Biomedical subjects

J Kim

Publications and source records attributed to J Kim.

At least 397 records · Page 22Linked to original sources

PubMed2000

Inhibitory effect of nitric oxide on voltage-dependent calcium currents in rat dorsal root ganglion cells.

The effect of nitric oxide (NO) on calcium current (I(Ca)) and intracellular calcium concentration ([Ca(2+)](i)) in primarily cultured dorsal root ganglion (DRG) neurons was investigated from neonatal rats. I(Ca) and [Ca(2+)](i) were simultaneously recorded using perforated-patch technique in combination with fluorescence measurement from single DRG neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), inhibited I(Ca) in small-diameter neurons without significant change in voltage-dependence of activation and activation time constants. SNP and SNAP also reduced the transient [Ca(2+)](i) peak accompanied by I(Ca). Inhibition by NO was reproducible, but gradually desensitized. In some DRG neurons, SNP and SNAP increased basal [Ca(2+)](i) in concentration of 10 microM with little effect on NO-induced inhibition of I(Ca). 8-Br-cGMP, a permeable cGMP analog, mimicked the effects of SNP and SNAP. These results suggest that, in DRG neurons, NO has inhibitory effect on I(Ca), which is independent of NO-induced increase of basal [Ca(2+)](i), through cGMP-dependent pathway.

Animals↗

PubMed2000

Localization of psoriasis-susceptibility locus PSORS1 to a 60-kb interval telomeric to HLA-C.

Recent genome scans have established the presence of a major psoriasis-susceptibility locus in the human leukocyte antigen (HLA) complex on chromosome 6p21.3. To narrow the interval for candidate gene testing, we performed a linkage-disequilibrium analysis of 339 families, with the use of 62 physically mapped microsatellite markers spanning the major histocompatibility complex (MHC). As detected by use of the transmission/disequilibrium test (TDT), individual markers yielded significant linkage disequilibrium across most of the MHC. However, the strongest evidence for marker-trait disequilibrium was found in an approximately 300-kb region extending from the MICA gene to the corneodesmosin gene. Maximum-likelihood haplotypes were constructed across the entire MHC in the original sample and across a 1.2-Mb region of the central MHC in an expanded sample containing 139 additional families. Short (two- to five-marker) haplotypes were subjected to the TDT using a "moving-window" strategy that reduced the variability of TDT P values relative to the single-locus results. Furthermore, the expanded sample yielded a sharp peak of evidence for linkage disequilibrium that spanned approximately 170 kb and that was centered 100 kb telomeric to HLA-C. The 1.2-Mb interval was further dissected by means of recombinant ancestral haplotype analysis. This analysis identified risk haplotype 1 (RH1), which is a 60-kb fragment of ancestral haplotype 57.1, on all identifiable HLA risk haplotypes. One of these haplotypes exhibits significant linkage disequilibrium with psoriasis but does not carry Cw6, which is the HLA allele most strongly associated with the disease. These results demonstrate that RH1 is highly likely to carry the disease allele at PSORS1, and they exclude HLA-C and corneodesmosin with a high degree of confidence.

Alleles↗

PubMed2000

Random changes of amino acid residues with expected frequency by saturated point mutagenesis.

The yeast transcriptional activator protein, Gcn4p from Saccharomyces cerevisiae binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control. A new random saturation mutagenesis method was developed to isolate Gcn4p derivatives with only one or two mutations in the DNA binding domain without using radioactive isotope. This will be used to identify the amino acids of Gcn4p involved in protein-protein interactions. Saturation mutagenesis in the DNA binding domain of Gcn4p was performed using spiked degenerate oligonucleotides containing randomized codon bases designed specifically for only one or two base changes in the mutagenized area. These oligonucleotides were synthesized to have two flanking restriction enzyme sites for cloning to the appropriate vector. The 3' ends were mutually primed after hybridization via the palindromic sequences of the restriction enzyme sites. These molecules were then converted to double stranded DNA upon treatment with DNA polymerase. Here, a library collection of 100,680 in an altered Gcn4p pool was generated by cloning a mixed-base oligonucleotide in the place of the sequence coding for the DNA binding domains. The quality of the library was examined by DNA sequencing and found to be in good agreement with the expected statistical values. Calculated mutation frequency was 66% of mutant nucleotide rate and actual sequencing data revealed 68% mutant nucleotide rates from the sequenced library. Thus, among 21 mutants, 16 had one point mutations and 5 had two point mutations. This approach appears to be an effective and general tool for creating proteins with one or two amino acid change(s) in their molecules.

