloning and sequence analysis of the estA gene encoding enzyme for producing (R)-beta-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001.

The estA gene encoding the enzyme that catalyzes the production of (R)-beta-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A + T and C + G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70-100 amino acids upstream of the G-X-S-X-G consensus sequence.

Journal Article↗

PubMed2000

A sequence-ready map for human chromosome 12q15-21.

Construction of sequence-ready clone map is an essential step toward sequencing the human genome. We chose a region that is frequently amplified in liposarcoma between D12S350 and D12S106 in chromosome 12q15-21 to build a PAC/BAC clone contig map. This region was spanned by 4 YACs and contained 30 STS on the YAC and radiation hybrid (RH) framework maps, providing an average STS spacing of 160 kb if each YAC is approximately 1.2 Mb in size. To convert a STS-based YAC map to a STS-based contig map of bacterial clones, 22 non-polymorphic STS markers were used as probes to screen the high density gridded arrays of PAC and BAC clones by filter hybridizations, followed by assembly of clones into contigs by marker content. Contigs have been extended and joined by direct end sequencing of appropriate clones, generating new STSs and rescreening the library as necessary. Using these approaches, we have constructed 5 contigs covering the region with the largest single contig being 1.4 Mb and a final size estimation of 3.6 Mb. The map is comprised of 17 YACs, 187 PACs, 160 BACs, and 17 cosmids; onto this, 6 polymorphic, 97 non-polymorphic, 24 ESTs, and 4 gene-based markers are now placed in a unique order, providing an average resolution of approximately 28 kb. Of a total of 131 markers, 97 were developed in the present study. The sequence-ready map should provide a framework to generate complete DNA sequence and ultimately gene map of this segment of chromosome 12.

Base Sequence↗

PubMed1999

Intracellular gene transfer in action: dual transcription and multiple silencings of nuclear and mitochondrial cox2 genes in legumes.

The respiratory gene cox2, normally present in the mitochondrion, was previously shown to have been functionally transferred to the nucleus during flowering plant evolution, possibly during the diversification of legumes. To search for novel intermediate stages in the process of intracellular gene transfer and to assess the evolutionary timing and frequency of cox2 transfer, activation, and inactivation, we examined nuclear and mitochondrial (mt) cox2 presence and expression in over 25 legume genera and mt cox2 presence in 392 genera. Transfer and activation of cox2 appear to have occurred during recent legume evolution, more recently than previously inferred. Many intermediate stages of the gene transfer process are represented by cox2 genes in the studied legumes. Nine legumes contain intact copies of both nuclear and mt cox2, although transcripts could not be detected for some of these genes. Both cox2 genes are transcribed in seven legumes that are phylogenetically interspersed with species displaying only nuclear or mt cox2 expression. Inactivation of cox2 in each genome has taken place multiple times and in a variety of ways, including loss of detectable transcripts or transcript editing and partial to complete gene loss. Phylogenetic evidence shows about the same number (3-5) of separate inactivations of nuclear and mt cox2, suggesting that there is no selective advantage for a mt vs. nuclear location of cox2 in plants. The current distribution of cox2 presence and expression between the nucleus and mitochondrion in the studied legumes is probably the result of chance mutations silencing either cox2 gene.

Cell Nucleus↗