Binding Sites↗

PubMed2000

Effects of saturation mutagenesis of the phage SP6 promoter on transcription activity, presented by activity logos.

A full set of SP6 promoter variants with all possible single substitutions at positions -17 to +5 was constructed. Transcription activities of these variants were individually measured in vivo and in vitro to determine the contribution of each base pair to the promoter activity. The in vivo activity was measured indirectly by transcriptional interference of the replication of promoter-bearing plasmids. This activity depends most highly on residues -11, -9, -8, -7, and +1 (initiation site). All substitutions at -11, -9, -8, and -7 abolished formation of closed complexes, except for A-8C. These residues are involved in base-specific interactions with the polymerase, and the substitutions exhibit the same strong inhibition in vitro. In contrast, the in vitro activities of some other variants, measured on linearized templates, were different from those in vivo. Some variants at -13, -4, and -2, among others, showed exceptionally higher activities in vivo than in vitro, supporting the possibility that these residues are involved in postbinding steps, including template melting and bending. The A-3T variant showed much lower activity in vivo than in vitro, but it bound to the polymerase 2-fold more than the consensus sequence and is possibly involved in polymerase binding. A quantitative hierarchy of all the base pairs is graphically displayed by activity logos, revealing the energetic contribution of each base pair to the activity.

Bacteriophages↗

PubMed2000

RNase P ribozymes selected in vitro to cleave a viral mRNA effectively inhibit its expression in cell culture.

An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G(224)G(225) --> AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k(cat)/K(m)) than that derived from the wild type ribozyme. Our results suggest that the mutated A(224)A(225) are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.

Animals↗

PubMed2000

Assembly of an A kinase-anchoring protein-beta(2)-adrenergic receptor complex facilitates receptor phosphorylation and signaling.

Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4] [5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the beta(2)-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the beta(2)-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the beta(2)-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance beta(2)-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor.

A Kinase Anchor Proteins↗

PubMed2000

The pattern of serum markers in patients with androgen-independent adenocarcinoma of the prostate.

In this exploratory study, our objectives were to correlate the serum and bone marrow concentrations of putative markers of prostate cancer progression in patients with advanced androgen-independent prostate cancer (AIPC), to assess the frequency and quantity of relative expression of these markers, and to correlate the expression of the markers with extent of disease (EOD) and overall survival. In a cohort of 50 patients with AIPC with bone metastases, we obtained serum and bone marrow samples and measured prostate specific antigen (PSA), serum interleukin-6 (sIL-6), bone marrow interleukin-6, serum chromogranin A (sCgA), bone marrow chromogranin A, and prostate specific membrane antigen (PSMA) by immunoassays. EOD was determined by quantifying identifiable bone lesions on radionuclide bone scans. Each variable was categorized into two groups (low and high) based on the median found in this cohort or on the cutoff based on normal limits when available. Analyses were performed in two subsets of patients with EOD either <20 or >/=20. Results showed that: (1) PSA is associated with EOD but not with outcome; (2) sIL-6 and sCgA may be intermediate markers of early progression in AIPC, because they are predictive of outcome only in patients with EOD <20; (3) elevated PSMA is associated with elevated sIL-6 but not with PSA, suggesting that PSMA may be a useful marker in AIPC; and (4) the ratio of PSA to putative markers of progression may reflect the complex clonal progression of prostate cancer. We conclude that patients with advanced AIPC exhibit one of two patterns of serologic marker expression: in some patients the disease status is reflected by PSA, and in others it is reflected by other markers. If these data are prospectively confirmed, this would help group patients with advanced AIPC into clinically relevant categories.

Journal Article↗

PubMed2000

Enhanced induction of Bax gene expression in H460 and H1299 cells with the combined treatment of cisplatin and adenovirus mediated wt-p53 gene transfer.

Cytotoxic effect of either cisplatin or p53 gene transfection of lung cancer cells may be different depending on the p53 status of cells. We investigated cytotoxic effects on the combined treatment of cisplatin and adenovirus mediated p53 gene transfer (Avp53) in both H460 and H1299 cells in vitro. The results showed the highest numbers of apoptotic cells in both H460 and H1299 cells following the combined treatment regardless of p53 status in comparison with either cisplatin or Avp53 alone. The expression levels of p53, p21, Bax and ICE were examined to understand a possible cellular signal path of the combined treatment. In western analyses, the patterns of phosphorylated p53 protein were different between Avp53 and combined treatment. The expressions of p21 and Bax were increased in combined treatment, whereas the cleaved form of ICE (20 kD) was not detected. These results suggest that cisplatin induced p53 protein phosphorylation and may activate the downstream of p53 gene expression such as p21 and Bax. The enhanced apoptosis of lung cancer cells by the combined treatment may be useful in the development of clinical therapeutic modality of lung tumors.

Adenoviridae↗

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

Dimer to monomer phase transition in alkali-metal fullerides: magnetic susceptibility changes

Ab initio calculations have been employed to investigate the peculiar change in magnetic property (from diamagnetic to paramagnetic) of the dianionic C60-dimer phase in a rapidly cooled AC60 samples ( A: alkali metal). We first note that the triplet state of (C60)-22 which was never considered previously is nearly degenerate with the singlet state, and the transition barrier between the two states is reasonably small. This explains the susceptibility increase with an increase in temperature and the magnetic phase transition in the process of the dimer to monomer phase transition.

Journal Article↗

PubMed2000

cAMP response element-binding protein-binding protein binds to human papillomavirus E2 protein and activates E2-dependent transcription.

cAMP response element-binding protein-binding protein (CBP) is a eucaryotic transcriptional co-activator that contains multiple protein-protein interaction domains for association with various transcription factors, components of the basal transcriptional apparatus, and other co-activator proteins. Here, we report that CBP is also a co-activator of the human papillomavirus (HPV) E2 protein, which is a sequence-specific transcription/replication factor. We provide biochemical, genetic, and functional evidence that CBP binds directly to HPV E2 in vivo and in vitro and activates E2-dependent transcription. Mutations in an amphipathic helix within HPV-18 E2 abolish its transcriptional activation properties and its ability to bind to CBP. Furthermore, the binding of CBP to E2 was shown to be necessary for E2-dependent transcription. Interestingly, the histone acetyltransferase activity of CBP plays a role in CBP activation of E2-dependent transcription.

Acetyltransferases↗

PubMed2000

Identification and developmental expression of par-6 gene in Xenopus laevis.

The par genes (partitioning defective) are required to establish polarity in the Caenorhabditis elegans embryo. We have identified the Xenopus homologue of C. elegans PAR-6 (XPAR-6). XPAR-6 is a protein of 377 amino acids with one PDZ domain which is involved in mediating protein-protein interactions. It shares 59% and 58% amino acid identity with the mouse and Drosophila PAR-6, respectively, and 54% overall identity with C. elegans PAR-6. Xpar-6 is expressed both maternally and zygotically. Xpar-6 is first detected in the animal half of the egg, and this pattern of expression persists into the cleavage and blastula stages. At the gastrula stage, the message is detected in animal pole area and in a broad domain of ventral region, but is excluded from dorsal region. With the onset of neurulation, the localized expression of Xpar-6 becomes more obvious, leading to it being enriched in the dorsolateral region along the lateral edges of neural plate and anterior presumptive head region surrounding the anterior border of neural plate. At late tailbud stage, Xpar-6 transcripts show localized expression throughout the head, labeling the branchial arches, eyes, otic vesicles and brain, while more posteriorly Xpar-6 labels the somites, pronephros, tail tip and proctodeum. Therefore, this analysis suggests that Xpar-6 has a regionalized pattern of expression during Xenopus early embryogenesis.

Amino Acid Sequence↗

PubMed2000

Tissue-specific chromatin structure of the phenobarbital-responsive unit and proximal promoter of CYP2B1/2 and modulation by phenobarbital.

Phenobarbital induction of transcription of CYP2B genes is mediated by an enhancer, termed a phenobarbital responsive unit (PBRU), approximately 2000 bp 5' of the transcription start site. To further delineate the mechanism of phenobarbital induction, protein binding in native chromatin and the nucleosomal structure of the PBRU and proximal promoter were examined in liver and kidney, in which the CYP2B1/2 genes are expressed and not expressed, respectively. Protein binding to the PBRU in kidney chromatin was not detected even though in vitro DNase I footprints were not detectably different with nuclear extracts from liver and kidney. Likewise, protein binding to regulatory motifs was not detected in the proximal promoter region in kidney chromatin. In liver chromatin, however, DNase I hypersensitivity and partial protection of the regulatory motifs from DNase I digestion or reaction with dimethyl sulfate was observed and phenobarbital treatment increased the hypersensitivity but only modestly affected protection. Low resolution Southern analysis of micrococcal nuclease-digested chromatin from untreated rats revealed micrococcal nuclease hypersensitive regions in the proximal promoter and PBRU regions in liver, but not in kidney. Phenobarbital treatment increased hyper-sensitivity in liver in both regions. Micrococcal nuclease hypersensitivity in the PBRU was largely restricted to a linker region between phased nucleosomes while in the proximal promoter hypersensitivity extended over approximately 200 bp suggesting disruption of a nucleosome in this region. These data indicate that in liver phenobarbital treatment substantially alters protein binding to regulatory motifs in the PBRU, while not greatly affecting such binding in the proximal promoter, and substantially alters chromatin structure in both regions, presumably as a result of chromatin modifying factors recruited to the PBRU. In the kidney, chromatin is probably in a closed conformation that prevents binding of regulatory factors.

Animals↗

PubMed2000

Diversity of the p70 killer cell inhibitory receptor (KIR3DL) family members in a single individual.

NK cells and some T cells express members of a multigenic family of killer cell inhibitory receptors (KIRs) including p70 KIR (KIR3DL) and p58 KIR (KIR2DL) family that recognize polymorphic class I MHC molecules on target cells and transmit an inhibitory signal to prevent killer cell-mediated cytoxicity. The cDNA sequences of p70 KIR family members reported so far suggest that the p70 KIR gene consists of a multigene complex and that each gene may exhibit certain degrees of polymorphism. However, it is not clear how diverse the repertoire of the p70 KIR family is, particularly in a single individual. To address this question in more detail and to determine some indication as to the origin of the diversity, we cloned p70 KIR cDNAs from a single individual. We identified nine new KIRs that are different from the previously reported ones. A comparison of the amino acid sequences with published sequences of p70 KIRs showed that two clones belonged to the KIR3DL1 family, five clones belonged to the KIR3DL2 family, one clone belonged to the KIR2DL4 family, and one clone appeared to be an alternatively spliced form of p70 KIR. These results suggested that the repertoire of p70 KIR family members in a single individual is highly diverse. It is not clear how the diverse receptors are generated in a single individual, but a comparison of amino acid sequences of p70 KIR family members suggested that some of them may be encoded by distinct genes or their alleles, while others may be generated by a recombination mechanism and/or an alternative splicing mechanism at the maturation of the mRNA transcripts.

Alternative Splicing↗

PubMed2000

Expression patterns of alpha-synuclein in human hematopoietic cells and in Drosophila at different developmental stages.

Alpha-synuclein, a presynaptic protein of the central nervous system, has been implicated in the synaptic events such as neuronal plasticity during development and learning, and neuronal degeneration under pathological conditions. As an effort to understand the biological function of alpha-synuclein, we examined the expression patterns of alpha-synuclein in various human hematopoietic cells, and in Drosophila at different developmental stages. The alpha-synuclein was ubiquitously expressed in all the tested hematopoietic cells including T cells, B cells, NK cells, and monocytes, as well as in the lymphoma cell lines, Jurkat and K562. A potential alpha-synuclein homologue was also expressed in Drosophila, and its expression appeared to be temporally and spatially regulated during development. Our data suggest that alpha-synuclein may function in invertebrates as well as in vertebrates and its function may not be restricted to the neuron.

Animals↗

PubMed2000

Effect of vehicles and pressure sensitive adhesives on the permeation of tacrine across hairless mouse skin.

The purpose of this study was to investigate the feasibility of developing transdermal drug delivery (TDD) system for tacrine used for treating the symptoms of Alzheimer's disease. The effects of various vehicles on the percutaneous absorption of tacrine in solution formulation and in pressure sensitive adhesive (PSA) matrix across the hairless mouse skin were evaluated using flow-through diffusion cell system at 37 degrees C. The permeation profiles of tacrine from solutions were different depending on vehicles used. The flux of tacrine increased significantly as its concentration in the solutions increased. The permeation rate of tacrine was higher in acrylic adhesives with hydroxyl functional group and without functional group than in polyisobutylene adhesive matrix. Incorporation of vehicles into the acrylic adhesive matrix significantly enhanced the permeation rate and shortened the lag time of tacrine. The maximum flux obtained from pressure sensitive adhesive matrix seemed to be high enough to obtain therapeutic effect.

Adhesives↗

PubMed2000

The itinerary of a vesicle component, Aut7p/Cvt5p, terminates in the yeast vacuole via the autophagy/Cvt pathways.

Aminopeptidase I (API) is delivered to the yeast vacuole by one of two alternative pathways, cytoplasm to vacuole targeting (Cvt) or autophagy, depending on nutrient conditions. Genetic, morphological, and biochemical studies indicate that the two pathways share many of the same molecular components. The Cvt pathway functions during vegetative growth, while autophagy is induced during starvation. Both pathways involve the formation of cytosolic vesicles that fuse with the vacuole. In either case, the mechanism of vesicle formation is not known. Autophagic uptake displays a greater capacity for cytosolic protein sequestration. This suggests the involvement of an inducible protein(s) that allows the vesicle-forming machinery to adapt to the increased degradative needs of the cell. We have analyzed the biosynthesis of Aut7p, a protein required for both pathways. We find Aut7p expression is induced by nitrogen starvation. Aut7p is degraded by a process dependent on both proteinase A and Cvt/autophagy components. Protease accessibility assays demonstrate that Aut7p is located within vesicles in strains defective in vesicle delivery or breakdown. Finally, the aut7/cvt5 mutant accumulates precursor API at a stage prior to vesicle completion. These data suggest that Aut7p is induced during autophagy and delivered to the vacuole together with precursor API by Cvt/autophagic vesicles.

Autophagy-Related Protein 8 Family↗

PubMed2000

Exon sharing of a novel human zinc-finger gene, ZIM2, and paternally expressed gene 3 (PEG3).

We have identified a novel human gene, ZIM2 (zinc-finger gene 2 from imprinted domain), located 25 kb downstream of PEG3 (paternally expressed gene 3). ZIM2 produces two different-size transcripts, 2.5 and 9.0 kb in length, with highest levels of expression in adult testis and modest levels in fetal kidney and brain. The 2.5-kb transcript of ZIM2 consists of 11 exons and encodes a Kruppel-type (C2H2) zinc-finger protein with a conserved Kruppel-associated box (KRAB) domain. Rapid amplification of cDNA ends and cDNA sequencing studies showed that ZIM2 and PEG3 transcripts share identical 5'-ends, composed of 7 small exons. Alternative splicing events connect these 7 exons either with the remaining 2 exons of PEG3 or with the remaining 4 exons of ZIM2. Interestingly, the third among the 7 shared exons exhibits sequence similarity to leucine-rich domains that are found at the N-terminal region of a subset of KRAB-containing zinc-finger genes. Sequencing of the 5'-termini of both transcripts indicates that ZIM2 and PEG3 share identical transcription start sites and may also share upstream regulatory elements, although the two genes show distinct patterns of tissue-specific expression.

Adult↗